Nucleic acid and corresponding protein entitled 213P1F11 useful in treatment and detection of cancer
A novel gene (designated 213P1F11) and its encoded protein, and variants thereof, are described wherein 213P1F11 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, 213P1F11 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 213P1F11 gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with 213P1F11 can be used in active or passive immunization.
[0001] Not applicable.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH[0002] Not applicable.
FIELD OF THE INVENTION[0003] The invention described herein relates to a gene and its encoded protein, termed 213P1F11, expressed in certain cancers, and to diagnostic and therapeutic methods and compositions useful in the management of cancers that express 213P1F11.
BACKGROUND OF THE INVENTION[0004] Cancer is the second leading cause of human death next to coronary disease. Worldwide, millions of people die from cancer every year. In the United States alone, as reported by the American Cancer Society, cancer causes the death of well over a half-million people annually, with over 1.2 million new cases diagnosed per year. While deaths from heart disease have been declining significantly, those resulting from cancer generally are on the rise. In the early part of the next century, cancer is predicted to become the leading cause of death.
[0005] Worldwide, several cancers stand out as the leading killers. In particular, carcinomas of the lung, prostate, breast, colon, pancreas, and ovary represent the primary causes of cancer death. These and virtually all other carcinomas share a common lethal feature. With very few exceptions, metastatic disease from a carcinoma is fatal. Moreover, even for those cancer patients who initially survive their primary cancers, common experience has shown that their lives are dramatically altered. Many cancer patients experience strong anxieties driven by the awareness of the potential for recurrence or treatment failure. Many cancer patients experience physical debilitations following treatment. Furthermore, many cancer patients experience a recurrence.
[0006] Worldwide, prostate cancer is the fourth most prevalent cancer in men. In North America and Northern Europe, it is by far the most common cancer in males and is the second leading cause of cancer death in men. In the United States alone, well over 30,000 men die annually of this disease—second only to lung cancer. Despite the magnitude of these figures, there is still no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy, surgical castration and chemotherapy continue to be the main treatment modalities. Unfortunately, these treatments are ineffective for many and are often associated with undesirable consequences.
[0007] On the diagnostic front, the lack of a prostate tumor marker that can accurately detect early-stage, localized tumors remains a significant limitation in the diagnosis and management of this disease. Although the serum prostate specific antigen (PSA) assay has been a very useful tool, however its specificity and general utility is widely regarded as lacking in several important respects.
[0008] Progress in identifying additional specific markers for prostate cancer has been improved by the generation of prostate cancer xenografts that can recapitulate different stages of the disease in mice. The LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immune deficient (SCID) mice and have exhibited the capacity to mimic the transition from androgen dependence to androgen independence (Klein et al., 1997, Nat. Med. 3:402). More recently identified prostate cancer markers include PCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res 1996 Sep 2 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad Sci USA. Dec. 7, 1999; 96(25): 14523-8) and prostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).
[0009] While previously identified markers such as PSA, PSM, PCTA and PSCA have facilitated efforts to diagnose and treat prostate cancer, there is need for the identification of additional markers and therapeutic largets for prostate and related cancers in order to further improve diagnosis and therapy.
[0010] Renal cell carcinoma (RCC) accounts for approximately 3 percent of adult malignancies. Once adenomas reach a diameter of 2 to 3 cm, malignant potential exists. In the adult, the two principal malignant renal tumors are renal cell adenocarcinoma and transitional cell carcinoma of the renal pelvis or ureter. The incidence of renal cell adenocarcinoma is estimated at more than 29,000 cases in the United States, and more than 11,600 patients died of this disease in 1998. Transitional cell carcinoma is less frequent, with an incidence of approximately 500 cases per year in the United States.
[0011] Surgery has been the primary therapy for renal cell adenocarcinoma for many decades. Until recently, metastatic disease has been refractory to any systemic therapy. With recent developments in systemic therapies, particularly immunotherapies, metastatic renal cell carcinoma may be approached aggressively in appropriate patients with a possibility of durable responses. Nevertheless, there is a remaining need for effective therapies for these patients.
[0012] Of all new cases of cancer in the United States, bladder cancer represents approximately 5 percent in men (fifth most common neoplasm) and 3 percent in women (eighth most common neoplasm). The incidence is increasing slowly, concurrent with an increasing older population. In 1998, there was an estimated 54,500 cases, including 39,500 in men and 15,000 in women. The age-adjusted incidence in the United States is 32 per 100,000 for men and 8 per 100,000 in women. The historic male/female ratio of 3:1 may be decreasing related to smoking patterns in women. There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800 in men and 3,900 in women). Bladder cancer incidence and mortality strongly increase with age and will be an increasing problem as the population becomes more elderly.
[0013] Most bladder cancers recur in the bladder. Bladder cancer is managed with a combination of transurethral resection of the bladder (TUR) and intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer points out the limitations of TUR. Most muscle-invasive cancers are not cured by TUR alone. Radical cystectomy and urinary diversion is the most effective means to eliminate the cancer but carry an undeniable impact on urinary and sexual function. There continues to be a significant need for treatment modalities that are beneficial for bladder cancer patients.
[0014] An estimated 130,200 cases of colorectal cancer occurred in 2000 in the United States, including 93,800 cases of colon cancer and 36,400 of rectal cancer. Colorectal cancers are the third most common cancers in men and women. Incidence rates declined significantly during 1992-1996 (−2.1% per year). Research suggests that these declines have been due to increased screening and polyp removal, preventing progression of polyps to invasive cancers. There were an estimated 56,300 deaths (47,700 from colon cancer, 8,600 from rectal cancer) in 2000, accounting for about 11% of all U.S. cancer deaths.
[0015] At present, surgery is the most common form of therapy for colorectal cancer, and for cancers that have not spread, it is frequently curative. Chemotherapy, or chemotherapy plus radiation, is given before or after surgery to most patients whose cancer has deeply perforated the bowel wall or has spread to the lymph nodes. A permanent colostomy (creation of an abdominal opening for elimination of body wastes) is occasionally needed for colon cancer and is infrequently required for rectal cancer. There continues to be a need for effective diagnostic and treatment modalities for colorectal cancer.
[0016] There were an estimated 164,100 new cases of lung and bronchial cancer in 2000, accounting for 14% of all U.S. cancer diagnoses. The incidence rate of lung and bronchial cancer is declining significantly in men, from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s, the rate of increase among women began to slow. In 1996, the incidence rate in women was 42.3 per 100,000.
[0017] Lung and bronchial cancer caused an estimated 156,900 deaths in 2000, accounting for 28% of all cancer deaths. During 1992-1996, mortality from lung cancer declined significantly among men (−1.7% per year) while rates for women were still significantly increasing (0.9% per year). Since 1987, more women have died each year of lung cancer than breast cancer, which, for over 40 years, was the major cause of cancer death in women. Decreasing lung cancer incidence and mortality rates most likely resulted from decreased smoking rates over the previous 30 years; however, decreasing smoking patterns among women lag behind those of men. Of concern, although the declines in adult tobacco use have slowed, tobacco use in youth is increasing again.
[0018] Treatment options for lung and bronchial cancer are determined by the type and stage of the cancer and include surgery, radiation therapy, and chemotherapy. For many localized cancers, surgery is usually the treatment of choice. Because the disease has usually spread by the time it is discovered, radiation therapy and chemotherapy are often needed in combination with surgery. Chemotherapy alone or combined with radiation is the treatment of choice for small cell lung cancer; on this regimen, a large percentage of patients experience remission, which in some cases is long lasting. There is however, an ongoing need for effective treatment and diagnostic approaches for lung and bronchial cancers.
[0019] An estimated 182,800 new invasive cases of breast cancer were expected to occur among women in the United States during 2000. Additionally, about 1,400 new cases of breast cancer were expected to be diagnosed in men in 2000. After increasing about 4% per year in the 1980s, breast cancer incidence rates in women have leveled off in the 1990s to about 110.6 cases per 100,000.
[0020] In the U.S. alone, there were an estimated 41,200 deaths (40,800 women, 400 men) in 2000 due to breast cancer. Breast cancer ranks second among cancer deaths in women. According to the most recent data, mortality rates declined significantly during 1992-1996 with the largest decreases in younger women, both white and black. These decreases were probably the result of earlier detection and improved treatment.
[0021] Taking into account the medical circumstances and the patient's preferences, treatment of breast cancer may involve lumpectomy (local removal of the tumor) and removal of the lymph nodes under the arm; mastectomy (surgical removal of the breast) and removal of the lymph nodes under the arm; radiation therapy; chemotherapy; or hormone therapy. Often, two or more methods are used in combination. Numerous studies have shown that, for early stage disease, long-term survival rates after lumpectomy plus radiotherapy are similar to survival rates after modified radical mastectomy. Significant advances in reconstruction techniques provide several options for breast reconstruction after mastectomy. Recently, such reconstruction has been done at the same time as the mastectomy.
[0022] Local excision of ductal carcinoma in situ (DCIS) with adequate amounts of surrounding normal breast tissue may prevent the local recurrence of the DCIS. Radiation to the breast and/or tamoxifen may reduce the chance of DCIS occurring in the remaining breast tissue. This is important because DCIS, if left untreated, may develop into invasive breast cancer. Nevertheless, there are serious side effects or sequelae to these treatments. There is, therefore, a need for efficacious breast cancer treatments.
[0023] There were an estimated 23,100 new cases of ovarian cancer in the United States in 2000. It accounts for 4% of all cancers among women and ranks second among gynecologic cancers. During 1992-1996, ovarian cancer incidence rates were significantly declining. Consequent to ovarian cancer, there were an estimated 14,000 deaths in 2000. Ovarian cancer causes more deaths than any other cancer of the female reproductive system.
[0024] Surgery, radiation therapy, and chemotherapy are treatment options for ovarian cancer. Surgery usually includes the removal of one or both ovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus (hysterectomy). In some very early tumors, only the involved ovary will be removed, especially in young women who wish to have children. In advanced disease, an attempt is made to remove all intra-abdominal disease to enhance the effect of chemotherapy. There continues to be an important need for effective treatment options for ovarian cancer.
[0025] There were an estimated 28,300 new cases of pancreatic cancer in the United States in 2000. Over the past 20 years, rates of pancreatic cancer have declined in men. Rates among women have remained approximately constant but may be beginning to decline. Pancreatic cancer caused an estimated 28,200 deaths in 2000 in the United States. Over the past 20 years, there has been a slight but significant decrease in mortality rates among men (about −0.9% per year) while rates have increased slightly among women.
[0026] Surgery, radiation therapy, and chemotherapy are treatment options for pancreatic cancer. These treatment options can extend survival and/or relieve symptoms in many patients but are not likely to produce a cure for most. There is a significant need for additional therapeutic and diagnostic options for pancreatic cancer.
SUMMARY OF THE INVENTION[0027] The present invention relates to a gene, designated 213P1F11, that has now been found to be over-expressed in the cancer(s) listed in Table I. Northern blot expression analysis of 213P1F11 gene expression in normal tissues shows a restricted expression pattern in adult tissues. The nucleotide (FIG. 2) and amino acid (FIG. 2, and FIG. 3) sequences of 213P1F11 are provided. The tissue-related profile of 213P1F11 in normal adult tissues, combined with the over-expression observed in the tissues listed in Table I, shows that 213P1F11 is aberrantly over-expressed in at least some cancers, and thus serves as a useful diagnostic, prophylactic, prognostic, and/or therapeutic target for cancers of the tissue(s) such as those listed in Table I.
[0028] The invention provides polynucleotides corresponding or complementary to all or part of the 213P1F11 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 213P1F11-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or more than 100 contiguous amino acids of a 213P1F11-related protein, as well as the peptides/proteins themselves; DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides or oligonucleotides complementary or having at least a 90% homology to the 213P1F11 genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 213P1F11 genes, mRNAs, or to 213P1F11-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding 213P1F11. Recombinant DNA molecules containing 213P1F11 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 213P1F11 gene products are also provided. The invention further provides antibodies that bind to 213P1F11 proteins and polypeptide fragments thereof, including polyclonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled with a detectable marker or therapeutic agent. In certain embodiments there is a proviso that the entire nucleic acid sequence of FIG. 2 is not encoded and/or the entire amino acid sequence of FIG. 2 is not prepared. In certain embodiments, the entire nucleic acid sequence of FIG. 2 is encoded and/or the entire amino acid sequence of FIG. 2 is prepared, either of which are in respective human unit dose forms.
[0029] The invention further provides methods for detecting the presence and status of 213P1F11 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 213P1F11. A typical embodiment of this invention provides methods for monitoring 213P1F11 gene products in a tissue or hematology sample having or suspected of having some form of growth dysregulation such as cancer.
[0030] The invention further provides various immunogenic or therapeutic compositions and strategies for treating cancers that express 213P1F11 such as cancers of tissues listed in Table I, including therapies aimed at inhibiting the transcription, translation, processing or function of 213P1F11 as well as cancer vaccines. In one aspect, the invention provides compositions, and methods comprising them, for treating a cancer that expresses 213P1F11 in a human subject wherein the composition comprises a carrier suitable for human use and a human unit dose of one or more than one agent that inhibits the production or function of 213P1F11. Preferably, the carrier is a uniquely human carrier. In another aspect of the invention, the agent is a moiety that is immunoreactive with 213P1F11 protein. Non-limiting examples of such moieties include, but are not limited to, antibodies (such as single chain, monoclonal, polyclonal, humanized, chimeric, or human antibodies), functional equivalents thereof (whether naturally occurring or synthetic), and combinations thereof. The antibodies can be conjugated to a diagnostic or therapeutic moiety. In another aspect, the agent is a small molecule as defined herein.
[0031] In another aspect, the agent comprises one or more than one peptide which comprises a cytotoxic T lymphocyte (CTL) epitope that binds an HLA class I molecule in a human to elicit a CTL response to 213P1F11 and/or one or more than one peptide which comprises a helper T lymphocyte (HTL) epitope which binds an HLA class II molecule in a human to elicit an HTL response. The peptides of the invention may be on the same or on one or more separate polypeptide molecules. In a further aspect of the invention, the agent comprises one or more than one nucleic acid molecule that expresses one or more than one of the CTL or HTL response stimulating peptides as described above. In yet another aspect of the invention, the one or more than one nucleic acid molecule may express a moiety that is immunologically reactive with 213P1F11 as described above. The one or more than one nucleic acid molecule may also be, or encodes, a molecule that inhibits production of 213P1F11. Non-limiting examples of such molecules include, but are not limited to, those complementary to a nucleotide sequence essential for production of 213P1F11 (e.g. antisense sequences or molecules that form a triple helix with a nucleotide double helix essential for 213P1F11 production) or a ribozyme effective to lyse 213P1F11 mRNA.
BRIEF DESCRIPTION OF THE FIGURES[0032] FIG. 1. The 213P1F11 SSH sequence of 166 nucleotides.
[0033] FIG. 2. The cDNA (SEQ ID. NO.:______) and amino acid sequence (SEQ ID. NO.:______) of 213P1F11 variant 1 clone CASP14-BrCl (also called “213P1F11 v.1” or “213P1F11 variant 1” or “213P1F11”) is shown in FIG. 2A. The start methionine is underlined. The open reading frame extends from nucleic acid 404-1132 including the stop codon. The cDNA (SEQ ID. NO.:______) and amino acid sequence (SEQ ID. NO.:______) of 213P1F11 variant 2 (also called “213P1F11 v.2”) is shown in FIG. 2B. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 409-1096 including the stop codon. The cDNA (SEQ ID. NO.:______) and amino acid sequence (SEQ ID. NO.:______) of 213P1F11 variant 3 (also called “213P1F11 v.3”) is shown in FIG. 2C. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 404-844 including the stop codon. The cDNA (SEQ ID. NO.:______) and amino acid sequence (SEQ ID. NO.:______) of 213P1F11 variant 4 (also called “213P1F11 v.4”) is shown in FIG. 2D. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 1-966 including the stop codon. The cDNA (SEQ ID. NO.:______) and amino acid sequence (SEQ ID. NO.:______) of 213P1F11 variant 5 (also called “213P1F11 v.5”) is shown in FIG. 2E. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 404-1132 including the stop codon. The cDNA (SEQ ID. NO.:______) and amino acid sequence (SEQ ID. NO.:______) of 213P1F11 variant 6 (also called “213P1F11 variant v.6”) is shown in FIG. 2F. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 404-1132 including the stop codon. The cDNA (SEQ ID. NO.:______) and amino acid sequence (SEQ ID. NO.:______) of 213P1F11 variant 7 (also called “213P1F11 v.7”) is shown in FIG. 2G. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 404-1132 including the stop codon. The cDNA (SEQ ID. NO.:______) and amino acid sequence (SEQ ID. NO.:______) of 213P1F11 variant 8 (also called “213P1F11 v.8”) is shown in FIG. 2H. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 404-1132 including the stop codon. As used herein, a reference to 213P1F11 includes all variants thereof, including those shown in FIG. 10.
[0034] FIG. 3. Amino acid sequence of 213P1F11 v.1 (SEQ ID. NO.:______) is shown in FIG. 3A; it has 242 amino acids. The amino acid sequence of 213P1F11 v.2 (SEQ ID. NO.:______) is shown in FIG. 3B; it has 230 amino acids. The amino acid sequence of 213P1F11 v.3 (SEQ ID. NO.:______) is shown in FIG. 3C; it has 146 amino acids. The amino acid sequence of 213P1F11 v.4 (SEQ ID. NO.:______) is shown in FIG. 3D; it has 321 amino acids. The amino acid sequence of 213P1F11 v.5 (SEQ ID. NO.:______) is shown in FIG. 3E; it has 242 amino acids. The amino acid sequence of 213P1F11 v.6 (SEQ ID. NO.:______) is shown in FIG. 3F; it has 242 amino acids. As used herein, a reference to 213P1F11 includes all variants thereof, including those shown in FIG. 11.
[0035] FIG. 4. The nucleic acid sequence alignment of 213P1F11 v.1 with human Caspase-14 (gi 6912286)precursor mRNA is shown in FIG. 4A. The amino acid sequence alignment of 213P1F11 v.1 with human Caspase-14 (gi 6912286) mRNA is shown in FIG. 4B. The amino acid sequence alignment of 213P1F11 v.1 with mouse Caspase-14 (gi 6753280) mRNA is shown in FIG. 4C. The amino acid sequence alignment of 213P1F11 v.2 with human Caspase-14 (gi 6912286) mRNA is shown in FIG. 4D. The amino acid sequence alignment of 213P1F11 v.3 with human Caspase-14 (gi 6912286) mRNA is shown in FIG. 4E. The amino acid sequence alignment of 213P1F11 v.2 with mouse caspase 14 (gi 6753280) mRNA is shown in FIG. 4F. The amino acid sequence alignment of 213P1F11 v.4 with human Caspase-14 (gi 6912286) mRNA is shown in FIG. 4G.
[0036] FIG. 5. Hydrophilicity amino acid profile of A) 213P1F11 variant 1, B) 213P1F11 variant 2, C) 213P1F11 variant 3 and D) 213P1F11 variant 4, determined by computer algorithm sequence analysis using the method of Hopp and Woods (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828) accessed on the Protscale website (www.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
[0037] FIG. 6. Hydropathicity amino acid profile of A) 213P1F11 variant 1, B) 213P1F11 variant 2, C) 213P1F11 variant 3 and D) 213P1F11 variant 4, determined by computer algorithm sequence analysis using the method of Kyte and Doolittle (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132) accessed on the ProtScale website (www.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
[0038] FIG. 7. Percent accessible residues amino acid profile of A) 213P1F11 variant 1, B) 213P1F11 variant 2, C) 213P1F11 variant 3 and D) 213P1F11 variant 4, determined by computer algorithm sequence analysis using the method of Janin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScale website (www.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
[0039] FIG. 8. Average flexibility amino acid profile of A) 213P1F11 variant 1, B) 213P1F11 variant 2, C) 213P1F11 variant 3 and D) 213P1F11 variant 4, determined by computer algorithm sequence analysis using the method of Bhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255) accessed on the ProtScale website (www.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
[0040] FIG. 9. Beta-turn amino acid profile of A) 213P1F11 variant 1, B) 213P1F11 variant 2, C) 213P1F11 variant 3 and D) 213P1F11 variant 4, determined by computer algorithm sequence analysis using the method of Deleage and Roux (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294) accessed on the ProtScale website (www.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
[0041] FIG. 10. Schematic display of nucleotide variants of 213P1F11. Variants 213P1F11 v.2 and v.3 are splice variants. Variant 213P1F11 v.4 is an alternative transcript. Others are Single Nucleotide Polymorphism (also called “SNP”) variants, which could also occur in any of the transcript variants that contains the base pairs. Numbers in “( )” underneath the box correspond to those of 213P1F11 v.1. The black boxes show the same sequence as 213P1F11 v.1. SNPs are indicated above the box.
[0042] FIG. 11. Schematic display of protein variants of 213P1F11. Nucleotide variants 213P1F11 v.1 though v.6 in FIG. 10 code for protein variants 213P1F11 v.1 through 213P1F11 v.6, respectively. Variants 213P1F11 v.7 through v.10 code the same protein as variant 213P1F11 v.1. Protein variants 213P1F11 v.5 and v.6 are variants with single amino acid variations, which may exist in transcript variants 213P1F11 v.2 through 4. The black boxes show the same sequence as 213P1F11 v.1. The numbers in “( )” underneath the box correspond to those of 213P1F11 v.1. Single amino acid differences are indicated above the box.
[0043] FIG. 12. Secondary structure prediction for 213P1F11 variants 1 through 4. The secondary structures of 213P1F11 variant 1 (A), variant 2 (B), variant 3 (C), and variant 4 (D) were predicted using the HNN—Hierarchical Neural Network method (Guermeur, 1997, http://pbil.ibcp.fr/cgi-bin/npsa automat.pl?page=npsa_nn.html), accessed from the ExPasy molecular biology server (http://www.expasy.ch/tools/). This method predicts the presence and location of alpha helices, extended strands, and random coils from the primary protein sequence. The percent of the protein in a given secondary structure is also listed for each variant.
[0044] FIG. 13. Exon compositions of transcript variants of 213P11F11. Variant 213P1F11 v.2 and v.3 are splice variants. Variant 213P1F11 v.4 is an alternative transcript. Compared with 213P1F11 v.1, 213P1F11 v.2 has a longer (+74 bp at 5′ end) exon 6 and variant 213P1F11 v.3 has a longer (+68 bp at 5′ end) exon 5. Variant 213P1F11 v.4 has three different exons. Relative locations of exons from all variants on the chromosome are shown at the bottom. Numbers in “( )” underneath the box correspond to those of 213P1F11 v.1. Black boxes show the same sequence as 213P1F11 v.1. Intron lengths are not proportional.
[0045] FIG. 14. Expression of 213P1F11 by RT-PCR. First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), LAPC xenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI), bladder cancer pool, breast cancer pool, and cancer metastasis pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 213P1F11, was performed at 26 and 30 cycles of amplification. Results show strong expression of 213P1F11 in bladder cancer pool, breast cancer pool, xenograft pool, and cancer metastasis pool.
[0046] FIG. 15. Expression of 213P1F11 v.1 compared to 213P1F11 v.2 in patient cancer samples by RT-PCR. To determine the relative expression of 213P1F11 v.1 compared to 213P1F11 v.2 in human cancers, primers were designed flanking the insertion in 213P1F11 v.2. Using these primers, amplification of 213P1F11 v.1 will generate a PCR fragment of 165 bp, whereas 213P1F11 v.2 will generate a PCR fragment of 249 bp as depicted in the figure. First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), bladder cancer pool, breast cancer pool, LAPC xenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI), and 213P1F11 v.1 plasmid control. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers depicted above, was performed at 35 cycles of amplification. Results show strong expression of 213P1F11 v.1 in bladder cancer pool, breast cancer pool, LAPC xenograft pool, and the plasmid positive control. A lower expression of the 249 bp 213P1F11 v.2 product was detected in breast cancer pool, LAPC xenograft pool, and to lower extent in bladder cancer pool.
[0047] FIG. 16. Expression of 213P1F11 in normal tissues. Three multiple tissue northern blots (A and B, Clontech; C, OriGene) with 2 ug of mRNA/lane were probed with the 213P1F11 SSH fragment. Size standards in kilobases (kb) are indicated on the side. Results show strong expression of 213P1F11 only in skin tissue. A weak transcript is detected in normal thymus but not in the other tissues tested.
[0048] FIG. 17. Expression of 213P1F11 in bladder cancer patient tissues. RNA was extracted from normal bladder (N), bladder cancer cell lines (UM-UC-3 and SCaBER), bladder cancer patient tumors (T) and normal tissue adjacent to bladder cancer (NAT). Northern blots with 10 ug of total RNA were probed with the 213P1F11 SSH fragment. Size standards in kilobases are indicated on the side. Results show strong expression of 213P1F11 in the bladder tumor tissues but not in normal bladder nor in the bladder cancer cell lines.
[0049] FIG. 18. Expression of 213P1F11 in prostate cancer xenografts. RNA was extracted from normal prostate, LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI prostate cancer xenografts. Northern blot with 10 &mgr;g of total RNA/lane was probed with 213P1F11 SSH sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of 213P1F11 in the LAPC-9AI xenograft, but not in the other xenografts nor in normal prostate.
[0050] FIG. 19. Expression of 213P1F11 in breast cancer patient tissues. RNA was extracted from normal breast (N), breast cancer cell lines (DU4475, MCF7 and CAMA-1), breast cancer patient tumors (T) and breast cancer metastasis to lymph node (Met). Northern blots with 10 ug of total RNA were probed with the 213P1F11 SSH fragment. Size standards in kilobases are indicated on the side. Results show strong expression of 213P1F11 in the breast tumor tissues as well as in the cancer metastasis specimen. Weak expression was also detected in the CAMA-1 cell line, but not in the other 2 breast cancer cell lines tested.
DETAILED DESCRIPTION OF THE INVENTION[0051] Outline of Sections
[0052] I.) Definitions
[0053] II.) 213P1F11 Polynucleotides
[0054] II.A.) Uses of 213P1F11 Polynucleotides
[0055] II.A.1.) Monitoring of Genetic Abnormalities
[0056] II.A.2.) Antisense Embodiments
[0057] II.A.3.) Primers and Primer Pairs
[0058] II.A.4.) Isolation of 213P1F11-Encoding Nucleic Acid Molecules
[0059] I.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
[0060] III.) 213P1F11-related Proteins
[0061] III.A.) Motif-bearing Protein Embodiments
[0062] III.B.) Expression of 213P1F11-related Proteins
[0063] III.C.) Modifications of 213P1F11-related Proteins
[0064] III.D.) Uses of 213P1F11-related Proteins
[0065] IV.) 213P1F11 Antibodies
[0066] V.) 213P1F11 Cellular Immune Responses
[0067] VI.) 213P1F11 Transgenic Animals
[0068] VII.) Methods for the Detection of 213P1F11
[0069] VIII.) Methods for Monitoring the Status of 213P1F11-related Genes and Their Products
[0070] IX.) Identification of Molecules That Interact With 213P1F11
[0071] X.) Therapeutic Methods and Compositions
[0072] X.A.) Anti-Cancer Vaccines
[0073] X.B.) 213P1F11 as a Target for Antibody-Based Therapy
[0074] X.C.) 213P1F11 as a Target for Cellular Immune Responses
[0075] X.C.1. Minigene Vaccines
[0076] X.C.2. Combinations of CTL Peptides with Helper Peptides
[0077] X.C.3. Combinations of CTL Peptides with T Cell Priming Agents
[0078] X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides
[0079] X.D.) Adoptive Immunotherapy
[0080] X.E.) Administration of Vaccines for Therapeutic or Prophylactic Purposes
[0081] XI.) Diagnostic and Prognostic Embodiments of 213P1F11.
[0082] XII.) Inhibition of 213P1F11 Protein Function
[0083] XII.A.) Inhibition of 213P1F11 With Intracellular Antibodies
[0084] XII.B.) Inhibition of 213P1F11 with Recombinant Proteins
[0085] XII.C.) Inhibition of 213P1F11 Transcription or Translation
[0086] XII.D.) General Considerations for Therapeutic Strategies
[0087] XIII.) KITS
[0088] I.) Definitions:
[0089] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.
[0090] The terms “advanced prostate cancer”, “locally advanced prostate cancer”, “advanced disease” and “locally advanced disease” mean prostate cancers that have extended through the prostate capsule, and are meant to include stage C disease under the American Urological Association (AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients having clinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically identified by palpable evidence of induration beyond the lateral border of the prostate, or asymmetry or induration above the prostate base. Locally advanced prostate cancer is presently diagnosed pathologically following radical prostatectomy if the tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades the seminal vesicles.
[0091] “Altering the native glycosylation paftem” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence 213P1F11 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence 213P1F11. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
[0092] The term “analog” refers to a molecule which is structurally similar or shares similar or corresponding attributes with another molecule (e.g. a 213P1F11-related protein). For example an analog of a 213P1F11 protein can be specifically bound by an antibody or T cell that specifically binds to 213P1F11.
[0093] The term “antibody” is used in the broadest sense. Therefore an “antibody” can be naturally occurring or man-made such as monoclonal antibodies produced by conventional hybridoma technology. Anti-213P1F11 antibodies comprise monoclonal and polyclonal antibodies as well as fragments containing the antigen-binding domain and/or one or more complementarity determining regions of these antibodies.
[0094] An “antibody fragment” is defined as at least a portion of the variable region of the immunoglobulin molecule that binds to its target, i.e., the antigen-binding region. In one embodiment it specifically covers single anti-213P1F11 antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-213P1F11 antibody compositions with polyepitopic specificity.
[0095] The term “codon optimized sequences” refers to nucleotide sequences that have been optimized for a particular host species by replacing any codons having a usage frequency of less than about 20%. Nucleotide sequences that have been optimized for expression in a given host species by elimination of spurious polyadenylation sequences, elimination of exon/intron splicing signals, elimination of transposon-like repeats and/or optimization of GC content in addition to codon optimization are referred to herein as an “expression enhanced sequences.”
[0096] The term “cytotoxic agent” refers to a substance that inhibits or prevents the expression activity of cells, function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof Examples of cytotoxic agents include, but are not limited to maytansinoids, yttrium, bismuth, ricin, ricin A-chain, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu. Antibodies may also be conjugated to an anti-cancer pro-drug activating enzyme capable of converting the pro-drug to its active form.
[0097] The term “homolog” refers to a molecule which exhibits homology to another molecule, by for example, having sequences of chemical residues that are the same or similar at corresponding positions.
[0098] “Human Leukocyte Antigen” or “HLA” is a human class I or class II Major Histocompatibility Complex (MHC) protein (see, e.g., Stites, et al., IMMUNOLOGY, 8TH ED., Lange Publishing, Los Altos, Calif. (1994).
[0099] The terms “hybridize”, “hybridizing”, “hybridizes” and the like, used in the context of polynucleotides, are meant to refer to conventional hybridization conditions, preferably such as hybridization in 50% formamide/6×SSC/0.1% SDS/100 &mgr;g/ml ssDNA, in which temperatures for hybridization are above 37 degrees C. and temperatures for washing in 0.1× SSC/0.1% SDS are above 55 degrees C.
[0100] The phrases “isolated” or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment. For example, a polynucleotide is said to be “isolated” when it is substantially separated from contaminant polynucleotides that correspond or are complementary to genes other than the 213P1F11 genes or that encode polypeptides other than 213P1F11 gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 213P1F11 polynucleotide. A protein is said to be “isolated,” for example, when physical, mechanical or chemical methods are employed to remove the 213P1F11 proteins from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated 213P1F11 protein. Alternatively, an isolated protein can be prepared by chemical means.
[0101] The term “mammal” refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.
[0102] The terms “metastatic prostate cancer” and “metastatic disease” mean prostate cancers that have spread to regional lymph nodes or to distant sites, and are meant to include stage D disease under the AUA system and stage T×N×M+ under the TNM system. As is the case with locally advanced prostate cancer, surgery is generally not indicated for patients with metastatic disease, and hormonal (androgen ablation) therapy is a preferred treatment modality. Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of treatment initiation. Approximately half of these androgen-refractory patients die within 6 months after developing that status. The most common site for prostate cancer metastasis is bone. Prostate cancer bone metastases are often osteoblastic rather than osteolytic (i.e., resulting in net bone formation). Bone metastases are found most frequently in the spine, followed by the femur, pelvis, rib cage, skull and humerus. Other common sites for metastasis include lymph nodes, lung, liver and brain. Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or bone lesion biopsy.
[0103] The term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts.
[0104] A “motif”, as in biological motif of a 213P1F11-related protein, refers to any pattern of amino acids forming part of the primary sequence of a protein, that is associated with a particular function (e.g. protein-protein interaction, protein-DNA interaction, etc) or modification (e.g. that is phosphorylated, glycosylated or amidated), or localization (e.g. secretory sequence, nuclear localization sequence, etc.) or a sequence that is correlated with being immunogenic, either humorally or cellularly. A motif can be either contiguous or capable of being aligned to certain positions that are generally correlated with a certain function or property. In the context of HLA motifs, “motif” refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule. Peptide motifs for HLA binding are typically different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor residues.
[0105] A “pharmaceutical excipient” comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservative, and the like.
[0106] “Pharmaceutically acceptable” refers to a non-toxic, inert, and/or composition that is physiologically compatible with humans or other mammals.
[0107] The term “polynucleotide” means a polymeric form of nucleotides of at least 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, and is meant to include single and double stranded forms of DNA and/or RNA. In the art, this term if often used interchangeably with “oligonucleotide”. A polynucleotide can comprise a nucleotide sequence disclosed herein wherein thymidine (T), as shown for example in FIG. 2, can also be uracil (U); this definition pertains to the differences between the chemical structures of DNA and RNA, in particular the observation that one of the four major bases in RNA is uracil (U) instead of thymidine (T).
[0108] The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or 8 amino acids. Throughout the specification, standard three letter or single letter designations for amino acids are used. In the art, this term is often used interchangeably with “peptide” or “protein”.
[0109] An HLA “primary anchor residue” is an amino acid at a specific position along a peptide sequence which is understood to provide a contact point between the immunogenic peptide and the HLA molecule. One to three, usually two, primary anchor residues within a peptide of defined length generally defines a “motif” for an immunogenic peptide. These residues are understood to fit in close contact with peptide binding groove of an HLA molecule, with their side chains buried in specific pockets of the binding groove. In one embodiment, for example, the primary anchor residues for an HLA class I molecule are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a 8, 9, 10, 11, or 12 residue peptide epitope in accordance with the invention. In another embodiment, for example, the primary anchor residues of a peptide that will bind an HLA class II molecule are spaced relative to each other, rather than to the termini of a peptide, where the peptide is generally of at least 9 amino acids in length. The primary anchor positions for each motif and supermotif are set forth in Table IV. For example, analog peptides can be created by altering the presence or absence of particular residues in the primary and/or secondary anchor positions shown in Table IV. Such analogs are used to modulate the binding affinity and/or population coverage of a peptide comprising a particular HLA motif or supermotif.
[0110] A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular manipulation in vitro.
[0111] Non-limiting examples of small molecules include compounds that bind or interact with 213P1F11, ligands including hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit 213P1F11 protein function. Such non-limiting small molecules preferably have a molecular weight of less than about 10 kDa, more preferably below about 9, about 8, about 7, about 6, about 5 or about 4 kDa. In certain embodiments, small molecules physically associate with, or bind, 213P1F11 protein; are not found in naturally occurring metabolic pathways; and/or are more soluble in aqueous than non-aqueous solutions “Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured nucleic acid sequences to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
[0112] “Stringent conditions” or “high stringency conditions”, as defined herein, are identified by, but not limited to, those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 &mgr;g/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium. citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C. “Moderately stringent conditions” are described by, but not limited to, those in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
[0113] An HLA “supermotif” is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles.
[0114] As used herein “to treat” or “therapeutic” and grammatically related terms, refer to any improvement of any consequence of disease, such as prolonged survival, less morbidity, and/or a lessening of side effects which are the byproducts of an alternative therapeutic modality; full eradication of disease is not required.
[0115] A “transgenic animal” (e.g., a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A “transgene” is a DNA that is integrated into the genome of a cell from which a transgenic animal develops.
[0116] As used herein, an HLA or cellular immune response “vaccine” is a composition that contains or encodes one or more peptides of the invention. There are numerous embodiments of such vaccines, such as a cocktail of one or more individual peptides; one or more peptides of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such individual peptides or polypeptides, e.g., a minigene that encodes a polyepitopic peptide. The “one or more peptides” can include any whole unit integer from 1-242 or more, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, or 242 or more peptides of the invention. The peptides or polypeptides can optionally be modified, such as by lipidation, addition of targeting or other sequences. HLA class I peptides of the invention can be admixed with, or linked to, HLA class II peptides, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes. HLA vaccines can also comprise peptide-pulsed antigen presenting cells, e.g., dendritic cells.
[0117] The term “variant” refers to a molecule that exhibits a variation from a described type or norm, such as a protein that has one or more different amino acid residues in the corresponding position(s) of a specifically described protein (e.g. the 213P1F11 protein shown in FIG. 2 or FIG. 3. An analog is an example of a variant protein. Splice isofomis and single nucleotides polymorphisms (SNPs) are further examples of variants.
[0118] The “213P1F11-related proteins” of the invention include those specifically identified herein, as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined herein or readily available in the art. Fusion proteins that combine parts of different 213P1F11 proteins or fragments thereof, as well as fusion proteins of a 213P1F11 protein and a heterologous polypeptide are also included. Such 213P1F11 proteins are collectively referred to as the 213P1F11-related proteins, the proteins of the invention, or 213P1F11. The term “213P1F11-related protein” refers to a polypeptide fragment or a 213P1F11 protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids; or, at least 30, 35,40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, or 242 or more amino acids.
[0119] II.) 213P1F11 Polynucleotides
[0120] One aspect of the invention provides polynucleotides corresponding or complementary to all or part of a 213P1F11 gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding a 213P1F11-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to a 213P1F11 gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to a 213P1F11 gene, mRNA, or to a 213P1F11 encoding polynucleotide (collectively, “213P1F11 polynucleotides”). In all instances when referred to in this section, T can also be U in FIG. 2.
[0121] Embodiments of a 213P1F11 polynucleotide include: a 213P1F11 polynucleotide having the sequence shown in FIG. 2, the nucleotide sequence of 213P1F11 as shown in FIG. 2 wherein T is U; at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2 where T is U. For example, embodiments of 213P1F11 nucleotides comprise, without limitation:
[0122] (I) a polynucleotide comprising, consisting essentially of, or consisting of a sequence as shown in FIG. 2 (SEQ ID NO:______), wherein T can also be U;
[0123] (II) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2 (SEQ ID NO:______), from nucleotide residue number 404 through nucleotide residue number 1132, including the stop codon, wherein T can also be U;
[0124] (III) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2B (SEQ ID NO:______), from nucleotide residue number 404 through nucleotide residue number 1096, including the stop codon, wherein T can also be U;
[0125] (IV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2C (SEQ ID NO:______), from nucleotide residue number 404 through nucleotide residue number 844, including the a stop codon, wherein T can also be U;
[0126] (V) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2D (SEQ ID NO:______), from nucleotide residue number 1 through nucleotide residue number 966, including the stop codon, wherein T can also be U;
[0127] (VI) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2E (SEQ ID NO:______), from nucleotide residue number 404 through nucleotide residue number 1132, including the stop codon, wherein T can also be U;
[0128] (VII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2F (SEQ ID NO:______), from nucleotide residue number 404 through nucleotide residue number 1132, including the stop codon, wherein T can also be U;
[0129] (VIII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2G (SEQ ID NO:______), from nucleotide residue number 404 through nucleotide residue number 1132, including the stop codon, wherein T can also be U;
[0130] (IX) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2H (SEQ ID NO:______), from nucleotide residue number 404 through nucleotide residue number 1132, including the stop codon, wherein T can also be U;
[0131] (X) a polynucleotide that encodes a 213P1F11-related protein that is at least 90% homologous to an entire amino acid sequence shown in FIGS. 2A-H (SEQ ID NO:______);
[0132] (XI) a polynucleotide that encodes a 213P1F11-related protein that is at least 90% identical to an entire amino acid sequence shown in FIGS. 2A-H (SEQ ID NO:______);
[0133] (XII) a polynucleotide that encodes at least one peptide set forth in Tables V-XIX;
[0134] (XIII) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of FIG. 3A in any whole number increment up to 242 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5A; or of FIG. 3B in any whole number increment up to 230 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5B; or of FIG. 3C in any whole number increment up to 146 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5C; or of FIG. 3D in any whole number increment up to 321 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5D;
[0135] (XIV) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of FIG. 3A in any whole number increment up to 242 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 6A; or of FIG. 3B in any whole number increment up to 230 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 6B; or of FIG. 3C in any whole number increment up to 146 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 6C; or of FIG. 3D in any whole number increment up to 321 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 6D;
[0136] (XV) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of FIG. 3A in any whole number increment up to 242 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7A; or of FIG. 3B in any whole number increment up to 230 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7B; or of FIG. 3C in any whole number increment up to 146 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7C; or of FIG. 3D in any whole number increment up to 321 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7D;
[0137] (XVI) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of FIG. 3A in any whole number increment up to 242 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile of FIG. 8A; or of FIG. 3B in any whole number increment up to 230 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile of FIG. 8B; or of FIG. 3C in any whole number increment up to 146 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile of FIG. 8C; or of FIG. 3D in any whole number increment up to 321 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile of FIG. 8D;
[0138] (XVII) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of FIG. 3A in any whole number increment up to 242 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIG. 9A; or of FIG. 3B in any whole number increment up to 230 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIG. 9B; or of FIG. 3C in any whole number increment up to 146 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIG. 9C; or of FIG. 3D in any whole number increment up to 321 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIG. 9D;
[0139] (XVIII) a polynucleotide that is fully complementary to a polynucleotide of any one of (I)-(XVII).
[0140] (XIX) a peptide that is encoded by any of (I)-(XVIII); and
[0141] (XXI) a polynucleotide of any of (I)-(XVIII) or peptide of (XIX) together with a pharmaceutical excipient and/or in a human unit dose form.
[0142] As used herein, a range is understood to specifically disclose all whole unit positions thereof.
[0143] Typical embodiments of the invention disclosed herein include 213P1F11 polynucleotides that encode specific portions of 213P1F11 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:
[0144] (a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 230, 235, 240, or 242 or more contiguous amino acids of 213P1F11.
[0145] (b) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, or 230 contiguous amino acids of variant 2;
[0146] (c) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 146 contiguous amino acids of variant 3; or
[0147] (d) 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 146 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, or 321 contiguous amino acids of variant 4.
[0148] For example, representative embodiments of the invention disclosed herein include: polynucleotides and their encoded peptides themselves encoding about amino acid 1 to about amino acid 10 of the 213P1F11 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 213P1F11 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 213P1F11 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 213P1F11 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the 213P1F11 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 50 to about amino acid 60 of the 213P1F11 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the 213P1F11 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 70 to about amino acid 80 of the 213P1F11 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 80 to about amino acid 90 of the 213P1F11 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90 to about amino acid 100 of the 213P1F11 protein shown in FIG. 2 or FIG. 3, in increments of about 10 amino acids, ending at the carboxyl terminal amino acid set forth in FIG. 2 or FIG. 3. Accordingly polynucleotides encoding portions of the amino acid sequence (of about 10 amino acids), of amino acids 100 through the carboxyl terminal amino acid of the 213P1F11 protein are embodiments of the invention. Wherein it is understood that each particular amino acid position discloses that position plus or minus five amino acid residues.
[0149] Polynucleotides encoding relatively long portions of a 213P1F11 protein are also within the scope of the invention. For example, polynucleotides encoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the 213P1F11 protein “or variant” shown in FIG. 2 or FIG. 3 can be generated by a variety of techniques well known in the art. These polynucleotide fragments can include any portion of the 213P1F11 sequence as shown in FIG. 2.
[0150] Additional illustrative embodiments of the invention disclosed herein include 213P1F11 polynucleotide fragments encoding one or more of the biological motifs contained within a 213P1F11 protein “or variant” sequence, including one or more of the motif-bearing subsequences of a 213P1F11 protein “or variant” set forth in Tables V-XIX.
[0151] Note that to determine the starting position of any peptide set forth in Tables V-XIX (collectively HLA Peptide Tables) respective to its parental protein, e.g., variant 1, variant 2, etc., reference is made to three factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Peptides in Table XXIX. Generally, a unique Search Peptide is used to obtain HLA peptides of a particular for a particular variant. The position of each Search Peptide relative to its respective parent molecule is listed in Table XXIX. Accordingly if a Search Peptide begins at position “X”, one must add the value “X−1” to each position in Tables V-XIX to obtain the actual position of the HLA peptides in their parental molecule. For example if a particular Search Peptide begins at position 150 of is parental molecule, one must add 150−1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.
[0152] One embodiment of the invention comprises an HLA peptide, that occurs at least twice in Tables V-XIX collectively, or an oligonucleotide that encodes the HLA peptide. Another embodiment of the invention comprises an HLA peptide that occurs at least once in Tables V-XVIII and at least once in table XIX, or an oligonucleotide that encodes the HLA peptide.
[0153] Another embodiment of the invention is antibody epitopes which comprise a peptide regions, or an oligonucleotide encoding the peptide region, that has one two, three, four, or five of the following characteristics:
[0154] i) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Hydrophilicity profile of FIG. 5;
[0155] ii) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equal to 0.0, in the Hydropathicity profile of FIG. 6;
[0156] iii) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Percent Accessible Residues profile of FIG. 7;
[0157] iv) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Average Flexibility profile of FIG. 8; or
[0158] v) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Beta-turn profile of FIG. 9.
[0159] In another embodiment, typical polynucleotide fragments of the invention encode one or more of the regions of 213P1F11 protein or variant that exhibit homology to a known molecule. In another embodiment of the invention, typical polynucleotide fragments can encode one or more of the 213P1F11 protein or variant N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylation sites or N-myristoylation site and amidation sites.
[0160] II.A.) Uses of 213P1F11 Polynucleotides
[0161] II.A.1.) Monitoring of Genetic Abnormalities
[0162] The polynucleotides of the preceding paragraphs have a number of different specific uses. The human 213P1F11 gene maps to the chromosomal location set forth in the Example entitled “Chromosomal Mapping of 213P1F11.” For example, because the 213P1F11 gene maps to this chromosome, polynucleotides that encode different regions of the 213P1F11 proteins are used to characterize cytogenetic abnormalities of this chromosomal locale, such as abnormalities that are identified as being associated with various cancers. In certain genes, a variety of chromosomal abnormalities including rearrangements have been identified as frequent cytogenetic abnormalities in a number of different cancers (see e.g. Krajinovic et al, Mutat. Res. 382(3-4): 81-83 (1998); Johansson et al, Blood 86(10): 3905-3914 (1995) and Finger et al, P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotides encoding specific regions of the 213P1F11 proteins provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes 213P1F11 that may contribute to the malignant phenotype. In this context, these polynucleotides satisfy a need in the art for expanding the sensitivity of chromosomal screening in order to identify more subtle and less common chromosomal abnormalities (see e.g. Evans et al., Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).
[0163] Furthermore, as 213P1F11 was shown to be highly expressed in bladder and other cancers, 213P1F11 polynucleotides are used in methods assessing the status of 213P1F11 gene products in normal versus cancerous tissues. Typically, polynucleotides that encode specific regions of the 213P1F11 proteins are used to assess the presence of perturbations (such as deletions, insertions, point mutations, or alterations resulting in a loss of an antigen etc.) in specific regions of the 213P1F11 gene, such as regions containing one or more motifs. Exemplary assays include both RT-PCR assays as well as single-strand conformation polymorphism (SSCP) analysis (see, e.g., Marrogi et al, J. Cutan. Pathol. 26(8): 369-378 (1999), both of which utilize polynucleotides encoding specific regions of a protein to examine these regions within the protein.
[0164] II.A.2.) Antisense Embodiments
[0165] Other specifically contemplated nucleic acid related embodiments of the invention disclosed herein are genomic DNA, cDNAs, ribozymes, and antisense molecules, as well as nucleic acid molecules based on an alternative backbone, or including alternative bases, whether derived from natural sources or synthesized, and include molecules capable of inhibiting the RNA or protein expression of 213P1F11. For example, antisense molecules can be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives, that specifically bind DNA or RNA in a base pair-dependent manner. A skilled artisan can readily obtain these classes of nucleic acid molecules using the 213P1F11 polynucleotides and polynucleotide sequences disclosed herein.
[0166] Antisense technology entails the administration of exogenous oligonucleotides that bind to a target polynucleotide located within the cells. The term “antisense” refers to the fact that such oligonucleotides are complementary to their intracellular targets, e.g., 213P1F11. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1: 1-5 (1988). The 213P1F11 antisense oligonucleotides of the present invention include derivatives such as S-oligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhanced cancer cell growth inhibitory action. S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of an oligonucleotide (O-oligo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom. The S-oligos of the present invention can be prepared by treatment of the corresponding O-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transfer reagent. See, e.g., Iyer, R. P. et al., J. Org. Chem. 55:4693-4698 (1990); and Iyer, R. P. et al, J. Am. Chem. Soc. 112:1253-1254 (1990). Additional 213P1F11 antisense oligonucleotides of the present invention include morpholino antisense oligonucleotides known in the art (see, e.g., Partridge et al, 1996, Antisense & Nucleic Acid Drug Development 6: 169-175).
[0167] The 213P1F11 antisense oligonucleotides of the present invention typically can be RNA or DNA that is complementary to and stably hybridizes with the first 100 5′ codons or last 100 3′ codons of a 213P1F11 genomic sequence or the corresponding mRNA. Absolute complementarity is not required, although high degrees of complementarity are preferred. Use of an oligonucleotide complementary to this region allows for the selective hybridization to 213P1F11 mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 213P1F11 antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 213P1F11 mRNA. Optionally, 213P1F11 antisense oligonucleotide is a 30-mer oligonucleotide that is complementary to a region in the first 10 5′ codons or last 10 3′ codons of 213P1F11. Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of 213P1F11 expression, see, e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).
[0168] II.A.3.) Primers and Primer Pairs
[0169] Further specific embodiments of this nucleotides of the invention include primers and primer pairs, which allow the specific amplification of polynucleotides of the invention or of any specific parts thereof, and probes that selectively or specifically hybridize to nucleic acid molecules of the invention or to any part thereof. Probes can be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme. Such probes and primers are used to detect the presence of a 213P1F11 polynucleotide in a sample and as a means for detecting a cell expressing a 213P1F11 protein.
[0170] Examples of such probes include polypeptides comprising all or part of the human 213P1F11 cDNA sequence shown in FIG. 2. Examples of primer pairs capable of specifically amplifying 213P1F11 mRNAs are also described in the Examples. As will be understood by the skilled artisan, a great many different primers and probes can be prepared based on the sequences provided herein and used effectively to amplify and/or detect a 213P1F11 mRNA.
[0171] The 213P1F11 polynucleotides of the invention are useful for a variety of purposes, including but not limited to their use as probes and primers for the amplification and/or detection of the 213P1F11 gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosis and/or prognosis of prostate cancer and other cancers; as coding sequences capable of directing the expression of 213P1F11 polypeptides; as tools for modulating or inhibiting the expression of the 213P1F11 gene(s) and/or translation of the 213P1F11 transcript(s); and as therapeutic agents.
[0172] The present invention includes the use of any probe as described herein to identify and isolate a 213P1F11 or 213P1F11 related nucleic acid sequence from a naturally occurring source, such as humans or other mammals, as well as the isolated nucleic acid sequence per se, which would comprise all or most of the sequences found in the probe used.
[0173] II.A.4.) Isolation of 213P1F11-Encoding Nucleic Acid Molecules
[0174] The 213P1F11 cDNA sequences described herein enable the isolation of other polynucleotides encoding 213P F11 gene product(s), as well as the isolation of polynucleotides encoding 213P1F11 gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of a 213P1F11 gene product as well as polynucleotides that encode analogs of 213P1F11-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding a 213P1F11 gene are well known (see, for example, Sambrook, J. et al, Molecular Cloning: A Laboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989; Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley and Sons, 1995). For example, lambda phage cloning methodologies can be conveniently employed, using commercially available cloning systems (e.g., Lambda ZAP Express, Stratagene). Phage clones containing 213P F11 gene cDNAs can be identified by probing with a labeled 213P1F11 cDNA or a fragment thereof. For example, in one embodiment, a 213P1F11 cDNA (e.g., FIG. 2) or a portion thereof can be synthesized and used as a probe to retrieve overlapping and fill-length cDNAs corresponding to a 213P1F11 gene. A 213P1F11 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 213P1F11 DNA probes or primers.
[0175] II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
[0176] The invention also provides recombinant DNA or RNA molecules containing a 213P1F11 polynucleotide, a fragment, analog or homologue thereof, including but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, as well as various viral and non-viral vectors well known in the art, and cells transformed or transfected with such recombinant DNA or RNA molecules. Methods for generating such molecules are well known (see, for example, Sambrook et al., 1989, supra).
[0177] The invention further provides a host-vector system comprising a recombinant DNA molecule containing a 213P1F11 polynucleotide, fragment, analog or homologue thereof within a suitable prokaryotic or eukaryotic host cell. Examples of suitable eukaryotic host cells include a yeast cell, a plant cell, or an animal cell, such as a mammalian cell or an insect cell (e.g., a baculovirus-infectible cell such as an Sf9 or HighFive cell). Examples of suitable mammalian cells include various prostate cancer cell lines such as DU145 and TsuPr1, other transfectable or transducible prostate cancer cell lines, primary cells (PrEC), as well as a number of mammalian cells routinely used for the expression of recombinant proteins (e.g., COS, CHO, 293, 293T cells). More particularly, a polynucleotide comprising the coding sequence of 213P1F11 or a fragment, analog or homolog thereof can be used to generate 213P1F11 proteins or fragments thereof using any number of host-vector systems routinely used and widely known in the art.
[0178] A wide range of host-vector systems suitable for the expression of 213P1F11 proteins or fragments thereof are available, see for example, Sambrook et al., 1989, supra; Current Protocols in Molecular Biology, 1995, supra). Preferred vectors for mammalian expression include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector pSRatkneo (Muller et al., 1991, MCB 11:1785). (Using these expression vectors, 213P1F11 can be expressed in several prostate cancer and non-prostate cell lines, including for example 293, 293T, rat-1, NIH 3T3 and TsuPr1. The host-vector systems of the invention are useful for the production of a 213P1F11 protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 213P1F11 and 213P1F11 mutations or analogs.
[0179] Recombinant human 213P1F11 protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 213P1F11-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding 213P1F11 or fragment, analog or homolog thereof, a 213P1F11-related protein is expressed in the 293T cells, and the recombinant 213P1F11 protein is isolated using standard purification methods (e.g., affinity purification using anti-213P1F11 antibodies). In another embodiment, a 213P1F11 coding sequence is subcloned into the retroviral vector pSR&agr;MSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 and rat-1 in order to establish 213P1F11 expressing cell lines. Various other expression systems well known in the art can also be employed. Expression constructs encoding a leader peptide joined in frame to a 213P1P11 coding sequence can be used for the generation of a secreted form of recombinant 213P1F11 protein.
[0180] As discussed herein, redundancy in the genetic code permits variation in 213P1F11 gene sequences. In particular, it is known in the art that specific host species often have specific codon preferences, and thus one can adapt the disclosed sequence as preferred for a desired host. For example, preferred analog codon sequences typically have rare codons (i.e., codons having a usage frequency of less than about 20% in known sequences of the desired host) replaced with higher frequency codons. Codon preferences for a specific species are calculated, for example, by utilizing codon usage tables available on the INTERNET such as at URL www.dna.affrc.gojp/˜nakamura/codon.html.
[0181] Additional sequence modifications are known to enhance protein expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and/or other such well-characterized sequences that are deleterious to gene expression. The GC content of the sequence is adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Where possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. Other useful modifications include the addition of a translational initiation consensus sequence at the start of the open reading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080 (1989). Skilled artisans understand that the general rule that eukaryotic ribosomes initiate translation exclusively at the 5′ proximal AUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS 92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).
[0182] III.) 213P1F11-Related Proteins
[0183] Another aspect of the present invention provides 213P1F11-related proteins. Specific embodiments of 213P1F11 proteins comprise a polypeptide having all or part of the amino acid sequence of human 213P1F11 as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of 213P1F11 proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 213P F11 shown in FIG. 2 or FIG. 3.
[0184] In general, naturally occurring allelic variants of human 213P1F11 share a high degree of structural identity and homology (e.g., 90% or more homology). Typically, allelic variants of a 213P1F11 protein contain conservative amino acid substitutions within the 213P1F11 sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 213P1F11. One class of 213P1F11 allelic variants are proteins that share a high degree of homology with at least a small region of a particular 213P1F11 amino acid sequence, but further contain a radical departure from the sequence, such as a non-conservative substitution, truncation, insertion or frame shift. In comparisons of protein sequences, the terms, similarity, identity, and homology each have a distinct meaning as appreciated in the field of genetics. Moreover, orthology and paralogy can be important concepts describing the relationship of members of a given protein family in one organism to the members of the same family in other organisms.
[0185] Amino acid abbreviations are provided in Table II. Conservative amino acid substitutions can frequently be made in a protein without altering either the conformation or the function of the protein. Proteins of the invention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 conservative substitutions. Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (O) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered “conservative” in particular environments (see, e.g. Table III herein; pages 13-15 “Biochemistry” 2nd ED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem May 19, 1995; 270(20):11882-6).
[0186] Embodiments of the invention disclosed herein include a wide variety of art-accepted variants or analogs of 213P1F11 proteins such as polypeptides having amino acid insertions, deletions and substitutions. 213P1F11 variants can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)) or other known techniques can be performed on the cloned DNA to produce the 213P1F11 variant DNA.
[0187] Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence that is involved in a specific biological activity such as a protein-protein interaction. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions (Creighton, The Proteins, (W. H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yield adequate amounts of variant, an isosteric amino acid can be used.
[0188] As defined herein, 213P1F11 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that is “cross reactive” with a 213P1F11 protein having an amino acid sequence of FIG. 3. As used in this sentence, “cross reactive” means that an antibody or T cell that specifically binds to a 213P1F11 variant also specifically binds to a 213P1F11 protein having an amino acid sequence set forth in FIG. 3. A polypeptide ceases to be a variant of a protein shown in FIG. 3, when it no longer contains any epitope capable of being recognized by an antibody or T cell that specifically binds to the starting 213P1F11 protein. Those skilled in the art understand that antibodies that recognize proteins bind to epitopes of varying size, and a grouping of the order of about four or five amino acids, contiguous or not, is regarded as a typical number of amino acids in a minimal epitope. See, e.g., Nair et al., J. Immunol 2000 165(12): 6949-6955; Hebbes et al., Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985) 135(4):2598-608.
[0189] Other classes of 213P1F11-related protein variants share 70%, 75%, 80%, 85% or 90% or more similarity with an amino acid sequence of FIG. 3, or a fragment thereof. Another specific class of 213P1F11 protein variants or analogs comprise one or more of the 213P1F11 biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of 213P1F11 fragments (nucleic or amino acid) that have altered functional (e.g. immunogenic) properties relative to the starting fragment. It is to be appreciated that motifs now or which become part of the art are to be applied to the nucleic or amino acid sequences of FIG. 2 or FIG. 3.
[0190] As discussed herein, embodiments of the claimed invention include polypeptides containing less than the full amino acid sequence of a 213P1F11 protein shown in FIG. 2 or FIG. 3. For example, representative embodiments of the invention comprise peptides/proteins having any 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a 213P1F11 protein shown in FIG. 2 or FIG. 3.
[0191] Moreover, representative embodiments of the invention disclosed herein include polypeptides consisting of about amino acid 1 to about amino acid 10 of a 213P1F11 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of a 213P1F11 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 20 to about amino acid 30 of a 213P1F11 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of a 213P1F11 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of a 213P1F11 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of a 213P1F11 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of a 213P1F11 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of a 213P1F11 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of a 213P1F11 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 to about amino acid 100 of a 213P1F11 protein shown in FIG. 2 or FIG. 3, etc. throughout the entirety of a 213P1F11 amino acid sequence. Moreover, polypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 130, or 140 or 150 etc.) of a 213P1F11 protein shown in FIG. 2 or FIG. 3 are embodiments of the invention. It is to be appreciated that the starting and stopping positions in this paragraph refer to the specified position as well as that position plus or minus 5 residues.
[0192] 213P1F11-related proteins are generated using standard peptide synthesis technology or using chemical cleavage methods well known in the art. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode a 213P1F11-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of a 213P1F11 protein (or variants, homologs or analogs thereof).
[0193] III.A.) Motif-Bearing Protein Embodiments
[0194] Additional illustrative embodiments of the invention disclosed herein include 213P1F11 polypeptides comprising the amino acid residues of one or more of the biological motifs contained within a 213P1F11 polypeptide sequence set forth in FIG. 2 or FIG. 3. Various motifs are known in the art, and a protein can be evaluated for the presence of such motifs by a number of publicly available Internet sites (see, e.g., URL addresses: pfam.wustl.edu/; http://searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html; psort.ims.u-tokyo.acjp/; www.cbs.dtu.dk/; www.ebi.ac.uk/interpro/scan.html; www.expasy.cb/tools/scnpsit1.html; Epimatrix™ and Epimer™, Brown University, www.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, bimas.dcrt.nih.gov/.).
[0195] Motif bearing subsequences of all 213P1F11 variant proteins are set forth and identified in Tables V-XIX.
[0196] Table XX sets forth several frequently occurring motifs based on pfam searches (see URL address pfam.wustl.edu/). The columns of Table XX list (1) motif name abbreviation, (2) percent identity found amongst the different member of the motif family, (3) motif name or description and (4) most common function; location information is included if the motif is relevant for location.
[0197] Polypeptides comprising one or more of the 213P1F11 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 213P1F11 motifs discussed above are associated with growth dysregulation and because 213P1F11 is overexpressed in certain cancers (See, e.g., Table I). Casein kinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C, for example, are enzymes known to be associated with the development of the malignant phenotype (see e.g. Chen et al., Lab Invest., 78(2): 165-174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338 (1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126 (1996); Peterziel et al., Oncogene 18(46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both glycosylation and myristoylation are protein modifications also associated with cancer and cancer progression (see e.g. Dennis et al., Biochem. Biophys. Acta 1473(1):21-34 (1999); Raju et al., Exp. Cell Res. 235(1): 145-154 (1997)). Amidation is another protein modification also associated with cancer and cancer progression (see e.g. Treston et al., J. Natl. Cancer Inst. Monogr. (13): 169-175 (1992)).
[0198] In another embodiment, proteins of the invention comprise one or more of the immunoreactive epitopes identified in accordance with art-accepted methods, such as the peptides set forth in Tables V-XIX. CTL epitopes can be determined using specific algorithms to identify peptides within a 213P1F11 protein that are capable of optimally binding to specified HLA alleles (e.g., Table IV; Epimatrix™ and Epimer™, Brown University, URL www.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, URL bimas.dcrt.nih.gov/.) Moreover, processes for identifying peptides that have sufficient binding affinity for HLA molecules and which are correlated with being immunogenic epitopes, are well known in the art, and are carried out without undue experimentation. In addition, processes for identifying peptides that are immunogenic epitopes, are well known in the art, and are carried out without undue experimentation either in vitro or in vivo.
[0199] Also known in the art are principles for creating analogs of such epitopes in order to modulate immunogenicity. For example, one begins with an epitope that bears a CTL or HTL motif (see, e.g., the HLA Class I and HLA Class II motifs/supermotifs of Table IV). The epitope is analoged by substituting out an amino acid at one of the specified positions, and replacing it with another amino acid specified for that position. For example, one can substitute out a deleterious residue in favor of any other residue, such as a preferred residue as defined in Table IV; substitute a less-preferred residue with a preferred residue as defined in Table IV; or substitute an originally-occurring preferred residue with another preferred residue as defined in Table IV. Substitutions can occur at primary anchor positions or at other positions in a peptide; see, e.g., Table IV.
[0200] A variety of references reflect the art regarding the identification and generation of epitopes in a protein of interest as well as analogs thereof. See, for example, WO 9733602 to Chesnut et al.; Sette, Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol. 2001 166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondo et al., Immunogenetics 1997 45(4): 249-258; Sidney et al, J. Immunol. 1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt et al, Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et at, J. Immunol. 152:163-75 (1994)); Kast et al, 1994 152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3): 266-278; Alexander et al, J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al, J. Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994 1(9): 751-761 and Alexander et al, Immunol. Res. 1998 18(2): 79-92.
[0201] Related embodiments of the invention include polypeptides comprising combinations of the different motifs set forth in Table XXI, and/or, one or more of the predicted CTL epitopes of Tables V-XIX, and/or, one or more of the T cell binding motifs known in the art. Preferred embodiments contain no insertions, deletions or substitutions either within the motifs or the intervening sequences of the polypeptides. In addition, embodiments which include a number of either N-terminal and/or C-terminal amino acid residues on either side of these motifs may be desirable (to, for example, include a greater portion of the polypeptide architecture in which the motif is located). Typically the number of N-terminal and/or C-terminal amino acid residues on either side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino acid residues.
[0202] 213P1F11-related proteins are embodied in many forms, preferably in isolated form A purified 213P1F11 protein molecule will be substantially free of other proteins or molecules that impair the binding of 213P1F11 to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 213P1F11-related proteins include purified 213P1F11-related proteins and functional, soluble 213P1F11-related proteins. In one embodiment, a functional, soluble 213P1F11 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.
[0203] The invention also provides 213P1F11 proteins comprising biologically active fragments of a 213P1F11 amino acid sequence shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the starting 213P1F11 protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the starting 213P1F11 protein; to be bound by such antibodies; to elicit the activation of HTL or CTL; and/or, to be recognized by HTL or CTL that also specifically bind to the starting protein.
[0204] 213P1F11-related polypeptides that contain particularly interesting structures can be predicted and/or identified using various analytical techniques well known in the art, including, for example, the methods of Chou-Fasman, Gamier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis, or on the basis of immunogenicity. Fragments that contain such structures are particularly useful in generating subunit-specific anti-213P1F11 antibodies, or T cells or in identifying cellular factors that bind to 213P1F11. For example, hydrophilicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can be generated, and immunogenic peptide fragments identified, using the method of Janin J., 1979, Nature 277:491-492. Average Flexibility profiles can be generated, and immunogenic peptide fragments identified, using the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generated, and immunogenic peptide fragments identified, using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294.
[0205] CTL epitopes can be determined using specific algorithms to identify peptides within a 213P1F11 protein that are capable of optimally binding to specified HLA alleles (e.g., by using the SYFPEITHI site at World Wide Web URL syfpeithi.bmi-heidelberg.com/; the listings in Table IV(A)-(E); Epimatrix™ and Epimer™, Brown University, URL (www.brown.edu/ResearcI/TB-HIV Lab/epimatrix/epimatrix.html); and BIMAS, URL bimas.dcrt.nih.gov/). Illustrating this, peptide epitopes from 213P1F11 that are presented in the context of human MHC Class I molecules, e.g., HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (Tables V-XIX). Specifically, the complete amino acid sequence of the 213P1F11 protein and relevant portions of other variants, i.e., for HLA Class I predictions 9 flanking redisues on either side of a point mutation, and for HLA Class II predictions 14 flanking residues on either side of a point mutation, were entered into the HLA Peptide Motif Search algorithm found in the Bioinformatics and Molecular Analysis Section (BIMAS) web site listed above; in addition to the site SYFPEITHI, at URL syfpeithi.bmi-heidelberg.com/.
[0206] The HLA peptide motif search algorithm was developed by Dr. Ken Parker based on binding of specific peptide sequences in the groove of HLA Class I molecules, in particular HLA-A2 (see, e.g., Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75 (1994)). This algorithm allows location and ranking of 8-mer, 9-mer (also refered to as “nonamer”), and 10-mer (also refered to as “decamer”) peptides from a complete protein sequence for predicted binding to HLA-A2 as well as numerous other HLA Class I molecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers. For example, for Class I HLA-A2, the epitopes preferably contain a leucine (L) or methionine (M) at position 2 and a valine (V) or leucine (L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7 (1992)). Selected results of 213P1F11 predicted binding peptides are shown in Tables V-XIX herein. In Tables V-XIX, the selected candidates, 9-mers and 10-mers, and 15-mers for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. The binding score corresponds to the estimated half time of dissociation of complexes containing the peptide at 37° C. at pH 6.5. Peptides with the highest binding score are predicted to be the most tightly bound to HLA Class I on the cell surface for the greatest period of time and thus represent the best immunogenic targets for T-cell recognition.
[0207] Actual binding of peptides to an HLA allele can be evaluated by stabilization of HLA expression on the antigen-processing defective cell line T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa et al., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides can be evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes (CTL) in the presence of antigen presenting cells such as dendritic cells.
[0208] It is to be appreciated that every epitope predicted by the BIMAS site, Epimer™ and Epimatrix™ sites, or specified by the HLA class I or class II motifs available in the art or which become part of the art such as set forth in Table IV (or determined using World Wide Web site URL syfpeithi.bmi-heidelberg.com/, or BIMAS, bimas.dcrt.nih.gov/) are to be “applied” to a 213P1F11 protein in accordance with the invention. As used in this context “applied” means that a 213P1F11 protein is evaluated, e.g., visually or by computer-based patterns finding methods, as appreciated by those of skill in the relevant art. Every subsequence of a 213P1F11 protein of 8, 9, 10, or 11 amino acid residues that bears an HLA Class I motif, or a subsequence of 9 or more amino acid residues that bear an HLA Class II motif are within the scope of the invention.
[0209] III.B.) Expression of 213P1F11-Related Proteins
[0210] In an embodiment described in the examples that follow, 213P1F11 can be conveniently expressed in cells (such as 293T cells) transfected with a commercially available expression vector such as a CMV-driven expression vector encoding 213P1F11 with a C-terminal 6×His and MYC tag (pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, Nashville Tenn.). The Tag5 vector provides an IgGK secretion signal that can be used to facilitate the production of a secreted 213P1F11 protein in transfected cells. The secreted HIS-tagged 213P1F11 in the culture media can be purified, e.g., using a nickel column using standard techniques.
[0211] III.C.) Modifications of 213P1F11-Related Proteins
[0212] Modifications of 213P1F11-related proteins such as covalent modifications are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a 213P1F11 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of a 213P1F11 protein. Another type of covalent modification of a 213P1F11 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of a protein of the invention. Another type of covalent modification of 213P1F11 comprises linking a 213P1F11 polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
[0213] The 213P1F11-related proteins of the present invention can also be modified to form a chimeric molecule comprising 213P1F11 fused to another, heterologous polypeptide or amino acid sequence. Such a chimeric molecule can be synthesized chemically or recombinantly. A chimeric molecule can have a protein of the invention fused to another tumor-associated antigen or fragment thereof. Alternatively, a protein in accordance with the invention can comprise a fusion of fragments of a 213P1F11 sequence (amino or nucleic acid) such that a molecule is created that is not, through its length, directly homologous to the amino or nucleic acid sequences shown in FIG. 2 or FIG. 3. Such a chimeric molecule can comprise multiples of the same subsequence of 213P1F11. A chimeric molecule can comprise a fusion of a 213P1F11-related protein with a polyhistidine epitope tag, which provides an epitope to which immobilized nickel can selectively bind, with cytokines or with growth factors. The epitope tag is generally placed at the amino- or carboxyl-terminus of a 213P1F11 protein. In an alternative embodiment, the chimeric molecule can comprise a fusion of a 213P1F11-related protein with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an “immunoadlesin”), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a 213P1F11 polypeptide in place of at least one variable region within an Ig molecule. In a preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgG1 molecule. For the production of immunoglobulin fusions see, e.g., U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
[0214] III.D.) Uses of 213P1F11-Related Proteins
[0215] The proteins of the invention have a number of different specific uses. As 213P1F11 is highly expressed in prostate and other cancers, 213P1F11-related proteins are used in methods that assess the status of 213P1F11 gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of a 213P1F11 protein are used to assess the presence of perturbations (such as deletions, insertions, point mutations etc.) in those regions (such as regions containing one or more motifs). Exemplary assays utilize antibodies or T cells targeting 213P1F11-related proteins comprising the amino acid residues of one or more of the biological motifs contained within a 213P1F11 polypeptide sequence in order to evaluate the characteristics of this region in normal versus cancerous tissues or to elicit an immune response to the epitope. Alternatively, 213P1F11-related proteins that contain the amino acid residues of one or more of the biological motifs in a 213P1F11 protein are used to screen for factors that interact with that region of 213P1F11.
[0216] 213P1F11 protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of a 213P1F11 protein), for identifying agents or cellular factors that bind to 213P1F11 or a particular structural domain thereof, and in various therapeutic and diagnostic contexts, including but not limited to diagnostic assays, cancer vaccines and methods of preparing such vaccines.
[0217] Proteins encoded by the 213P1F11 genes, or by analogs, homologs or fragments thereof, have a variety of uses, including but not limited to generating antibodies and in methods for identifying ligands and other agents and cellular constituents that bind to a 213P1F11 gene product. Antibodies raised against a 213P1F11 protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 213P1F11 protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. 213P1F11-related nucleic acids or proteins are also used in generating HTL or CTL responses.
[0218] Various immunological assays useful for the detection of 213P1F11 proteins are used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical methods, and the like. Antibodies can be labeled and used as immunological imaging reagents capable of detecting 213P1F11-expressing cells (e.g., in radioscintigrapbic imaging methods). 213P1F11 proteins are also particularly useful in generating cancer vaccines, as further described herein.
[0219] IV.) 213P1F11 Antibodies
[0220] Another aspect of the invention provides antibodies that bind to 213P1F11-related proteins. Preferred antibodies specifically bind to a 213P1F11-related protein and do not bind (or bind weakly) to peptides or proteins that are not 213P1F11-related proteins. For example, antibodies that bind 213P1F11 can bind 213P1F11-related proteins such as the homologs or analogs thereof.
[0221] 213P1F11 antibodies of the invention are particularly useful in cancer (see, e.g., Table I) diagnostic and prognostic assays, and imaging methodologies. Similarly, such antibodies are useful in the treatment, diagnosis, and/or prognosis of other cancers, to the extent 213P1F11 is also expressed or overexpressed in these other cancers. Moreover, intracellularly expressed antibodies (e.g., single chain antibodies) are therapeutically useful in treating cancers in which the expression of 213P1F11 is involved, such as advanced or metastatic prostate cancers.
[0222] The invention also provides various immunological assays useful for the detection and quantification of 213P1F11 and mutant 213P1F11-related proteins. Such assays can comprise one or more 213P1F11 antibodies capable of recognizing and binding a 213P1F11-related protein, as appropriate. These assays are performed within various immunological assay formats well known in the art, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.
[0223] Immunological non-antibody assays of the invention also comprise T cell inmmunogenicity assays (inhibitory or stimulatory) as well as major histocompatibility complex (MHC) binding assays.
[0224] In addition, immunological imaging methods capable of detecting prostate cancer and other cancers expressing 213P1F11 are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled 213P1F11 antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of 213P1F11 expressing cancers such as prostate cancer.
[0225] 213P1F11 antibodies are also used in methods for purifying a 213P1F11-related protein and for isolating 213P1F11 homologues and related molecules. For example, a method of purifying a 213P1F11-related protein comprises incubating a 213P1F11 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 213P1F11-related protein under conditions that permit the 213P1F11 antibody to bind to the 213P1F11-related protein; washing the solid matrix to eliminate impurities; and eluting the 213P1F11-related protein from the coupled antibody. Other uses of 213P1F11 antibodies in accordance with the invention include generating anti-idiotypic antibodies that mimic a 213P1F11 protein.
[0226] Various methods for the preparation of antibodies are well known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host using a 213P1F11-related protein, peptide, or fragment, in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). In addition, fusion proteins of 213P1F11 can also be used, such as a 213P1F11 GST-fusion protein. In a particular embodiment, a GST fusion protein comprising all or most of the amino acid sequence of FIG. 2 or FIG. 3 is produced, then used as an immunogen to generate appropriate antibodies. In another embodiment, a 213P1F11-related protein is synthesized and used as an immunogen.
[0227] In addition, naked DNA immunization techniques known in the art are used (with or without purified 213P1F11-related protein or 213P1F11 expressing cells) to generate an immune response to the encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).
[0228] The amino acid sequence of a 213P1F11 protein as shown in FIG. 2 or FIG. 3 can be analyzed to select specific regions of the 213P1F11 protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of a 213P1F11 amino acid sequence are used to identify hydrophilic regions in the 213P1F11 structure. Regions of a 213P1F11 protein that show immunogenic structure, as well as other regions and domains, can readily be identified using various other methods known in the art, such as Chou-Fasman, Gamier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can be generated using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated using the method of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can be generated using the method of Janin J., 1979, Nature 277:491-492. Average Flexibility profiles can be generated using the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generated using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294. Thus, each region identified by any of these programs or methods is within the scope of the present invention. Methods for the generation of 213P1F11 antibodies are further illustrated by way of the examples provided herein. Methods for preparing a protein or polypeptide for use as an immunogen are well known in the art. Also well known in the art are methods for preparing immunogenic conjugates of a protein with a carrier, such as BSA, KLH or other carrier protein. In some circumstances, direct conjugation using, for example, carbodiimide reagents are used; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, Ill., are effective. Administration of a 213P1F11 immunogen is often conducted by injection over a suitable time period and with use of a suitable adjuvant, as is understood in the art. During the immunization schedule, titers of antibodies can be taken to determine adequacy of antibody formation.
[0229] 213P1F11 monoclonal antibodies can be produced by various means well known in the art. For example, immortalized cell lines that secrete a desired monoclonal antibody are prepared using the standard hybridoma technology of Kohler and Milstein or modifications that immortalize antibody-producing B cells, as is generally known. Immortalized cell lines that secrete the desired antibodies are screened by immunoassay in which the antigen is a 213P1F11-related protein. When the appropriate immortalized cell culture is identified, the cells can be expanded and antibodies produced either from in vitro cultures or from ascites fluid.
[0230] The antibodies or fragments of the invention can also be produced, by recombinant means. Regions that bind specifically to the desired regions of a 213P1F11 protein can also be produced in the context of chimeric or complementarity determining region (CDR) grafted antibodies of multiple species origin. Humanized or human 213P1F11 antibodies can also be produced, and are preferred for use in therapeutic contexts. Methods for humanizing murine and other non-human antibodies, by substituting one or more of the non-human antibody CDRs for corresponding human antibody sequences, are well known (see for example, Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen et al., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Simns et al., 1993, J. Immunol. 151: 2296.
[0231] Methods for producing fully human monoclonal antibodies include phage display and transgenic methods (for review, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539). Fully human 213P1F11 monoclonal antibodies can be generated using cloning technologies employing large human Ig gene combinatorial libraries (i.e., phage display) (Griffiths and Hoogenboom, Building an in vitro immune system: human antibodies from phage display libraries. In: Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, Clark, M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatorial libraries. Id., pp 65-82). Fully human 213P1F11 monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application WO98/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614; U.S. Pat. No. 6,162,963 issued Dec. 19, 2000; U.S. Pat. No. 6,150,584 issued Nov. 12, 2000; and, U.S. Pat. No. 6,11,4598 issued Sep. 5, 2000). This method avoids the in vitro manipulation required with phage display technology and efficiently produces high affinity authentic human antibodies.
[0232] Reactivity of 213P1F11 antibodies with a 213P1F11-related protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, 213P1F11-related proteins, 213P1F11-expressing cells or extracts thereof. A 213P1F11 antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme. Further, bi-specific antibodies specific for two or more 213P1F11 epitopes are generated using methods generally known in the art. Homodimeric antibodies can also be generated by cross-linking techniques known in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).
[0233] V.) 213P1F11 Cellular Immune Responses
[0234] The mechanism by which T cells recognize antigens has been delineated. Efficacious peptide epitope vaccine compositions of the invention induce a therapeutic or prophylactic immune responses in very broad segments of the world-wide population. For an understanding of the value and efficacy of compositions of the invention that induce cellular immune responses, a brief review of immunology-related technology is provided.
[0235] A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. and Bodimer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev. Immunol. 11:403, 1993). Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues that correspond to motifs required for specific binding to HLA antigen molecules have been identified and are set forth in Table IV (see also, e.g., Southwood, et al., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics 41:178, 1995; Rammensee et al., SYFPEITHI, access via World Wide Web at URL syfpeithi.bmi-heidelberg.com/; Sette, A. and Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J. Immunol. 155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and Sidney, J. Immunogenetics November 1999; 50(3-4):201-12, Review).
[0236] Furthermore, x-ray crystallographic analyses of HLA-peptide complexes have revealed pockets within the peptide binding cleft/groove of HLA molecules which accommodate, in an allele-specific mode, residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present. (See, e.g., Madden, D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203, 1996; Fremont et al., Immunity 8:305, 1998; Stem et al., Structure 2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science 257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol. Biol. 219:277, 1991.)
[0237] Accordingly, the definition of class I and class II allele-specific HLA binding motifs, or class I or class II supermotifs allows identification of regions within a protein that are correlated with binding to particular HLA antigen(s).
[0238] Thus, by a process of HLA motif identification, candidates for epitope-based vaccines have been identified; such candidates can be further evaluated by HLA-peptide binding assays to determine binding affinity and/or the time period of association of the epitope and its corresponding HLA molecule. Additional confirmatory work can be performed to select, amongst these vaccine candidates, epitopes with preferred characteristics in terms of population coverage, and/or immunogenicity.
[0239] Various strategies can be utilized to evaluate cellular immunogenicity, including:
[0240] 1) Evaluation of primary T cell cultures from normal individuals (see, e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J. Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1, 1998). This procedure involves the stimulation of peripheral blood lymphocytes (PBL) from normal subjects with a test peptide in the presence of antigen presenting cells in vitro over a period of several weeks. T cells specific for the peptide become activated during this time and are detected using, e.g., a lymphokine- or 51Cr-release assay involving peptide sensitized target cells.
[0241] 2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. et al., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol. 8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997). For example, in such methods peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice. Several weeks following immunization, splenocytes are removed and cultured in vitro in the presence of test peptide for approximately one week. Peptide-specific T cells are detected using, e.g., a 51Cr-release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.
[0242] 3) Demonstration of recall T cell responses from immune individuals who have been either effectively vaccinated and/or from chronically ill patients (see, e.g., Rehermann, B. et al, J. Exp. Med. 181:1047, 1995; Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin. Invest. 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648, 1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly, recall responses are detected by culturing PBL from subjects that have been exposed to the antigen due to disease and thus have generated an immune response “naturally”, or from patients who were vaccinated against the antigen. PBL from subjects are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of “memory” T cells, as compared to “naive” T cells. At the end of the culture period, T cell activity is detected using assays including 51Cr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release.
[0243] VI.) 213P1F11 Transgenic Animals
[0244] Nucleic acids that encode a 213P1F11-related protein can also be used to generate either transgenic animals or “knock out” animals that, in turn, are useful in the development and screening of therapeutically useful reagents. In accordance with established techniques, cDNA encoding 213P1F11 can be used to clone genomic DNA that encodes 213P1F11. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 213P1F11. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Pat. No. 4,736,866 issued Apr. 12, 1988, and U.S. Pat. No. 4,870,009 issued Sep. 26, 1989. Typically, particular cells would be targeted for 213P1F11 transgene incorporation with tissue-specific enhancers.
[0245] Transgenic animals that include a copy of a transgene encoding 213P1F11 can be used to examine the effect of increased expression of DNA that encodes 213P1F11. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this aspect of the invention, an animal is treated with a reagent and a reduced incidence of a pathological condition, compared to untreated animals that bear the transgene, would indicate a potential therapeutic intervention for the pathological condition.
[0246] Alternatively, non-human homologues of 213P1F11 can be used to construct a 213P1F11 “knock out” animal that has a defective or altered gene encoding 213P1F11 as a result of homologous recombination between the endogenous gene encoding 213P1F11 and altered genomic DNA encoding 213P1F11 introduced into an embryonic cell of the animal. For example, cDNA that encodes 213P F11 can be used to clone genomic DNA encoding 213P1F11 in accordance with established techniques. A portion of the genomic DNA encoding 213P1F11 can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see, e.g., Li et al, Cell, 69:915 (1992)). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras (see, e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal, and the embryo brought to term to create a “knock out” animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knock out animals can be characterized, for example, for their ability to defend against certain pathological conditions or for their development of pathological conditions due to absence of a 213P1F11 polypeptide.
[0247] VII.) Methods for the Detection of 213P1F11
[0248] Another aspect of the present invention relates to methods for detecting 213P1F11 polynucleotides and 213P1F11-related proteins, as well as methods for identifying a cell that expresses 213P1F11. The expression profile of 213P1F11 makes it a diagnostic marker for metastasized disease. Accordingly, the status of 213P1F11 gene products provides information useful for predicting a variety of factors including susceptibility to advanced stage disease, rate of progression, and/or tumor aggressiveness. As discussed in detail herein, the status of 213P1F11 gene products in patient samples can be analyzed by a variety protocols that are well known in the art including immunohistochemical analysis, the variety of Northern blotting techniques including in situ hybridization, RT-PCR analysis (for example on laser capture micro-dissected samples), Western blot analysis and tissue array analysis.
[0249] More particularly, the invention provides assays for the detection of 213P1F11 polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like. Detectable 213P1F11 polynucleotides include, for example, a 213P1F11 gene or fragment thereof, 213P1F11 mRNA, alternative splice variant 213P1F11 mRNAs, and recombinant DNA or RNA molecules that contain a 213P1F11 polynucleotide. A number of methods for amplifying and/or detecting the presence of 213P1F11 polynucleotides are well known in the art and can be employed in the practice of this aspect of the invention.
[0250] In one embodiment, a method for detecting a 213P1F11 mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using a 213P1F11 polynucleotides as sense and antisense primers to amplify 213P1F11 cDNAs therein; and detecting the presence of the amplified 213P1F11 cDNA. Optionally, the sequence of the amplified 213P1F11 cDNA can be determined.
[0251] In another embodiment, a method of detecting a 213P1F11 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 213P1F11 polynucleotides as sense and antisense primers; and detecting the presence of the amplified 213P1F11 gene. Any number of appropriate sense and antisense probe combinations can be designed from a 213P1F11 nucleotide sequence (see, e.g., FIG. 2) and used for this purpose.
[0252] The invention also provides assays for detecting the presence of a 213P1F11 protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like. Methods for detecting a 213P1F11-related protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELISA, ELIFA and the like. For example, a method of detecting the presence of a 213P1F11-related protein in a biological sample comprises first contacting the sample with a 213P1F11 antibody, a 213P1F11-reactive fragment thereof, or a recombinant protein containing an antigen binding region of a 213P1F11 antibody; and then detecting the binding of 213P1F11-related protein in the sample.
[0253] Methods for identifying a cell that expresses 213P1F11 are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a 213P1F11 gene comprises detecting the presence of 213P1F11 mRNA in the cell. Methods for the detection of particular mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled 213P1F11 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 213P1F11, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like). Alternatively, an assay for identifying a cell that expresses a 213P1F11 gene comprises detecting the presence of 213P1F11-related protein in the cell or secreted by the cell. Various methods for the detection of proteins are well known in the art and are employed for the detection of 213P1F11-related proteins and cells that express 213P1F11-related proteins.
[0254] 213P1F11 expression analysis is also useful as a tool for identifying and evaluating agents that modulate 213P1F11 gene expression. For example, 213P1F11 expression is significantly upregulated in prostate cancer, and is expressed in cancers of the tissues listed in Table I. Identification of a molecule or biological agent that inhibits 213P1F11 expression or over-expression in cancer cells is of therapeutic value. For example, such an agent can be identified by using a screen that quantifies 213P1F11 expression by RT-PCR, nucleic acid hybridization or antibody binding.
[0255] VIII.) Methods for Monitoring the Status of 213P1F11-related Genes and Their Products
[0256] Oncogenesis is known to be a multistep process where cellular growth becomes progressively dysregulated and cells progress from a normal physiological state to precancerous and then cancerous states (see, e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al., Cancer Surv. 23: 19-32 (1995)). In this context, examining a biological sample for evidence of dysregulated cell growth (such as aberrant 213P1F11 expression in cancers) allows for early detection of such aberrant physiology, before a pathologic state such as cancer has progressed to a stage that therapeutic options are more limited and or the prognosis is worse. In such examinations, the status of 213P1F11 in a biological sample of interest can be compared, for example, to the status of 213P1F11 in a corresponding normal sample (e.g. a sample from that individual or alternatively another individual that is not affected by a pathology). An alteration in the status of 213P1F11 in the biological sample (as compared to the normal sample) provides evidence of dysregulated cellular growth. In addition to using a biological sample that is not affected by a pathology as a normal sample, one can also use a predetermined normative value such as a predetermined normal level of mRNA expression (see, e.g., Grever et al., J. Comp. Neurol. Dec. 9, 1996; 376(2): 306-14 and U.S. Pat. No. 5,837,501) to compare 213P1F11 status in a sample.
[0257] The term “status” in this context is used according to its art accepted meaning and refers to the condition or state of a gene and its products. Typically, skilled artisans use a number of parameters to evaluate the condition or state of a gene and its products. These include, but are not limited to the location of expressed gene products (including the location of 213P1F11 expressing cells) as well as the level, and biological activity of expressed gene products (such as 213P1F11 mRNA, polynucleotides and polypeptides). Typically, an alteration in the status of 213P1F11 comprises a change in the location of 213P1F11 and/or 213P1F11 expressing cells and/or an increase in 213P1F11 mRNA and/or protein expression.
[0258] 213P1F11 status in a sample can be analyzed by a number of means well known in the art, including without limitation, immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser capture micro-dissected samples, Western blot analysis, and tissue array analysis. Typical protocols for evaluating the status of a 213P1F11 gene and gene products are found, for example in Ausubel et al. eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus, the status of 213P1F11 in a biological sample is evaluated by various methods utilized by skilled artisans including, but not limited to genomic Southern analysis (to examine, for example perturbations in a 213P1F11 gene), Northern analysis and/or PCR analysis of 213P1F11 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 213P1F11 mRNAs), and, Western and/or immunohistochemical analysis (to examine, for example alterations in polypeptide sequences, alterations in polypeptide localization within a sample, alterations in expression levels of 213P1F11 proteins and/or associations of 213P1F11 proteins with polypeptide binding partners). Detectable 213P1F11 polynucleotides include, for example, a 213P1F11 gene or fragment thereof, 213P1F11 mRNA, alternative splice variants, 213P1F11 mRNAs, and recombinant DNA or RNA molecules containing a 213P1F11 polynucleotide.
[0259] The expression profile of 213P1F11 makes it a diagnostic marker for local and/or metastasized disease, and provides information on the growth or oncogenic potential of a biological sample. In particular, the status of 213P1F11 provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining 213P1F11 status and diagnosing cancers that express 213P1F11, such as cancers of the tissues listed in Table I. For example, because 213P1F11 mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue, assays that evaluate the levels of 213P1F11 mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 213P1F11 dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.
[0260] The expression status of 213P1F11 provides information including the presence, stage and location of dysplastic, precancerous and cancerous cells, predicting susceptibility to various stages of disease, and/or for gauging tumor aggressiveness. Moreover, the expression profile makes it useful as an imaging reagent for metastasized disease. Consequently, an aspect of the invention is directed to the various molecular prognostic and diagnostic methods for examining the status of 213P1F11 in biological samples such as those from individuals suffering from, or suspected of suffering from a pathology characterized by dysregulated cellular growth, such as cancer.
[0261] As described above, the status of 213P1F11 in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of 213P1F11 in a biological sample taken from a specific location in the body can be examined by evaluating the sample for the presence or absence of 213P1F11 expressing cells (e.g. those that express 213P1F11 mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when 213P1F11-expressing cells are found in a biological sample that does not normally contain such cells (such as a lymph node), because such alterations in the status of 213P1F11 in a biological sample are often associated with dysregulated cellular growth. Specifically, one indicator of dysregulated cellular growth is the metastases of cancer cells from an organ of origin (such as the prostate) to a different area of the body (such as a lymph node). In this context, evidence of dysregulated cellular growth is important for example because occult lymph node metastases can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, e.g., Murphy et al., Prostate 42(4): 315-317 (2000);Su et al., Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J Urol August 1995 154(2 Pt 1):474-8).
[0262] In one aspect, the invention provides methods for monitoring 213P1F11 gene products by determining the status of 213P1F11 gene products expressed by cells from an individual suspected of having a disease associated with dysregulated cell growth (such as hyperplasia or cancer) and then comparing the status so determined to the status of 213P1F11 gene products in a corresponding normal sample. The presence of aberrant 213P1F11 gene products in the test sample relative to the normal sample provides an indication of the presence of dysregulated cell growth within the cells of the individual.
[0263] In another aspect, the invention provides assays useful in determining the presence of cancer in an individual, comprising detecting a significant increase in 213P1F11 mRNA or protein expression in a test cell or tissue sample relative to expression levels in the corresponding normal cell or tissue. The presence of 213P1F11 mRNA can, for example, be evaluated in tissues including but not limited to those listed in Table I. The presence of significant 213P1F11 expression in any of these tissues is useful to indicate the emergence, presence and/or severity of a cancer, since the corresponding normal tissues do not express 213P1F11 mRNA or express it at lower levels.
[0264] In a related embodiment, 213P1F11 status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of 213P1F11 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 213P1F11 expressed in a corresponding normal sample. In one embodiment, the presence of 213P1F11 protein is evaluated, for example, using immunohistochemical methods. 213P1F11 antibodies or binding partners capable of detecting 213P1F11 protein expression are used in a variety of assay formats well known in the art for this purpose.
[0265] In a further embodiment, one can evaluate the status of 213P1F11 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules. These perturbations can include insertions, deletions, substitutions and the like. Such evaluations are useful because perturbations in the nucleotide and amino acid sequences are observed in a large number of proteins associated with a growth dysregulated phenotype (see, e.g., Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, a mutation in the sequence of 213P1F11 may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in 213P1F11 indicates a potential loss of function or increase in tumor growth.
[0266] A wide variety of assays for observing perturbations in nucleotide and amino acid sequences are well known in the art. For example, the size and structure of nucleic acid or amino acid sequences of 213P1F11 gene products are observed by the Northern, Southern, Western, PCR and DNA sequencing protocols discussed herein. In addition, other methods for observing perturbations in nucleotide and amino acid sequences such as single strand conformation polymorphism analysis are well known in the art (see, e.g., U.S. Pat. No. 5,382,510 issued Sep. 7, 1999, and U.S. Pat. No. 5,952,170 issued Jan. 17, 1995).
[0267] Additionally, one can examine the methylation status of a 213P1F11 gene in a biological sample. Aberrant demethylation and/or hypermethylation of CpG islands in gene 5′ regulatory regions frequently occurs in immortalized and transformed cells, and can result in altered expression of various genes. For example, promoter hypermethylation of the pi-class glutathione S-transferase (a protein expressed in normal prostate but not expressed in >90% of prostate carcinomas) appears to permanently silence transcription of this gene and is the most frequently detected genomic alteration in prostate carcinomas (De Marzo et al., Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration is present in at least 70% of cases of high-grade prostatic intraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers Prev., 1998, 7:531-536). In another example, expression of the LAGE-I tumor specific gene (which is not expressed in normal prostate but is expressed in 25-50% of prostate cancers) is induced by deoxy-azacytidine in lymphoblastoid cells, suggesting that tumoral expression is due to demethylation (Lethe et al., Int. J. Cancer 76(6): 903-908 (1998)). A variety of assays for examining methylation status of a gene are well known in the art. For example, one can utilize, in Southern hybridization approaches, methylation-sensitive restriction enzymes that cannot cleave sequences that contain methylated CpG sites to assess the methylation status of CpG islands. In addition, MSP (methylation specific PCR) can rapidly profile the methylation status of all the CpG sites present in a CpG island of a given gene. This procedure involves initial modification of DNA by sodium bisulfite (which will convert all umethylated cytosines to uracil) followed by amplification using primers specific for methylated versus unmethylated DNA. Protocols involving methylation interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel et al. eds., 1995.
[0268] Gene amplification is an additional method for assessing the status of 213P1F11. Gene amplification is measured in a sample directly, for example, by conventional Southern blotting or Northern blotting to quantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies are employed that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn are labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
[0269] Biopsied tissue or peripheral blood can be conveniently assayed for the presence of cancer cells using for example, Northern, dot blot or RT-PCR analysis to detect 213P1F11 expression. The presence of RT-PCR amplifiable 213P1F11 mRNA provides an indication of the presence of cancer. RT-PCR assays are well known in the art. RT-PCR detection assays for tumor cells in peripheral blood are currently being evaluated for use in the diagnosis and management of a number of human solid tumors. In the prostate cancer field, these include RT-PCR assays for the detection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25:373-384; Ghossein et al, 1995, J. Clin. Oncol. 13:1195-2000; Heston et al., 1995, Clin. Chem. 41:1687-1688).
[0270] A further aspect of the invention is an assessment of the susceptibility that an individual has for developing cancer. In one embodiment, a method for predicting susceptibility to cancer comprises detecting 213P1F11 mRNA or 213P1F11 protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 213P1F11 mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of 213P1F11 in prostate or other tissue is examined, with the presence of 213P1F11 in the sample providing an indication of prostate cancer susceptibility (or the emergence or existence of a prostate tumor). Similarly, one can evaluate the integrity 213P1F11 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations in 213P1F11 gene products in the sample is an indication of cancer susceptibility (or the emergence or existence of a tumor).
[0271] The invention also comprises methods for gauging tumor aggressiveness. In one embodiment, a method for gauging aggressiveness of a tumor comprises determining the level of 213P1F11 mRNA or 213P1F11 protein expressed by tumor cells, comparing the level so determined to the level of 213P1F11 mRNA or 213P1F11 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 213P1F11 mRNA or 213P1F11 protein expression in the tumor sample relative to the normal sample indicates the degree of aggressiveness. In a specific embodiment, aggressiveness of a tumor is evaluated by determining the extent to which 213P1F11 is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of 213P1F11 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations indicates more aggressive tumors.
[0272] Another embodiment of the invention is directed to methods for observing the progression of a malignancy in an individual over time. In one embodiment, methods for observing the progression of a malignancy in an individual over time comprise determining the level of 213P1F11 mRNA or 213P1F11 protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 213P1F11 mRNA or 213P1F11 protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 213P1F11 mRNA or 213P1F11 protein expression in the tumor sample over time provides information on the progression of the cancer. In a specific embodiment, the progression of a cancer is evaluated by determining 213P1F11 expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 213P1F11 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like, where the presence of one or more perturbations indicates a progression of the cancer.
[0273] The above diagnostic approaches can be combined with any one of a wide variety of prognostic and diagnostic protocols known in the art. For example, another embodiment of the invention is directed to methods for observing a coincidence between the expression of 213P1F11 gene and 213P1F11 gene products (or perturbations in 213P1F11 gene and 213P1F11 gene products) and a factor that is associated with malignancy, as a means for diagnosing and prognosticating the status of a tissue sample. A wide variety of factors associated with malignancy can be utilized, such as the expression of genes associated with malignancy (e.g. PSA, PSCA and PSM expression for prostate cancer etc.) as well as gross cytological observations (see, e.g., Bocking et al, 1984, Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al, 1998, Mod. Pathol. 11(6):543-51; Baisden et al, 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods for observing a coincidence between the expression of 213P1F11 gene and 213P1F11 gene products (or perturbations in 213P1F11 gene and 213P1F11 gene products) and another factor that is associated with malignancy are useful, for example, because the presence of a set of specific factors that coincide with disease provides information crucial for diagnosing and prognosticating the status of a tissue sample.
[0274] In one embodiment, methods for observing a coincidence between the expression of 213P1F11 gene and 213P1F11 gene products (or perturbations in 213P1F11 gene and 213P1F11 gene products) and another factor associated with malignancy entails detecting the overexpression of 213P1F11 mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSM expression), and observing a coincidence of 213P1F11 mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment, the expression of 213P1F11 and PSA mRNA in prostate tissue is examined, where the coincidence of 213P1F11 and PSA mRNA overexpression in the sample indicates the existence of prostate cancer, prostate cancer susceptibility or the emergence or status of a prostate tumor.
[0275] Methods for detecting and quantifying the expression of 213P1F11 mRNA or protein are described herein, and standard nucleic acid and protein detection and quantification technologies are well known in the art. Standard methods for the detection and quantification of 213P1F11 mRNA include in situ hybridization using labeled 213P1F11 riboprobes, Northern blot and related techniques using 213P1F11 polynucleotide probes, RT-PCR analysis using primers specific for 213P1F11, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like. In a specific embodiment, semi-quantitative RT-PCR is used to detect and quantify 213P1F11 mRNA expression. Any number of primers capable of amplifying 213P1F11 can be used for this purpose, including but not limited to the various primer sets specifically described herein. In a specific embodiment, polyclonal or monoclonal antibodies specifically reactive with the wild-type 213P1F11 protein can be used in an immunohistochemical assay of biopsied tissue.
[0276] IX.) Identification of Molecules That Interact With 213P1F11
[0277] The 213P1F11 protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 213P1F11, as well as pathways activated by 213P1F11 via any one of a variety of art accepted protocols. For example, one can utilize one of the so-called interaction trap systems (also referred to as the “two-hybrid assay”). In such systems, molecules interact and reconstitute a transcription factor which directs expression of a reporter gene, whereupon the expression of the reporter gene is assayed. Other systems identify protein-protein interactions in vivo through reconstitution of a eukaryotic transcriptional activator, see, e.g., U.S. Pat. No. 5,955,280 issued Sep. 21, 1999, U.S. Pat. No. 5,925,523 issued Jul. 20, 1999, U.S. Pat. No. 5,846,722 issued Dec. 8, 1998 and 6,004,746 issued Dec. 21, 1999. Algorithms are also available in the art for genome-based predictions of protein function (see, e.g., Marcotte, et al., Nature 402: Nov. 4, 1999, 83-86).
[0278] Alternatively one can screen peptide libraries to identify molecules that interact with 213P1F11 protein sequences. In such methods, peptides that bind to 213P1F11 are identified by screening libraries that encode a random or controlled collection of amino acids. Peptides encoded by the libraries are expressed as fusion proteins of bacteriophage coat proteins, the bacteriophage particles are then screened against the 213P1F11 protein(s).
[0279] Accordingly, peptides having a wide variety of uses, such as therapeutic, prognostic or diagnostic reagents, are thus identified without any prior information on the structure of the expected ligand or receptor molecule. Typical peptide libraries and screening methods that can be used to identify molecules that interact with 213P1F11 protein sequences are disclosed for example in U.S. Pat. No. 5,723,286 issued Mar. 3, 1998 and U.S. Pat. No. 5,733,731 issued Mar. 31, 1998.
[0280] Alternatively, cell lines that express 213P1F11 are used to identify protein-protein interactions mediated by 213P1F11. Such interactions can be examined using immunoprecipitation techniques (see, e.g., Hamilton B. J., et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). 213P1F11 protein can be immunoprecipitated from 213P1F11-expressing cell lines using anti-213P1F11 antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express fusions of 213P1F11 and a His-tag (vectors mentioned above). The immunoprecipitated complex can be examined for protein association by procedures such as Western blotting, 35S-methionine labeling of proteins, protein microsequencing, silver staining and two-dimensional gel electrophoresis.
[0281] Small molecules and ligands that interact with 213P1F11 can be identified through related embodiments of such screening assays. For example, small molecules can be identified that interfere with protein function, including molecules that interfere with 213P1F11's ability to mediate phosphorylation and de-phosphorylation, interaction with DNA or RNA molecules as an indication of regulation of cell cycles, second messenger signaling or tumorigenesis. Similarly, small molecules that modulate 213P1F11-related ion channel, protein pump, or cell communication functions are identified and used to treat patients that have a cancer that expresses 213P1F11 (see, e.g., Hille, B., Ionic Channels of Excitable Membranes 2nd Ed., Sinauer Assoc., Sunderland, Mass., 1992). Moreover, ligands that regulate 213P1F11 function can be identified based on their ability to bind 213P1F11 and activate a reporter construct. Typical methods are discussed for example in U.S. Pat. No. 5,928,868 issued Jul. 27, 1999, and include methods for forming hybrid ligands in which at least one ligand is a small molecule. In an illustrative embodiment, cells engineered to express a fusion protein of 213P1F11 and a DNA-binding protein are used to co-express a fusion protein of a hybrid ligand/small molecule and a cDNA library transcriptional activator protein. The cells further contain a reporter gene, the expression of which is conditioned on the proximity of the first and second fusion proteins to each other, an event that occurs only if the hybrid ligand binds to target sites on both hybrid proteins. Those cells that express the reporter gene are selected and the unknown small molecule or the unknown ligand is identified. This method provides a means of identifying modulators which activate or inhibit 213P1F11.
[0282] An embodiment of this invention comprises a method of screening for a molecule that interacts with a 213P1F11 amino acid sequence shown in FIG. 2 or FIG. 3, comprising the steps of contacting a population of molecules with a 213P1F11 amino acid sequence, allowing the population of molecules and the 213P1F11 amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 213P1F11 amino acid sequence, and then separating molecules that do not interact with the 213P1F11 amino acid sequence from molecules that do. In a specific embodiment, the method further comprises purifying, characterizing and identifying a molecule that interacts with the 213P1F11 amino acid sequence. The identified molecule can be used to modulate a function performed by 213P1F11. In a preferred embodiment, the 213P1F11 amino acid sequence is contacted with a library of peptides.
[0283] X.) Therapeutic Methods and Compositions
[0284] The identification of 213P1F11 as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in prostate and other cancers, opens a number of therapeutic approaches to the treatment of such cancers. As contemplated herein, 213P1F11 functions as a transcription factor involved in activating tumor-promoting genes or repressing genes that block tumorigenesis.
[0285] Accordingly, therapeutic approaches that inhibit the activity of a 213P1F11 protein are useful for patients suffering from a cancer that expresses 213P1F11. These therapeutic approaches generally fall into two classes. One class comprises various methods for inhibiting the binding or association of a 213P1F11 protein with its binding partner or with other proteins. Another class comprises a variety of methods for inhibiting the transcription of a 213P1F11 gene or translation of 213P1F11 mRNA.
[0286] X.A.) Anti-Cancer Vaccines
[0287] The invention provides cancer vaccines comprising a 213P1F11-related protein or 213P1F11-related nucleic acid. In view of the expression of 213P1F11, cancer vaccines prevent and/or treat 213P1F11-expressing cancers with minimal or no effects on non-target tissues. The use of a tumor antigen in a vaccine that generates humoral and/or cell-mediated immune responses as anti-cancer therapy is well known in the art and has been employed in prostate cancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong et al, 1997, J. Immunol. 159:3113-3117).
[0288] Such methods can be readily practiced by employing a 213P1F11-related protein, or a 213P1F11-encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the 213P1F11 immunogen (which typically comprises a number of antibody or T cell epitopes). Skilled artisans understand that a wide variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, e.g., Heryln et al., Ann Med February 1999 31(1):66-78; Maruyama et al., Cancer Immunol Immunother June 2000 49(3):123-32) Briefly, such methods of generating an immune response (e.g. humoral and/or cell-mediated) in a mammal, comprise the steps of: exposing the mammal's immune system to an immunoreactive epitope (e.g. an epitope present in a 213P1F11 protein shown in FIG. 3 or analog or homolog thereof) so that the mammal generates an immune response that is specific for that epitope (e.g. generates antibodies that specifically recognize that epitope). In a preferred method, a 213P1F11 immunogen contains a biological motif, see e.g., Tables V-XIX, or a peptide of a size range from 213P1F11 indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.
[0289] The entire 213P1F11 protein, immunogenic regions or epitopes thereof can be combined and delivered by various means. Such vaccine compositions can include, for example, lipopeptides (e.g., Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin Exp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs) (see e.g., Tarn, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J. P., J. Immunol. Methods 196:17-32, 1996), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Perkus, M. E. et al., In: Concepts in vaccine development, Kauftnann, S. H. E., ed., p. 379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology 4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al., Virology 175:535, 1990), particles of viral or synthetic origin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. et al., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al., Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol. 148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993; Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993; Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.) may also be used.
[0290] In patients with 213P1F11-associated cancer, the vaccine compositions of the invention can also be used in conjunction with other treatments used for cancer, e.g., surgery, chemotherapy, drug therapies, radiation therapies, etc. including use in combination with immune adjuvants such as IL-2, IL-12, GM-CSF, and the like.
[0291] Cellular Vaccines:
[0292] CTL epitopes can be determined using specific algorithms to identify peptides within 213P1F11 protein that bind corresponding HLA alleles (see e.g., Table IV; Epimer™ and Epimatrix™, Brown University (URL www.brown.edu/Researcb/TB-HIV_Lab/epimatrix/epimatrix.html); and, BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI at URL syfpeithi.bmi-heidelberg.com/). In a preferred embodiment, a 213P1F11 immunogen contains one or more amino acid sequences identified using techniques well known in the art, such as the sequences shown in Tables V-XIX, or a peptide of 8, 9, 10 or 11 amino acids specified by an HLA Class I motif/supermotif (e.g., Table IV (A), Table IV (D), or Table IV (E)) and/or a peptide of at least 9 amino acids that comprises an HLA Class II motif/supermotif (e.g., Table IV (B) or Table IV (C)). As is appreciated in the art, the HLA Class I binding groove is essentially closed ended so that peptides of only a particular size range can fit into the groove and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLA Class II binding groove is essentially open ended; therefore a peptide of about 9 or more amino acids can be bound by an HLA Class II molecule. Due to the binding groove differences between HLA Class I and II, HLA Class I motifs are length specific, i.e., position two of a Class I motif is the second amino acid in an amino to carboxyl direction of the peptide. The amino acid positions in a Class II motif are relative only to each other, not the overall peptide, i.e., additional amino acids can be attached to the amino and/or carboxyl termini of a motif-bearing sequence. HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long, or longer than 25 amino acids.
[0293] Antibody-Based Vaccines
[0294] A wide variety of methods for generating an immune response in a mammal are known in the art (for example as the first step in the generation of hybridomas). Methods of generating an immune response in a mammal comprise exposing the mammal's immune system to an immunogenic epitope on a protein (e.g. a 213P1F11 protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to 213P1F11 in a host, by contacting the host with a sufficient amount of at least one 213P1F11 B cell or cytotoxic T-cell epitope or analog thereof, and at least one periodic interval thereafter re-contacting the host with the 213P1F11 B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a 213P1F11-related protein or a man-made multiepitopic peptide comprising: administering 213P1F11 immunogen (e.g. a 213P1F11 protein or a peptide fragment thereof, a 213P1F11 fusion protein or analog etc.) in a vaccine preparation to a human or another mammal. Typically, such vaccine preparations further contain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or a universal helper epitope such as a PADRE™ peptide (Epimmune Inc., San Diego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92). An alternative method comprises generating an immune response in an individual against a 213P1F11 immunogen by: administering in vivo to muscle or skin of the individual's body a DNA molecule that comprises a DNA sequence that encodes a 213P1F11 immunogen, the DNA sequence operatively linked to regulatory sequences which control the expression of the DNA sequence; wherein the DNA molecule is taken up by cells, the DNA sequence is expressed in the cells and an immune response is generated against the immunogen (see, e.g., U.S. Pat. No. 5,962,428). Optionally a genetic vaccine facilitator such as anionic lipids; saponins; lectins; estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; and urea is also administered. In addition, an antiidiotypic antibody can be administered that mimics 213P1F11, in order to generate a response to the target antigen.
[0295] Nucleic Acid Vaccines:
[0296] Vaccine compositions of the invention include nucleic acid-mediated modalities. DNA or RNA that encode protein(s) of the invention can be administered to a patient. Genetic immunization methods can be employed to generate prophylactic or therapeutic humoral and cellular immune responses directed against cancer cells expressing 213P1F11. Constructs comprising DNA encoding a 213P1F11-related protein/immunogen and appropriate regulatory sequences can be injected directly into muscle or skin of an individual, such that the cells of the muscle or skin take-up the construct and express the encoded 213P1F11 protein/immunogen. Alternatively, a vaccine comprises a 213P1F11-related protein. Expression of the 213P1F11-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear a 213P1F11 protein. Various prophylactic and therapeutic genetic immunization techniques known in the art can be used (for review, see information and references published at Internet address www.genweb.com). Nucleic acid-based delivery is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).
[0297] For therapeutic or prophylactic immunization purposes, proteins of the invention can be expressed via viral or bacterial vectors. Various viral gene delivery systems that can be used in the practice of the invention include, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus, and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol. 8:658-663; Tsang et al. J. Natl. Cancer Inst. 87:982-990 (1995)). Non-viral delivery systems can also be employed by introducing naked DNA encoding a 213P1F11-related protein into the patient (e.g., intramuscularly or intradermally) to induce an anti-tumor response.
[0298] Vaccinia virus is used, for example, as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into a host, the recombinant vaccinia virus expresses the protein immunogenic peptide, and thereby elicits a host immune response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein.
[0299] Thus, gene delivery systems are used to deliver a 213P1F11-related nucleic acid molecule. In one embodiment, the full-length human 213P1F11 cDNA is employed. In another embodiment, 213P1F11 nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopes are employed.
[0300] Ex Vivo Vaccines
[0301] Various ex vivo strategies can also be employed to generate an immune response. One approach involves the use of antigen presenting cells (APCs) such as dendritic cells (DC) to present 213P1F11 antigen to a patient's immune system. Dendritic cells express MHC class I and II molecules, B7 co-stimulator, and IL-12, and are thus highly specialized antigen presenting cells. In prostate cancer, autologous dendritic cells pulsed with peptides of the prostate-specific membrane antigen (PSMA) are being used in a Phase I clinical trial to stimulate prostate cancer patients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphy et al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used to present 213P1F11 peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with 213P1F11 peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete 213P1F11 protein. Yet another embodiment involves engineering the overexpression of a 213P1F11 gene in dendritic cells using various implementing vectors known in the art, such as adenovirus (Arthur et al., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al., 1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNA transfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), or tumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med. 186:1177-1182). Cells that express 213P1F11 can also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents.
[0302] X.B.) 213P1F11 as a Target for Antibody-based Therapy
[0303] 213P1F11 is an attractive target for antibody-based therapeutic strategies. A number of antibody strategies are known in the art for targeting both extracellular and intracellular molecules (see, e.g., complement and ADCC mediated killing as well as the use of intrabodies). Because 213P1F11 is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of 213P1F11-immunoreactive compositions are prepared that exhibit excellent sensitivity without toxic, non-specific and/or non-target effects caused by binding of the immunoreactive composition to non-target organs and tissues. Antibodies specifically reactive with domains of 213P1F11 are useful to treat 213P1F11-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.
[0304] 213P1F11 antibodies can be introduced into a patient such that the antibody binds to 213P1F11 and modulates a function, such as an interaction with a binding partner, and consequently mediates destruction of the tumor cells and/or inhibits the growth of the tumor cells. Mechanisms by which such antibodies exert a therapeutic effect can include complement-mediated cytolysis, antibody-dependent cellular cytotoxicity, modulation of the physiological function of 213P1F11, inhibition of ligand binding or signal transduction pathways, modulation of tumor cell differentiation, alteration of tumor angiogenesis factor profiles, and/or apoptosis.
[0305] Those skilled in the art understand that antibodies can be used to specifically target and bind immunogenic molecules such as an immunogenic region of a 213P1F11 sequence shown in FIG. 2 or FIG. 3. In addition, skilled artisans understand that it is routine to conjugate antibodies to cytotoxic agents (see, e.g., Slevers et al. Blood 93:11 3678-3684 (Jun. 1, 1999)). When cytotoxic and/or therapeutic agents are delivered directly to cells, such as by conjugating them to antibodies specific for a molecule expressed by that cell (e.g. 213P1F11), the cytotoxic agent will exert its known biological effect (i.e. cytotoxicity) on those cells.
[0306] A wide variety of compositions and methods for using antibody-cytotoxic agent conjugates to kill cells are known in the art. In the context of cancers, typical methods entail administering to an animal having a tumor a biologically effective amount of a conjugate comprising a selected cytotoxic and/or therapeutic agent linked to a targeting agent (e.g. an anti-213P1F11 antibody) that binds to a marker (e.g. 213P1F11) expressed, accessible to binding or localized on the cell surfaces. A typical embodiment is a method of delivering a cytotoxic and/or therapeutic agent to a cell expressing 213P1F11, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 213P1F11 epitope, and, exposing the cell to the antibody-agent conjugate. Another illustrative embodiment is a method of treating an individual suspected of suffering from metastasized cancer, comprising a step of administering parenterally to said individual a pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent.
[0307] Cancer immunotherapy using anti-213P1F11 antibodies can be done in accordance with various approaches that have been successfully employed in the treatment of other types of cancer, including but not limited to colon cancer (Arlen et al, 1998, Crit. Rev. Immunol. 18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari et al., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al, 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al, 1994, Cancer Res. 54:6160-6166; Velders et al, 1995, Cancer Res. 55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin. Immunol. 11:117-127). Some therapeutic approaches involve conjugation of naked antibody to a toxin or radioisotope, such as the conjugation of Y91 or I131 to anti-CD20 antibodies (e.g., Zevalin™, IDEC Pharmaceuticals Corp. or Bexxar™, Coulter Pharmaceuticals), while others involve co-administration of antibodies and other therapeutic agents, such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). The antibodies can be conjugated to a therapeutic agent. To treat prostate cancer, for example, 213P1F11 antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation. Also, antibodies can be conjugated to a toxin such as calicheamicin (e.g., Mylotarg™, Wyeth-Ayerst, Madison, N.J., a recombinant humanized IgG4 kappa antibody conjugated to antitumor antibiotic calicheamicin) or a maytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform, ImmunoGen, Cambridge, Mass., also see e.g., U.S. Pat. No. 5,416,064).
[0308] Although 213P1F11 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well. Fan et al. (Cancer Res. 53:4637-4642, 1993), Prewett et al. (International J. of Onco. 9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991) describe the use of various antibodies together with chemotherapeutic agents.
[0309] Although 213P1F11 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well.
[0310] Cancer patients can be evaluated for the presence and level of 213P1F11 expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 213P1F11 imaging, or other techniques that reliably indicate the presence and degree of 213P1F11 expression. Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose. Methods for immunohistochemical analysis of tumor tissues are well known in the art.
[0311] Anti-213P1F11 monoclonal antibodies that treat prostate and other cancers include those that initiate a potent immune response against the tumor or those that are directly cytotoxic. In this regard, anti-213P1F11 monoclonal antibodies (mAbs) can elicit tumor cell lysis by either complement-mediated or antibody-dependent cell cytotoxicity (ADCC) mechanisms, both of which require an intact Fc portion of the immunoglobulin molecule for interaction with effector cell Fc receptor sites on complement proteins. In addition, anti-213P1F11 mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 213P1F11. Mechanisms by which directly cytotoxic mAbs act include: inhibition of cell growth, modulation of cellular differentiation, modulation of tumor angiogenesis factor profiles, and the induction of apoptosis. The mechanism(s) by which a particular anti-213P1F11 mAb exerts an anti-tumor effect is evaluated using any number of in vitro assays that evaluate cell death such as ADCC, ADMMC, complement-mediated cell lysis, and so forth, as is generally known in the art.
[0312] In some patients, the use of murine or other non-human monoclonal antibodies, or human/mouse chimeric mAbs can induce moderate to strong immune responses against the non-human antibody. This can result in clearance of the antibody from circulation and reduced efficacy. In the most severe cases, such an immune response can lead to the extensive formation of immune complexes which, potentially, can cause renal failure. Accordingly, preferred monoclonal antibodies used in the therapeutic methods of the invention are those that are either fully human or humanized and that bind specifically to the target 213P1F11 antigen with high affinity but exhibit low or no antigenicity in the patient.
[0313] Therapeutic methods of the invention contemplate the administration of single anti-213P1F11 mabs as well as combinations, or cocktails, of different mAbs. Such mAb cocktails can have certain advantages inasmuch as they contain mAbs that target different epitopes, exploit different effector mechanisms or combine directly cytotoxic mAbs with mAbs that rely on immune effector functionality. Such mAbs in combination can exhibit synergistic therapeutic effects. In addition, anti-213P1F11 mAbs can be administered concomitantly with other therapeutic modalities, including but not limited to various chemotherapeutic agents, androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery or radiation. The anti-213P1F11 mAbs are administered in their “naked” or unconjugated form, or can have a therapeutic agent(s) conjugated to them.
[0314] Anti-213P1F11 antibody formulations are administered via any route capable of delivering the antibodies to a tumor cell. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like. Treatment generally involves repeated administration of the anti-213P1F11 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of 10-1000 mg mAb per week are effective and well tolerated.
[0315] Based on clinical experience with the Herceptin™ mAb in the treatment of metastatic breast cancer, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti-213P1F11 mAb preparation represents an acceptable dosing regimen. Preferably, the initial loading dose is administered as a 90 minute or longer infusion. The periodic maintenance dose is administered as a 30 minute or longer infusion, provided the initial dose was well tolerated. As appreciated by those of skill in the art, various factors can influence the ideal dose regimen in a particular case. Such factors include, for example, the binding affinity and half life of the Ab or mAbs used, the degree of 213P1F11 expression in the patient, the extent of circulating shed 213P1F11 antigen, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient.
[0316] Optionally, patients should be evaluated for the levels of 213P1F11 in a given sample (e.g. the levels of circulating 213P1F11 antigen and/or 213P1F11 expressing cells) in order to assist in the determination of the most effective dosing regimen, etc. Such evaluations are also used for monitoring purposes throughout therapy, and are useful to gauge therapeutic success in combination with the evaluation of other parameters (for example, urine cytology and/or ImmunoCyt levels in bladder cancer therapy, or by analogy, serum PSA levels in prostate cancer therapy).
[0317] Anti-idiotypic anti-213P1F11 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 213P1F11-related protein. In particular, the generation of anti-idiotypic antibodies is well known in the art; this methodology can readily be adapted to generate anti-idiotypic anti-213P1F11 antibodies that mimic an epitope on a 213P1F11-related protein (see, for example, Wagner et al, 1997, Hybridoma 16: 33-40; Foon et al, 1995, J. Clin. Invest. 96:334-342; Herlyn et al, 1996, Cancer Immunol. Immunother. 43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccine strategies.
[0318] X.C.) 213P1F11 as a Target for Cellular Immune Responses
[0319] Vaccines and methods of preparing vaccines that contain an immunogenically effective amount of one or more HLA-binding peptides as described herein are further embodiments of the invention. Furthermore, vaccines in accordance with the invention encompass compositions of one or more of the claimed peptides. A peptide can be present in a vaccine individually. Alternatively, the peptide can exist as a homopolymer comprising multiple copies of the same peptide, or as a heteropolymer of various peptides. Polymers have the advantage of increased immunological reaction and, where different peptide epitopes are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with different antigenic determinants of the pathogenic organism or tumor-related peptide targeted for an immune response. The composition can be a naturally occurring region of an antigen or can be prepared, e.g., recombinantly or by chemical synthesis.
[0320] Carriers that can be used with vaccines of the invention are well known in the art, and include, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, and the like. The vaccines can contain a physiologically tolerable (i.e., acceptable) diluent such as water, or saline, preferably phosphate buffered saline. The vaccines also typically include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Additionally, as disclosed herein, CTL responses can be primed by conjugating peptides of the invention to lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS). Moreover, an adjuvant such as a synthetic cytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotides has been found to increase CTL responses 10- to 100-fold. (see, e.g. Davila and Celis, J. Immunol. 165:539-547 (2000))
[0321] Upon immunization with a peptide composition in accordance with the invention, via injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the vaccine by producing large amounts of CTLs and/or HTLs specific for the desired antigen. Consequently, the host becomes at least partially immune to later development of cells that express or overexpress 213P1F11 antigen, or derives at least some therapeutic benefit when the antigen was tumor-associated.
[0322] In some embodiments, it may be desirable to combine the class I peptide components with components that induce or facilitate neutralizing antibody and or helper T cell responses directed to the target antigen. A preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention. An alternative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a cross reactive HTL epitope such as PADRE™ (Epimmune, San Diego, Calif.) molecule (described e.g., in U.S. Pat. No. 5,736,142).
[0323] A vaccine of the invention can also include antigen-presenting cells (APC), such as dendritic cells (DC), as a vehicle to present peptides of the invention. Vaccine compositions can be created in vitro, following dendritic cell mobilization and harvesting, whereby loading of dendritic cells occurs in vitro. For example, dendritic cells are transfected, e.g., with a minigene in accordance with the invention, or are pulsed with peptides. The dendritic cell can then be administered to a patient to elicit immune responses in vivo. Vaccine compositions, either DNA- or peptide-based, can also be administered in vivo in combination with dendritic cell mobilization whereby loading of dendritic cells occurs in vivo.
[0324] Preferably, the following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition for use in a vaccine, or for selecting discrete epitopes to be included in a vaccine and/or to be encoded by nucleic acids such as a minigene. It is preferred that each of the following principles be balanced in order to make the selection. The multiple epitopes to be incorporated in a given vaccine composition may be, but need not be, contiguous in sequence in the native antigen from which the epitopes are derived.
[0325] 1.) Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with tumor clearance. For HLA Class I this includes 3-4 epitopes that come from at least one tumor associated antigen (TAA). For HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one TAA (see, e.g., Rosenberg et al, Science 278:1447-1450). Epitopes from one TAA may be used in combination with epitopes from one or more additional TAAs to produce a vaccine that targets tumors with varying expression patterns of frequently-expressed TAAs.
[0326] 2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC50 of 500 nM or less, often 200 nM or less; and for Class II an IC50 of 1000 nM or less.
[0327] 3.) Sufficient supermotif bearing-peptides, or a sufficient array of allele-specific motif-bearing peptides, are selected to give broad population coverage. For example, it is preferable to have at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess the breadth, or redundancy of, population coverage.
[0328] 4.) When selecting epitopes from cancer-related antigens it is often useful to select analogs because the patient may have developed tolerance to the native epitope.
[0329] 5.) Of particular relevance are epitopes referred to as “nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A nested peptide sequence can comprise B cell, HLA class I and/or HLA class II epitopes. When providing nested epitopes, a general objective is to provide the greatest number of epitopes per sequence. Thus, an aspect is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing a multi-epitopic sequence, such as a sequence comprising nested epitopes, it is generally important to screen the sequence in order to insure that it does not have pathological or other deleterious biological properties.
[0330] 6.) If a polyepitopic protein is created, or when creating a minigene, an objective is to generate the smallest peptide that encompasses the epitopes of interest. This principle is similar, if not the same as that employed when selecting a peptide comprising nested epitopes. However, with an artificial polyepitopic peptide, the size minimization objective is balanced against the need to integrate any spacer sequences between epitopes in the polyepitopic protein. Spacer amino acid residues can, for example, be introduced to avoid junctional epitopes (an epitope recognized by the immune system, not present in the target antigen, and only created by the man-made juxtaposition of epitopes), or to facilitate cleavage between epitopes and thereby enhance epitope presentation. Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that non-native epitope. Of particular concern is a junctional epitope that is a “dominant epitope.” A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.
[0331] 7.) Where the sequences of multiple variants of the same target protein are present, potential peptide epitopes can also be selected on the basis of their conservancy. For example, a criterion for conservancy may define that the entire sequence of an HLA class I binding peptide or the entire 9-mer core of a class II binding peptide be conserved in a designated percentage of the sequences evaluated for a specific protein antigen.
[0332] X.C.1. Minigene Vaccines
[0333] A number of different approaches are available which allow simultaneous delivery of multiple epitopes. Nucleic acids encoding the peptides of the invention are a particularly useful embodiment of the invention. Epitopes for inclusion in a minigene are preferably selected according to the guidelines set forth in the previous section. A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding a peptide comprising one or multiple epitopes of the invention.
[0334] The use of multi-epitope minigenes is described below and in, Ishioka et al., J. Immunol 162:3915-3925, 1999; An, L. and Whitton, J. L., J. Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol 157:822, 1996; Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid encoding supermotif- and/or motif-bearing epitopes derived 213P1F11, the PADRE® universal helper T cell epitope or multiple HTL epitopes from 213P1F11 (see e.g., Tables V-XIX), and an endoplasmic reticulum-translocating signal sequence can be engineered. A vaccine may also comprise epitopes that are derived from other TAAs.
[0335] The immunogenicity of a multi-epitopic minigene can be confirmed in transgenic mice to evaluate the magnitude of CTL induction responses against the epitopes tested. Further, the immunogenicity of DNA-encoded epitopes in vivo can be correlated with the in vitro responses of specific CTL lines against target cells transfected with the DNA plasmid. Thus, these experiments can show that the minigene serves to both: 1.) generate a CTL response and 2.) that the induced CTLs recognized cells expressing the encoded epitopes.
[0336] For example, to create a DNA sequence encoding the selected epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes may be reverse translated. A human codon usage table can be used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, antibody epitopes, a ubiquitination signal sequence, and/or an endoplasmic reticulum targeting signal. In addition, HLA presentation of CTL and HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes; these larger peptides comprising the epitope(s) are within the scope of the invention.
[0337] The minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the epitope polypeptide, can then be cloned into a desired expression vector.
[0338] Standard regulatory sequences well known to those of skill in the art are preferably included in the vector to ensure expression in the target cells. Several vector elements are desirable: a promoter with a down-stream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.
[0339] Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing minigene expression.
[0340] Once an expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.
[0341] In addition, immunostimulatory sequences (ISSs or CpGs) appear to play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence, if desired to enhance immunogenicity.
[0342] In some embodiments, a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (included to enhance or decrease immunogenicity) can be used. Examples of proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, or for HTL responses, pan-DR binding proteins (PADRE™, Epimmune, San Diego, Calif.). Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-&bgr;) may be beneficial in certain diseases.
[0343] Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker flasks or a bioreactor according to well-known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.
[0344] Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as “naked DNA,” is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available. Cationic lipids, glycolipids, and fusogenic liposomes can also be used in the formulation (see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.
[0345] Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes. For example, the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays. The transfection method used will be dependent on the final formulation. Electroporation can be used for “naked” DNA, whereas cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 (51Cr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 51Cr release, indicates both production of, and HLA presentation of, minigene-encoded CTL epitopes. Expression of HTL epitopes may be evaluated in an analogous manner using assays to assess HTL activity.
[0346] In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product. The dose and route of administration are formulation dependent (e.g., IM for DNA in PBS, intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for one week in the presence of peptides encoding each epitope being tested. Thereafter, for CTL effector cells, assays are conducted for cytolysis of peptide-loaded, 51Cr-labeled target cells using standard techniques. Lysis of target cells that were sensitized by HLA loaded with peptide epitopes, corresponding to minigene-encoded epitopes, demonstrates DNA vaccine function for in vivo induction of CTLs. Immunogenicity of HTL epitopes is confirmed in transgenic mice in an analogous manner.
[0347] Alternatively, the nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253. Using this technique, particles comprised solely of DNA are administered. In a further alternative embodiment, DNA can be adhered to particles, such as gold particles.
[0348] Minigenes can also be delivered using other bacterial or viral delivery systems well known in the art, e.g., an expression construct encoding epitopes of the invention can be incorporated into a viral vector such as vaccinia.
[0349] X.C.2. Combinations of CTL Peptides with Helper Peptides
[0350] Vaccine compositions comprising CTL peptides of the invention can be modified, e.g., analoged, to provide desired attributes, such as improved serum half life, broadened population coverage or enhanced immunogenicity.
[0351] For instance, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. Although a CTL peptide can be directly linked to a T helper peptide, often CTL epitope/HTL epitope conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues and sometimes 10 or more residues. The CTL peptide epitope can be linked to the T helper peptide epitope either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.
[0352] In certain embodiments, the T helper peptide is one that is recognized by T helper cells present in a majority of a genetically diverse population. This can be accomplished by selecting peptides that bind to many, most, or all of the HLA class II molecules. Examples of such amino acid bind many HLA Class II molecules include sequences from antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO:______), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO:______), and Streptococcus 18 kD protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO:______). Other examples include peptides bearing a DR 1-4-7 supermotif, or either of the DR3 motifs.
[0353] Alternatively, it is possible to prepare synthetic peptides capable of stimulating T helper lymphocytes, in a loosely HLA-restricted fashion, using amino acid sequences not found in nature (see, e.g., PCT publication WO 95/07707). These synthetic compounds called Pan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego, Calif.) are designed to most preferably bind most HLA-DR (human HLA class II) molecules. For instance, a pan-DR-binding epitope peptide having the formula: aKXVAAWTLKAAa (SEQ ID NO:______), where “X” is either cyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanine or L-alanine, has been found to bind to most HLA-DR alleles, and to stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type. An alternative of a pan-DR binding epitope comprises all “L” natural amino acids and can be provided in the form of nucleic acids that encode the epitope.
[0354] HTL peptide epitopes can also be modified to alter their biological properties. For example, they can be modified to include D-amino acids to increase their resistance to proteases and thus extend their serum half life, or they can be conjugated to other molecules such as lipids, proteins, carbohydrates, and the like to increase their biological activity. For example, a T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.
[0355] X.C.3. Combinations of CTL Peptides with T Cell Priming Agents
[0356] In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes B lymphocytes or T lymphocytes. Lipids have been identified as agents capable of priming CTL in vivo. For example, palmitic acid residues can be attached to the &egr;-and &agr;-amino groups of a lysine residue and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidated peptide can then be administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. In a preferred embodiment, a particularly effective immunogenic composition comprises palmitic acid attached to &egr;- and &agr;-amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.
[0357] As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide (see, e.g., Deres, et al., Nature 342:561, 1989). Peptides of the invention can be coupled to P3CSS, for example, and the lipopeptide administered to an individual to specifically prime an immune response to the target antigen. Moreover, because the induction of neutralizing antibodies can also be primed with P3CSS-conjugated epitopes, two such compositions can be combined to more effectively elicit both humoral and cell-mediated responses.
[0358] X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides
[0359] An embodiment of a vaccine composition in accordance with the invention comprises ex vivo administration of a cocktail of epitope-bearing peptides to PBMC, or isolated DC therefrom, from the patient's blood. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin™ (Pharmacia-Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC with peptides and prior to reinfusion into patients, the DC are washed to remove unbound peptides. In this embodiment, a vaccine comprises peptide-pulsed DCs which present the pulsed peptide epitopes complexed with HLA molecules on their surfaces.
[0360] The DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL responses to 213P1F11. Optionally, a helper T cell (HTL) peptide, such as a natural or artificial loosely restricted HLA Class II peptide, can be included to facilitate the CTL response. Thus, a vaccine in accordance with the invention is used to treat a cancer which expresses or overexpresses 213P1F11.
[0361] X.D. Adoptive Immunotherapy
[0362] Antigenic 213P1F11-related peptides are used to elicit a CTL and/or HTL response ex vivo, as well. The resulting CTL or HTL cells, can be used to treat tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention. Ex vivo CTL or HTL responses to a particular antigen are induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptide. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cell (e.g., a tumor cell). Transfected dendritic cells may also be used as antigen presenting cells.
[0363] X.E. Administration of Vaccines for Therapeutic or Prophylactic Purposes
[0364] Pharmaceutical and vaccine compositions of the invention are typically used to treat and/or prevent a cancer that expresses or overexpresses 213P1F11. In therapeutic applications, peptide and/or nucleic acid compositions are administered to a patient m an amount sufficient to elicit an effective B cell, CTL and/or HTL response to the antigen and to cure or at least partially arrest or slow symptoms and/or complications. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.
[0365] For pharmaceutical compositions, the immunogenic peptides of the invention, or DNA encoding them, are generally administered to an individual already bearing a tumor that expresses 213P1F 11. The peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences. Patients can be treated with the immunogenic peptides separately or in conjunction with other treatments, such as surgery, as appropriate.
[0366] For therapeutic use, administration should generally begin at the first diagnosis of 213P1F11-associated cancer. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. The embodiment of the vaccine composition (i.e., including, but not limited to embodiments such as peptide cocktails, polyepitopic polypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells) delivered to the patient may vary according to the stage of the disease or the patient's health status. For example, in a patient with a tumor that expresses 213P1F11, a vaccine comprising 213P1F11-specific CTL may be more efficacious in killing tumor cells in patient with advanced disease than alternative embodiments.
[0367] It is generally important to provide an amount of the peptide epitope delivered by a mode of administration sufficient to effectively stimulate a cytotoxic T cell response; compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the invention.
[0368] The dosage for an initial therapeutic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1,000 &mgr;g and the higher value is about 10,000; 20,000; 30,000; or 50,000 &mgr;g. Dosage values for a human typically range from about 500 &mgr;g to about 50,000 &mgr;g per 70 kilogram patient. Boosting dosages of between about 1.0 &mgr;g to about 50,000 &mgr;g of peptide pursuant to a boosting regimen over weeks to months may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from the patient's blood. Administration should continue until at least clinical symptoms or laboratory tests indicate that the neoplasia, has been eliminated or reduced and for a period thereafter. The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.
[0369] In certain embodiments, the peptides and compositions of the present invention are employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of the peptides in preferred compositions of the invention, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions relative to these stated dosage amounts.
[0370] The vaccine compositions of the invention can also be used purely as prophylactic agents. Generally the dosage for an initial prophylactic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 &mgr;g and the higher value is about 10,000; 20,000; 30,000; or 50,000 &mgr;g. Dosage values for a human typically range from about 500 &mgr;g to about 50,000 &mgr;g per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 &mgr;g to about 50,000 fig of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine. The immunogenicity of the vaccine can be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patient's blood.
[0371] The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, nasal, intrathecal, or local (e.g. as a cream or topical ointment) administration. Preferably, the pharmaceutical compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
[0372] A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
[0373] The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
[0374] The concentration of peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
[0375] A human unit dose form of a composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, in one embodiment an aqueous carrier, and is administered in a volume/quantity that is known by those of skill in the art to be used for administration of such compositions to humans (see, e.g., Remington's Pharmaceutical Sciences, 17th Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pa., 1985). For example a peptide dose for initial immunization can be from about 1 to about 50,000 &mgr;g, generally 100-5,000 &mgr;g, for a 70 kg patient. For example, for nucleic acids an initial immunization may be performed using an expression vector in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 &mgr;g) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-107 to 5×109 pfu.
[0376] For antibodies, a treatment generally involves repeated administration of the anti-213P1F11 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1 to about 10 mg/kg body weight. In general, doses in the range of 10-500 mg mAb per week are effective and well tolerated. Moreover, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti-213P1F11 mAb preparation represents an acceptable dosing regimen. As appreciated by those of skill in the art, various factors can influence the ideal dose in a particular case. Such factors include, for example, half life of a composition, the binding affinity of an Ab, the immunogenicity of a substance, the degree of 213P1F11 expression in the patient, the extent of circulating shed 213P1F11 antigen, the desired steady-state concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient. Non-limiting preferred human unit doses are, for example, 500 &mgr;g-1 mg, 1 mg-50 mg, 50 mg-100 mg, 100 mg-200 mg, 200 mg-300 mg, 400 mg-500 mg, 500 mg-600 mg, 600 mg-700 mg, 700 mg-800 mg, 800 mg-900 mg, 900 mg-1 g, or 1 mg-700 mg. In certain embodiments, the dose is in a range of 2-5 mg/kg body weight, e.g., with follow on weekly doses of 1-3 mg/kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg body weight followed, e.g., in two, three or four weeks by weekly doses; 0.5-10 mg/kg body weight, e.g., followed in two, three or four weeks by weekly doses; 225, 250, 275, 300, 325, 350, 375, 400 mg m2 of body area weekly; 1-600 mg m2 of body area weekly; 225-400 mg m2 of body area weekly; these does can be followed by weekly doses for 2, 3, 4, 5, 6, 7, 8, 9, 19, 11, 12 or more weeks.
[0377] In one embodiment, human unit dose forms of polynucleotides comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art a therapeutic effect depends on a number of factors, including the sequence of the polynucleotide, molecular weight of the polynucleotide and route of administration. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. Generally, for a polynucleotide of about 20 bases, a dosage range may be selected from, for example, an independently selected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to an independently selected upper limit, greater than the lower limit, of about 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dose may be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200 to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to 500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg. Generally, parenteral routes of administration may require higher doses of polynucleotide compared to more direct application to the nucleotide to diseased tissue, as do polynucleotides of increasing length.
[0378] In one embodiment, human unit dose forms of T-cells comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art, a therapeutic effect depends on a number of factors. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. A dose may be about 104 cells to about 106 cells, about 106 cells to about 108 cells, about 108 to about 1011 cells, or about 108 to about 5×1010 cells. A dose may also about 106 cells/m2 to about 1010 cells/m2, or about 106 cells/m2 to about 108 cells/m2.
[0379] Proteins(s) of the invention, and/or nucleic acids encoding the protein(s), can also be administered via liposomes, which may also serve to: 1) target the proteins(s) to a particular tissue, such as lymphoid tissue; 2) to target selectively to diseases cells; or, 3) to increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al, Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
[0380] For targeting cells of the immune system, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.
[0381] For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.
[0382] For aerosol administration, immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are about 0.01%-20% by weight, preferably about 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from about 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute about 0.1%-20% by weight of the composition, preferably about 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
[0383] XI.) Diagnostic and Prognostic Embodiments of 213P1F11.
[0384] As disclosed herein, 213P1F11 polynucleotides, polypeptides, reactive cytotoxic T cells (CTL), reactive helper T cells (HTL) and anti-polypeptide antibodies are used in well known diagnostic, prognostic and therapeutic assays that examine conditions associated with dysregulated cell growth such as cancer, in particular the cancers listed in Table I (see, e.g., both its specific pattern of tissue expression as well as its overexpression in certain cancers as described for example in the Example entitled “Expression Analysis of 213P1F11 in Normal Tissues and patient Specimens”).
[0385] 213P1F11 can be analogized to a prostate associated antigen PSA, the archetypal marker that has been used by medical practitioners for years to identify and monitor the presence of prostate cancer (see, e.g., Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J. Urol. August; 162(2):293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91(19): 1635-1640(1999)). A variety of other diagnostic markers are also used in similar contexts including p53 and K-ras (see, e.g., Tulchinsky et al., Int J Mol Med July 1999 4(1):99-102 and Minimoto et al., Cancer Detect Prev 2000;24(1):1-12). Therefore, this disclosure of 213P1F11 polynucleotides and polypeptides (as well as 213P1F11 polynucleotide probes and anti-213P1F11 antibodies used to identify the presence of these molecules) and their properties allows skilled artisans to utilize these molecules in methods that are analogous to those used, for example, in a variety of diagnostic assays directed to examining conditions associated with cancer.
[0386] Typical embodiments of diagnostic methods which utilize the 213P1F11 polynucleotides, polypeptides, reactive T cells and antibodies are analogous to those methods from well-established diagnostic assays which employ, e.g., PSA polynucleotides, polypeptides, reactive T cells and antibodies. For example, just as PSA polynucleotides are used as probes (for example in Northern analysis, see, e.g., Sharief et al., Biochem. Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example in PCR analysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190 (2000)) to observe the presence and/or the level of PSA mRNAs in methods of monitoring PSA overexpression or the metastasis of prostate cancers, the 213P1F11 polynucleotides described herein can be utilized in the same way to detect 213P1F11 overexpression or the metastasis of prostate and other cancers expressing this gene. Alternatively, just as PSA polypeptides are used to generate antibodies specific for PSA which can then be used to observe the presence and/or the level of PSA proteins in methods to monitor PSA protein overexpression (see, e.g., Stephan et al., Urology 55(4):560-3 (2000)) or the metastasis of prostate cells (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the 213P1F11 polypeptides described herein can be utilized to generate antibodies for use in detecting 213P1F11 overexpression or the metastasis of prostate cells and cells of other cancers expressing this gene.
[0387] Specifically, because metastases involves the movement of cancer cells from an organ of origin (such as the lung or prostate gland etc.) to a different area of the body (such as a lymph node), assays which examine a biological sample for the presence of cells expressing 213P1F11 polynucleotides and/or polypeptides can be used to provide evidence of metastasis. For example, when a biological sample from tissue that does not normally contain 213P1F11-expressing cells (lymph node) is found to contain 213P1F11-expressing cells such as the 213P1F11 expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.
[0388] Alternatively 213P1F11 polynucleotides and/or polypeptides can be used to provide evidence of cancer, for example, when cells in a biological sample that do not normally express 213P1F11 or express 213P1F11 at a different level are found to express 213P1F11 or have an increased expression of 213P1F11 (see, e.g., the 213P1F11 expression in the cancers listed in Table I and in patient samples etc. shown in the accompanying Figures). In such assays, artisans may further wish to generate supplementary evidence of metastasis by testing the biological sample for the presence of a second tissue restricted marker (in addition to 213P1F11) such as PSA, PSCA etc. (see, e.g., Alanen et al, Pathol. Res. Pract. 192(3): 233-237 (1996)).
[0389] Just as PSA polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA, 213P1F11 polynucleotide fragments and polynucleotide variants are used in an analogous manner. In particular, typical PSA polynucleotides used in methods of monitoring PSA are probes or primers which consist of fragments of the PSA cDNA sequence. Illustrating this, primers used to PCR amplify a PSA polynucleotide must include less than the whole PSA sequence to function in the polymerase chain reaction. In the context of such PCR reactions, skilled artisans generally create a variety of different polynucleotide fragments that can be used as primers in order to amplify different portions of a polynucleotide of interest or to optimize amplification reactions (see, e.g., Caetano-Anolles, G. Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al, Methods Mol. Biol. 98:121-154 (1998)). An additional illustration of the use of such fragments is provided in the Example entitled “Expression Analysis of 213P1F11 in Normal Tissues and Patient Specimens,” where a 213P1F11 polynucleotide fragment is used as a probe to show the expression of 213P1F11 RNAs in cancer cells. In addition, variant polynucleotide sequences are typically used as primers and probes for the corresponding mRNAs in PCR and Northern analyses (see, e.g., Sawai et al., Fetal Diagn. Ther. 1996 Nov-Dec 11(6):407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al eds., 1995)). Polynucleotide fragments and variants are useful in this context where they are capable of binding to a target polynucleotide sequence (e.g., a 213P1F11 polynucleotide shown in FIG. 2 or variant thereof) under conditions of high stringency.
[0390] Furthermore, PSA polypeptides which contain an epitope that can be recognized by an antibody or T cell that specifically binds to that epitope are used in methods of monitoring PSA. 213P1F11 polypeptide fragments and polypeptide analogs or variants can also be used in an analogous manner. This practice of using polypeptide fragments or polypeptide variants to generate antibodies (such as anti-PSA antibodies or T cells) is typical in the art with a wide variety of systems such as fusion proteins being used by practitioners (see, e.g., Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubel et al. eds., 1995). In this context, each epitope(s) functions to provide the architecture with which an antibody or T cell is reactive. Typically, skilled artisans create a variety of different polypeptide fragments that can be used in order to generate immune responses specific for different portions of a polypeptide of interest (see, e.g., U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it may be preferable to utilize a polypeptide comprising one of the 213P1F11 biological motifs discussed herein or a motif-bearing subsequence which is readily identified by one of skill in the art based on motifs available in the art. Polypeptide fragments, variants or analogs are typically useful in this context as long as they comprise an epitope capable of generating an antibody or T cell specific for a target polypeptide sequence (e.g. a 213P1F11 polypeptide shown in FIG. 3).
[0391] As shown herein, the 213P1F11 polynucleotides and polypeptides (as well as the 213P1F11 polynucleotide probes and anti-213P1F11 antibodies or T cells used to identify the presence of these molecules) exhibit specific properties that make them useful in diagnosing cancers such as those listed in Table I. Diagnostic assays that measure the presence of 213P1F11 gene products, in order to evaluate the presence or onset of a disease condition described herein, such as prostate cancer, are used to identify patients for preventive measures or further monitoring, as has been done so successfully with PSA. Moreover, these materials satisfy a need in the art for molecules having similar or complementary characteristics to PSA in situations where, for example, a definite diagnosis of metastasis of prostatic origin cannot be made on the basis of a test for PSA alone (see, e.g., Alanen et al, Pathol. Res. Pract. 192(3): 233-237 (1996)), and consequently, materials such as 213P1F11 polynucleotides and polypeptides (as well as the 213P1F11 polynucleotide probes and anti-213P1F11 antibodies used to identify the presence of these molecules) need to be employed to confirm a metastases of prostatic origin.
[0392] Finally, in addition to their use in diagnostic assays, the 213P1F11 polynucleotides disclosed herein have a number of other utilities such as their use in the identification of oncogenetic associated chromosomal abnormalities in the chromosomal region to which the 213P1F11 gene maps (see the Example entitled “Chromosomal Mapping of 213P1F11” below). Moreover, in addition to their use in diagnostic assays, the 213P1F11-related proteins and polynucleotides disclosed herein have other utilities such as their use in the forensic analysis of tissues of unknown origin (see, e.g., Takahama K Forensic Sci Int Jun. 28, 1996;80(1-2): 63-9).
[0393] Additionally, 213P1F11-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of 213P1F11. For example, the amino acid or nucleic acid sequence of FIG. 2 or FIG. 3, or fragments of either, can be used to generate an immune response to a 213P1F11 antigen. Antibodies or other molecules that react with 213P1F11 can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.
[0394] XII.) Inhibition of 213P1F11 Protein Function
[0395] The invention includes various methods and compositions for inhibiting the binding of 213P1F11 to its binding partner or its association with other protein(s) as well as methods for inhibiting 213P1F11 function.
[0396] XII.A.) Inhibition of 213P1F11 With Intracellular Antibodies
[0397] In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 213P1F11 are introduced into 213P1F11 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti-213P1F11 antibody is expressed intracellularly, binds to 213P1F11 protein, and thereby inhibits its function. Methods for engineering such intracellular single chain antibodies are well known. Such intracellular antibodies, also known as “intrabodies”, are specifically targeted to a particular compartment within the cell, providing control over where the inhibitory activity of the treatment is focused. This technology has been successfully applied in the art (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors (see, e.g., Richardson et al., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al, 1994, J. Biol. Chem. 289: 23931-23936; Deshane et al, 1994, Gene Ther. 1: 332-337).
[0398] Single chain antibodies comprise the variable domains of the heavy and light chain joined by a flexible linker polypeptide, and are expressed as a single polypeptide. Optionally, single chain antibodies are expressed as a single chain variable region fragment joined to the light chain constant region. Well-known intracellular trafficking signals are engineered into recombinant polynucleotide vectors encoding such single chain antibodies in order to precisely target the intrabody to the desired intracellular compartment. For example, intrabodies targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and, optionally, a C-terminal ER retention signal, such as the KDEL amino acid motif. Intrabodies intended to exert activity in the nucleus are engineered to include a nuclear localization signal. Lipid moieties are joined to intrabodies in order to tether the intrabody to the cytosolic side of the plasma membrane. Intrabodies can also be targeted to exert function in the cytosol. For example, cytosolic intrabodies are used to sequester factors within the cytosol, thereby preventing them from being transported to their natural cellular destination.
[0399] In one embodiment, intrabodies are used to capture 213P1F11 in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engineered into such 213P1F11 intrabodies in order to achieve the desired targeting. Such 213P1F11 intrabodies are designed to bind specifically to a particular 213P1F11 domain. In another embodiment, cytosolic intrabodies that specifically bind to a 213P1F11 protein are used to prevent 213P1F P11 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g., preventing 213P1F11 from forming transcription complexes with other factors).
[0400] In order to specifically direct the expression of such intrabodies to particular cells, the transcription of the intrabody is placed under the regulatory control of an appropriate tumor-specific promoter and/or enhancer. In order to target intrabody expression specifically to prostate, for example, the PSA promoter and/or promoter/enhancer can be utilized (See, for example, U.S. Pat. No. 5,919,652 issued Jul. 6, 1999).
[0401] XII.B.) Inhibition of 213P1F11 with Recombinant Proteins
[0402] In another approach, recombinant molecules bind to 213P1F11 and thereby inhibit 213P1F11 function. For example, these recombinant molecules prevent or inhibit 213P1F11 from accessing/binding to its binding partner(s) or associating with other protein(s). Such recombinant molecules can, for example, contain the reactive part(s) of a 213P1F11 specific antibody molecule. In a particular embodiment, the 213P1F11 binding domain of a 213P1F11 binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 213P1F11 ligand binding domains linked to the Fc portion of a human IgG, such as human IgG1. Such IgG portion can contain, for example, the CH2 and CH3 domains and the hinge region, but not the CH1 domain. Such dimeric fusion proteins are administered in soluble form to patients suffering from a cancer associated with the expression of 213P1F11, whereby the dimeric fusion protein specifically binds to 213P1F11 and blocks 213P1F11 interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.
[0403] XII.C.) Inhibition of 213P1F11 Transcription or Translation
[0404] The present invention also comprises various methods and compositions for inhibiting the transcription of the 213P1F11 gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 213P1F11 mRNA into protein.
[0405] In one approach, a method of inhibiting the transcription of the 213P1F11 gene comprises contacting the 213P1F11 gene with a 213P1F11 antisense polynucleotide. In another approach, a method of inhibiting 213P1F11 mRNA translation comprises contacting a 213P1F11 mRNA with an antisense polynucleotide. In another approach, a 213P1F11 specific ribozyme is used to cleave a 213P1F11 message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 213P1F11 gene, such as 213P1F11 promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a 213P1F11 gene transcription factor are used to inhibit 213P1F11F11 mRNA transcription. The various polynucleotides and compositions useful in the aforementioned methods have been described above. The use of antisense and ribozyme molecules to inhibit transcription and translation is well known in the art.
[0406] Other factors that inhibit the transcription of 213P1F11 by interfering with 213P1F11 transcriptional activation are also useful to treat cancers expressing 213P1F11. Similarly, factors that interfere with 213P1F11 processing are useful to treat cancers that express 213P1F11. Cancer treatment methods utilizing such factors are also within the scope of the invention.
[0407] XII.D.) General Considerations for Therapeutic Strategies
[0408] Gene transfer and gene therapy technologies can be used to deliver therapeutic polynucleotide molecules to tumor cells synthesizing 213P1F11 (i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other 213P1F11 inhibitory molecules). A number of gene therapy approaches are known in the art. Recombinant vectors encoding 213P1F11 antisense polynucleotides, ribozymes, factors capable of interfering with 213P1F11 transcription, and so forth, can be delivered to target tumor cells using such gene therapy approaches.
[0409] The above therapeutic approaches can be combined with any one of a wide variety of surgical, chemotherapy or radiation therapy regimens. The therapeutic approaches of the invention can enable the use of reduced dosages of chemotherapy (or other therapies) and/or less frequent administration, an advantage for all patients and particularly for those that do not tolerate the toxicity of the chemotherapeutic agent well.
[0410] The anti-tumor activity of a particular composition (e.g., antisense, ribozyme, intrabody), or a combination of such compositions, can be evaluated using various in vitro and in vivo assay systems. In vitro assays that evaluate therapeutic activity include cell growth assays, soft agar assays and other assays indicative of tumor promoting activity, binding assays capable of determining the extent to which a therapeutic composition will inhibit the binding of 213P1F11 to a binding partner, etc.
[0411] In vivo, the effect of a 213P1F11 therapeutic composition can be evaluated in a suitable animal model. For example, xenogenic prostate cancer models can be used, wherein human prostate cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine 3: 402-408). For example, PCT Patent Application WO98/16628 and U.S. Pat. No. 6,107,540 describe various xenograft models of human prostate cancer capable of recapitulating the development of primary tumors, micrometastasis, and the formation of osteoblastic metastases characteristic of late stage disease. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.
[0412] In vivo assays that evaluate the promotion of apoptosis are useful in evaluating therapeutic compositions. In one embodiment, xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.
[0413] The therapeutic compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).
[0414] Therapeutic formulations can be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like. A preferred formulation for intravenous injection comprises the therapeutic composition in a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinylchloride or polyethylene bags containing 0.9% sterile Sodium Chloride for Injection, USP. Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing for example, benzyl alcohol preservative) or in sterile water prior to injection.
[0415] Dosages and administration protocols for the treatment of cancers using the foregoing methods will vary with the method and the target cancer, and will generally depend on a number of other factors appreciated in the art.
[0416] XIII.) Kits
[0417] For use in the diagnostic and therapeutic applications described herein, kits are also within the scope of the invention. Such kits can comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method. For example, the container(s) can comprise a probe that is or can be detectably labeled. Such probe can be an antibody or polynucleotide specific for a 213P1F11-related protein or a 213P1F11 gene or message, respectively. Where the method utilizes nucleic acid hybridization to detect the target nucleic acid, the kit can also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label. The kit can include all or part of the amino acid sequence of FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acid molecules that encodes such amino acid sequences.
[0418] The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
[0419] A label can be present on the container to indicate that the composition is used for a specific therapy or non-therapeutic application, and can also indicate directions for either in vivo or in vitro use, such as those described above. Directions and or other information can also be included on an insert which is included with the kit.
EXAMPLES[0420] Various aspects of the invention are further described and illustrated by way of the several examples that follow, none of which are intended to limit the scope of the invention.
Example 1 SSH-Generated Isolation of a cDNA Fragment of the 213P1F11 Gene[0421] To isolate genes that are over-expressed in bladder cancer, Suppression Subtractive Hybridization (SSH) procedure using cDNA derived from bladder cancer tissues was performed, including invasive transitional cell carcinoma. The 213P1F11 SSH cDNA sequence was derived from a bladder cancer pool minus cDNAs derived from 9 normal tissues. The 213P1F11 cDNA was identified as highly expressed in the bladder cancer tissue pool, with no expression detected in normal tissues.
[0422] The SSH DNA sequence of 166 bp (FIG. 1) did not show homology to any known gene. 213P1F11v.1 of 3336 bp was identified and the open reading frame cloned from bladder cancer cDNA, revealing an ORF of 242 amino acids (FIG. 2 and FIG. 3). Other variants of 213P1F11, were also identified and these are listed in FIGS. 2 and 3. 213P1F11 v.1 reveals 100% identity to caspase-14 precursor apoptosis-related cysteine protease protein (FIG. 4).
[0423] Materials and Methods
[0424] Human Tissues:
[0425] The patient cancer and normal tissues were purchased from different sources such as the NDRI (Philadelphia, Pa.). mRNA for some-normal tissues were purchased from Clontech, Palo Alto, Calif.
[0426] RNA Isolation:
[0427] Tissues were homogenized in Trizol reagent (Life Technologies, Gibco BRL) using 10 ml/g tissue isolate total RNA. Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Total and mRNA were quantified by spectrophotometric analysis (O.D. 260/280 nm) and analyzed by gel electrophoresis.
[0428] Oligonucleotides:
[0429] The following HPLC purified oligonucleotides were used. 1 DPNCDN (cDNA synthesis primer): (SEQ ID NO: 707) 5′TTTTGATCAAGCTT303′ Adaptor 1: (SEQ ID NO: 708) 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ ID NO: 708) 3′GGCCCGTCCTAG5′ Adaptor 2: (SEQ ID NO: 709) 5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO: 710) 3′CGGCTCCTAG5′ PCR primer 1: (SEQ ID NO: 711) 5′CTAATACGACTCACTATAGGGC3′ Nested primer (NP)1: (SEQ ID NO: 712) 5′TCGAGCGGCCGCCCGGGCAGGA3′ Nested primer (NP)2: (SEQ ID NO: 713) 5′AGCGTGGTCGCGGCCGAGGA3′
[0430] Suppression Subtractive Hybridization:
[0431] Suppression Subtractive Hybridization (SSH) was used to identify cDNAs corresponding to genes that may be differentially expressed in bladder cancer. The SSH reaction utilized cDNA from bladder cancer and normal tissues.
[0432] The gene 213P1F11 sequence was derived from a bladder cancer pool minus normal tissue cDNA subtraction. The SSH DNA sequence (FIG. 1) was identified.
[0433] The cDNA derived from of pool of normal tissues was used as the source of the “driver” cDNA, while the cDNA from a pool of bladder cancer tissues was used as the source of the “tester” cDNA. Double stranded cDNAs corresponding to tester and driver cDNAs were synthesized from 2 &mgr;g of poly(A)+ RNA isolated from the relevant xenograft tissue, as described above, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- and second-strand synthesis were carried out as described in the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3 hrs at 37° C. Digested cDNA was extracted with phenol/chloroform (1:1) and ethanol precipitated.
[0434] Driver cDNA was generated by combining in a 1:1 ratio Dpn II digested cDNA from the relevant tissue source (see above) with a mix of digested cDNAs derived from the nine normal tissues: stomach, skeletal muscle, lung, brain, liver, kidney, pancreas, small intestine, and heart.
[0435] Tester cDNA was generated by diluting 1 &mgr;l of Dpn II digested cDNA from the relevant tissue source (see above) (400 ng) in 5 it of water. The diluted cDNA (2 &mgr;l, 160 ng) was then ligated to 2 &mgr;l of Adaptor I and Adaptor 2 (10 &mgr;M), in separate ligation reactions, in a total volume of 10 &mgr;l at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation was terminated with 1 &mgr;l of 0.2 M EDTA and heating at 72° C. for 5 min.
[0436] The first hybridization was performed by adding 1.5 &mgr;l (600 ng) of driver cDNA to each of two tubes containing 1.5 &mgr;l (20 ng) Adaptor 1- and Adaptor 2-ligated tester cDNA. In a final volume of 4 &mgr;l, the samples were overlaid with mineral oil, denatured in an MJ Research thermal cycler at 98° C. for 1.5 minutes, and then were allowed to hybridize for 8 hrs at 68° C. The two hybridizations were then mixed together with an additional 1 &mgr;l of fresh denatured driver cDNA and were allowed to hybridize overnight at 68° C. The second hybridization was then diluted in 200 &mgr;l of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA, heated at 70° C. for 7 min. and stored at −20° C.
[0437] PCR Amplification. Cloning and Sequencing of Gene Fragments Generated from SSH:
[0438] To amplify gene fragments resulting from SSH reactions, two PCR amplifications were performed. In the primary PCR reaction 1 &mgr;l of the diluted final hybridization mix was added to 1 &mgr;l of PCR primer 1 (10 &mgr;M), 0.5 &mgr;l dNTP mix (10 &mgr;M), 2.5 &mgr;l 10× reaction buffer (CLONTECH) and 0.5 &mgr;l 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25 &mgr;l. PCR 1 was conducted using the following conditions: 75° C. for 5 min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C. for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions were performed for each experiment. The products were pooled and diluted 1:10 with water. For the secondary PCR reaction, 1 &mgr;l from the pooled and diluted primary PCR reaction was added to the same reaction mix as used for PCR 1, except that primers NP1 and NP2 (10 &mgr;M) were used instead of PCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10 sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR products were analyzed using 2% agarose gel electrophoresis.
[0439] The PCR products were inserted into pCR2.1 using the T/A vector cloning kit (Invitrogen). Transformed E. coli were subjected to blue/white and ampicillin selection. White colonies were picked and arrayed into 96 well plates and were grown in liquid culture overnight. To identify inserts, PCR amplification was performed on 1 ml of bacterial culture using the conditions of PCR1 and NP1 and NP2 as primers. PCR products were analyzed using 2% agarose gel electrophoresis.
[0440] Bacterial clones were stored in 20% glycerol in a 96 well format. Plasmid DNA was prepared, sequenced, and subjected to nucleic acid homology searches of the GenBank, dBest, and NCI-CGAP databases.
[0441] RT-PCR Expression Analysis:
[0442] First strand cDNAs can be generated from 1 &mgr;g of mRNA with oligo (dT) 12-18 priming using the Gibco-BRL Superscript Preamplification system. The manufacturer's protocol was used which included an incubation for 50 min at 42° C. with reverse transcriptase followed by RNAse H treatment at 37° C. for 20 min. After completing the reaction, the volume can be increased to 200 &mgr;l with water prior to normalization. First strand cDNAs from 16 different normal human tissues can be obtained from Clontech.
[0443] Normalization of the first strand cDNAs from multiple tissues was performed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO: 714) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 715) to amplify &bgr;-actin. First strand cDNA (5 &mgr;l) were amplified in a total volume of 50 &mgr;l containing 0.4 &mgr;M primers, 0.2 &mgr;M each dNTPs, 1×PCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCl2, 50 mM KCl, pH 8.3) and 1× Klentaq DNA polymerase (Clontech). Five &mgr;l of the PCR reaction can be removed at 18, 20, and 22 cycles and used for agarose gel electrophoresis. PCR was performed using an MJ Research thermal cycler under the following conditions: Initial denaturation can be at 94° C. for 15 sec, followed by a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C. for 5 sec. A final extension at 72° C. was carried out for 2 min. After agarose gel electrophoresis, the band intensities of the 283 b.p. &bgr;-actin bands from multiple tissues were compared by visual inspection. Dilution factors for the first strand cDNAs were calculated to result in equal &bgr;-actin band intensities in all tissues after 22 cycles of PCR. Three rounds of normalization can be required to achieve equal band intensities in all tissues after 22 cycles of PCR.
[0444] To determine expression levels of the 213P1F11 gene, 5 &mgr;l of normalized first strand cDNA were analyzed by PCR using 26, and 30 cycles of amplification. Semi-quantitative expression analysis can be achieved by comparing the PCR products at cycle numbers that give light band intensities. The primers used for RT-PCR were designed using the 213P1F11 SSH sequence and are listed below: 2 213P1F11.1 5′-GGATACCAGGGAACGCTTGGAG-3′ (SEQ ID NO: ) 213P1F11.2 5′-TTTGACCTTTCCTGCTCAAGTAACC-3′ (SEQ ID NO: )
[0445] A typical RT-PCR expression analysis is shown in FIG. 14. First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), LAPC xenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI), bladder cancer pool, breast cancer pool, and cancer metastasis pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 213P1F11, was performed at 26 and 30 cycles of amplification. Results show strong expression of 213P1F11 in bladder cancer pool, breast cancer pool, xenograft pool, and cancer metastasis pool, but not in the vital pools.
Example 2 Full Length Cloning of 213P1F11[0446] The 213P1F11 SSH cDNA sequence was derived from a bladder cancer pool minus normal tissues cDNA subtraction. The SSH cDNA sequence (FIG. 1) was designated 213P1F1.
[0447] The SSH DNA sequence of 166 bp (FIG. 1) did not show homology to any known gene. The full-length cDNA 213P1F11 was cloned from bladder cancer cDNA. Variants of 213P1F11 were identified and these are listed in FIGS. 2 and 3. 213P1F11 v.1 reveals 100% identity to caspase-14 precursor apoptosis-related cysteine protease protein (FIG. 4).
Example 3 Chromosomal Mapping of 213P1F11[0448] Chromosomal localization can implicate genes in disease pathogenesis. Several chromosome mapping approaches are available including fluorescent in situ hybridization (FISH), human/hamster radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics 7:22; Research Genetics, Huntsville Ala.), human-rodent somatic cell hybrid panels such as is available from the Coriell Institute (Camden, N.J.), and genomic viewers utilizing BLAST homologies to sequenced and mapped genomic clones (NCBI, Bethesda, Md.).
[0449] 213P1F11 maps to chromosome 19p13.1 using 213P1F11 sequence and the NCBI BLAST tool: (http:Hwww.ncbi.nlm.nih.gov/genome/seq/page.cgi?F-HsBlast.html&&ORG=Hs).
Example 4 Expression Analysis of 213P1F11 in Normal Tissues and Patient Specimens[0450] Expression analysis by RT-PCR demonstrated that 213P1F11 is strongly expressed in bladder cancer patient specimens (FIG. 14). First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), LAPC xenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI), bladder cancer pool, breast cancer pool, and cancer metastasis pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 213P1F11, was performed at 26 and 30 cycles of amplification. Results show strong expression of 213P1F11 in bladder cancer pool, breast cancer pool, xenograft pool, and cancer metastasis pool, but not in the vital pools.
[0451] To determine the relative expression of 213P1F11 v.1 compared to 213P1F11 v.2 in human cancers, primers were designed flanking the insertion in 213P1F11 v.2 (FIG. 15). Using these primers, amplification of 213P1F11 v.1 will generate a PCR fragment of 165 bp, whereas 213P1F11 v.2 will generate a PCR fragment of 249 bp as depicted in FIG. 15. The PCR product of 165 bp will also correspond to the variants 213P1F11 v.3, v.4, v.5, v.6 and v.7. First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), bladder cancer pool, breast cancer pool, LAPC xenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI), and 213P1F11 v.1 plasmid control. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers depicted above, was performed at 35 cycles of amplification. Results show strong expression of 213P1F11 v.1 in bladder cancer pool, breast cancer pool, LAPC xenograft pool, and the plasmid positive control. A lower expression of the 249 bp 213P1F11 v.2 product was detected in breast cancer pool, LAPC xenograft pool, and to lower extent in bladder cancer pool. Altogether these data show that expression of 213P1F11 v.1 is more abundant than 213P1F11 v.2 in human patient cancer samples.
[0452] Extensive northern blot analysis of 213P1F11 in multiple human normal tissues is shown in FIG. 16. Strong expression was only detected in skin tissue. A weak transcript is detected in normal thymus but not in the other tissues tested.
[0453] RNA was extracted from normal prostate, LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI prostate cancer xenografts. Northern blot with 10 &mgr;g of total RNA/lane was probed with 213P1F11 SSH sequence (FIG. 18). Results show expression of 213P1F11 in the LAPC-9AI xenograft, but not in the other xenografts nor in normal prostate.
[0454] Expression of 213P1F11 in patient bladder cancer specimens is shown in FIG. 17. RNA was extracted from normal bladder (N), bladder cancer cell lines (UM-UC-3 and SCaBER), bladder cancer patient tumors (T) and normal tissue adjacent to bladder cancer (NAT). Northern blots with 10 ug of total RNA were probed with the 213P1F11 SSH fragment. Size standards in kilobases are indicated on the side. Results show strong expression of 213P1F11 in the bladder tumor tissues but not in normal bladder, nor in the bladder cancer cell lines.
[0455] FIG. 19 shows that 213P1F11 was expressed in breast cancer patient tissues. RNA was extracted from normal breast (N), breast cancer cell lines (DU4475, MCF7 and CAMA-1), breast cancer patient tumors (T) and breast cancer metastasis to lymph node (Met). Northern blots with 10 ug of total RNA were probed with the 213P1F11 SSH fragment. Results show strong expression of 213P1F11 in the breast tumor tissues as well as in the cancer metastasis specimen. Weak expression was also detected in the CAMA-1 cell line, but not in the other 2 breast cancer cell lines tested.
[0456] The restricted expression of 213P1F11 in normal tissues and the expression detected in bladder cancer, breast cancer, prostate cancer xenograft, and cancer metastases suggest that 213P1F11 is a potential therapeutic target and a diagnostic marker for human cancers.
Example 5 Transcript Variants of 213P1F11[0457] Transcript variants are variants of matured mRNA from the same gene by alternative transcription or alternative splicing. Alternative transcripts are transcripts from the same gene but start transcription at different points. Splice variants are mRNA variants spliced differently from the same transcript. In eukaryotes, when a multi-exon gene is transcribed from genomic DNA, the initial RNA is spliced to produce functional mRNA, which has only exons and is used for translation into an amino acid sequence. Accordingly, a given gene can have zero to many alternative transcripts and each transcript can have zero to many splice variants. Each transcript variant has a unique exon makeup, and can have different coding and/or non-coding (5′ or 3′ end) portions, from the original transcript. Transcript variants can code for similar or different proteins with the same or a similar function or may encode proteins with different functions, and may be expressed in the same tissue at the same time, or at different tissue, or at different times, proteins encoded by transcript variants can have similar or different cellular or extracellular localizations, i.e., be secreted.
[0458] Transcript variants are identified by a variety of art-accepted methods. For example, alternative transcripts and splice variants are identified in a full-length cloning experiment, or by use of full-length transcript and EST sequences. First, all human ESTs were grouped into clusters which show direct or indirect identity with each other. Second, ESTs in the same cluster were further grouped into sub-clusters and assembled into a consensus sequence. The original gene sequence is compared to the consensus sequence(s) or other full-length sequences. Each consensus sequence is a potential splice variant for that gene (see, e.g., http://www.doubletwist.com/products/c11_agentsOverview.jhtml). Even when a variant is identified that is not a full-length clone, that portion of the variant is very useful for antigen generation and for further cloning of the full-length splice variant, using techniques known in the art.
[0459] Moreover, computer programs are available in the art that identify transcript variants based on genomic sequences. Genomic-based transcript variant identification programs include FgenesH (A. Salamov and V. Solovyev, “Ab initio gene finding in Drosophila genomic DNA,” Genome Research. April 2000;10(4):516-22); Grail (http://compbio.oml.gov/Grail-bin/EmptyGrailForm) and GenScan (http://genes.mit.edu/GENSCAN.html). For a general discussion of splice variant identification protocols see., e.g., Southan, C., A genomic perspective on human proteases, FEBS Lett. Jun. 8, 2001; 498(2-3):214-8; de Souza, S. J., et al., Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags, Proc. Natl. Acad Sci USA. Nov. 7, 2000; 97(23):12690-3.
[0460] To further confirm the parameters of a transcript variant, a variety of techniques are available in the art, such as full-length cloning, proteomic validation, PCR-based validation, and 5′ RACE validation, etc. (see e.g., Proteomic Validation: Brennan, S. O., et at, Albumin banks peninsula: a new termination variant characterized by electrospray mass spectrometry, Biochem Biophys Acta. Aug. 17, 1999;1433(1-2):321-6; Ferranti P, et al, Differential splicing of pre-messenger RNA produces multiple forms of mature caprine alpha(sl)-casein, Eur J. Biochem. Oct. 1, 1997;249(1):1-7. For PCR-based Validation: Wellmann S, et al., Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology, Clin Chem. April 2001;47(4):654-60; Jia, H. P., et al., Discovery of new human beta-defensins using a genomics-based approach, Gene. Jan. 24, 2001; 263(1-2):211-8. For PCR-based and 5′ RACE Validation: Brigle, K. E., et at, Organization of the murine reduced folate carrier gene and identification of variant splice forms, Biochem Biophys Acta. Aug. 7, 1997; 1353(2): 191-8).
[0461] It is known in the art that genomic regions are modulated in cancers. When the genomic region, to which a gene maps, is modulated in a particular cancer, the alternative transcripts or splice variants of the gene are modulated as well. Disclosed herein is that 213P1 E 1 has a particular expression profile related to cancer. Alternative transcripts and splice variants of 213P F11 may also be involved in cancers in the same or different tissues, thus serving as tumor-associated markers/antigens.
[0462] The exon composition of the original transcript, designated as 213P1F11 v.1, is shown in Table XXIIIA. Using the full-length gene and EST sequences, two splice variants were identified, designated as 213P1F11 v.2 and 213P1F11 v.3. Compared with 213P1F11 v.1, splice variant 213P1F11 v.2 had a longer exon 6 while 213P1F11 had a longer exon 5. Using the computer program GenScan, one alternative transcript was identified, designated as 213P1F11 v.4. This alternative transcript had three different leading exons in place of the first two exons of 213P1F11 v.1. The exon composition of the alternative transcript 213P1F11 v.4 is shown in Table XXIIIB. Since 213P1F11 v.4 shares the same exons 5 and 6 as 213P1F11 v.1, splice variants of this alternative transcript with a longer exon 5 or a longer exon 6, or both, may exist in human tissues. In fact, each different combination of exons in spatial order, e.g., exons 1, 2, 3, 4 and 7, is a potential splice variant. FIG. 13 shows the schematic alignment of exons of the two transcripts (in addition to variants 2 and 3).
[0463] Tables XXIV through XXVII are set forth herein on a variant-by-variant basis. Table XXIV shows the nucleotide sequences of transcript variant 2 through variant 4. Table XXV shows the alignment of transcript variant 2 through variant 4, each with the nucleic acid sequence of 213P1F11 variant 1. Table XXVI lays out amino acid translation of transcript variant 2 through variant 4 for the identified reading frame orientation. Table XXVII displays alignments of the amino acid sequences encoded by splice variant 2 through variant 4, each with that of 213P1F11 variant 1. Table XXVIII displays clustal alignments of 213P1F11 protein variant 1 through variant 6.
Example 6 Single Nucleotide Polymorphisms of 213P1F11[0464] Single Nucleotide Polymorphism (SNP) is a single base pair variation in nucleotide sequences. At a specific point of the genome, there are four possible nucleotide base pairs: A/T, C/G, G/C and T/A. Genotype refers to the base pair make-up of one or more spots in the genome of an individual, while haplotype refers to base pair make-up of more than one varied spots on the same DNA molecule (chromosome in higher organism). SNPs that occur on a cDNA are called cSNPs. These cSNPs may change amino acids of the protein encoded by the gene and thus change the functions of the protein. Some SNPs cause inherited diseases and some others contribute to quantitative variations in phenotype and reactions to environmental factors including diet and drugs among individuals. Therefore, SNPs and/or combinations of alleles (called haplotypes) have many applications including diagnosis of inherited diseases, determination of drug reactions and dosage, identification of genes responsible for disearses and discovery of genetic relationship between individuals (P. Nowotny, J. M. Kwon and A. M. Goate, “SNP analysis to dissect human traits,” Curr. Opin. Neurobiol. October 2001; 11(5):637-641; M. Pirmohamed and B. K. Park, “Genetic susceptibility to adverse drug reactions,” Trends Pharmacol. Sci. June 2001; 22(6):298-305; J. H. Riley, C. J. Allan, E. Lai and A. Roses, “The use of single nucleotide polymorphisms in the isolation of common disease genes,” Pharmacogenomics. February 2000; 1(1):39-47; R. Judson, J. C. Stephens and A. Windemuth, “The predictive power of haplotypes in clinical response,” Pharmacogenomics. February 2000; 1(1):15-26).
[0465] SNPs are identified by a variety of art-accepted methods (P. Bean, “The promising voyage of SNP target discovery,” Am. Clin. Lab. October-November 2001; 20(9):18-20; K. M. Weiss, “In search of human variation,” Genome Res. July 1998; 8(7):691-697; M. M. She, “Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies,” Clin. Chem. February 2001; 47(2):164-172). For example, SNPs are identified by sequencing DNA fragments that show polymorphism by gel-based methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). They can also be discovered by direct sequencing of DNA samples pooled from different individuals or by comparing sequences from different DNA samples. With the rapid accumulation of sequence data in public and private databases, one can discover SNPs by comparing sequences using computer programs (Z. Gu, L. Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting in cyberspace,” Hum. Mutat. 1998; 12(4):221-225). SNPs can be verified and genotype or haplotype of an individual can be determined by a variety of methods including direct sequencing and high throughput microarrays (P. Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu. Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K. Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft, “High-throughput SNP genotyping with the Masscode system,” Mol. Diagn. December 2000; 5(4):329-340).
[0466] Using the methods described above, six SNPs were identified in the original transcript, 213P1F11 v.1, at positions 473 (T/C), 737 (C/A), 2027 (C/T), 2037 (T/C), 2268 (A/G) and 3196 (A/T). The transcripts or proteins with alternative alleles were designated as variants 213P1F11 v.5, v.6, v.7, v.8, v.9, and v.10. FIG. 10 shows the schematic alignment of the nucleotide variants. FIG. 11 shows the schematic alignment of protein variants, corresponding to nucleotide variants. Nucleotide variants that code for the same amino acid sequence as variant 1 are not shown in FIG. 11. These alleles of the SNPs, though shown separately here, can occur in different combinations (haplotypes) and in any one of the transcript variants that contains the sequence context of the SNPs, e.g., 213P1F11 v.2, 213P1F11 v.3 or 213P1f11 v.4.
Example 7 Production of Recombinant 213P1F11 in Prokaryotic Systems[0467] To express recombinant 213P1F11 and 213P1F11 variants in prokaryotic cells, the full or partial length 213P1F11 and 213P1F11 variant cDNA sequences are cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 213P1F11 or 213P1F11 variants are expressed in these constructs, amino acids 1 to 242 of 213P1F11 variant 1, amino acids 1-230 of variant 2, amino acids 1-146 of variant 3, amino acids 1-321 of variant 4; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 213P1F11, variants, or analogs thereof.
[0468] A. In vitro Transcription and Translation Constructs:
[0469] pCRII: To generate 213P1F11 sense and anti-sense RNA probes for RNA in situ investigations, pCRII constructs (Invitrogen, Carlsbad Calif.) are generated encoding either all or fragments of the 213P1F11 cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 213P1F11 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of 213P1F11 at the RNA level. Transcribed 213P1F11 RNA representing the cDNA amino acid coding region of the 213P1F11 gene is used in in vitro translation systems such as the TnT™ Coupled Reticulolysate Sytem (Promega, Corp., Madison, Wis.) to synthesize 213P1F11 protein.
[0470] B. Bacterial Constructs:
[0471] pGEX Constructs: To generate recombinant 213P1F11 proteins in bacteria that are fused to the Glutathione S-transferase (GST) protein, all or parts of the T-fusion vector of the pGEX family (Amersham Pharmacia Biotech, Piscataway, N.J.). These constructs allow controlled expression of recombinant 213P1F11 protein sequences with GST fused at the amino-terminus and a six histidine epitope (6×His) at the carboxyl-terminus. The GST and 6×His tags permit purification of the recombinant fusion protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-GST and anti-His antibodies. The 6×His tag is generated by adding 6 histidine codons to the cloning primer at the 3′ end, e.g., of the open reading frame (ORF). A proteolytic cleavage site, such as the PreScission™ recognition site in pGEX-6P-1, may be employed such that it permits cleavage of the GST tag from 213P1F11-related protein. The ampicillin resistance gene and pBR322 origin permits selection and maintenance of the pGEX plasmids in E. coli.
[0472] pMAL Constructs: To generate, in bacteria, recombinant 213P1F11 proteins that are fused to maltose-binding protein (MBP), all or parts of the 213P1F11 cDNA protein coding sequence are fused to the MBP gene by cloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs, Beverly, Mass.). These constructs allow controlled expression of recombinant 213P1F11 protein sequences with MBP fused at the amino-terminus and a 6×His epitope tag at the carboxyl-terminus. The MBP and 6×His tags permit purification of the recombinant protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-MBP and anti-His antibodies. The 6×His epitope tag is generated by adding 6 histidine codons to the 3′ cloning primer. A Factor Xa recognition site permits cleavage of the pMAL tag from 213P1F11. The pMAL-c2X and pMAL-p2X vectors are optimized to express the recombinant protein in the cytoplasm or periplasm respectively. Periplasm expression enhances folding of proteins with disulfide bonds.
[0473] pET Constructs: To express 213P1F11 in bacterial cells, all or parts of the 213P1F11 cDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, Wis.). These vectors allow tightly controlled expression of recombinant 213P1F11 protein in bacteria with and without fusion to proteins that enhance solubility, such as NusA and thioredoxin (Trx), and epitope tags, such as 6×His and S-Tag™ that aid purification and detection of the recombinant protein. For example, constructs are made utilizing pET NusA fusion system 43.1 such that regions of the 213P1F11 protein are expressed as amino-terminal fusions to NusA.
[0474] C. Yeast Constructs:
[0475] pESC Constructs: To express 213P1F11 in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of the 213P1F11 cDNA protein coding sequence are cloned into the pESC family of vectors each of which contain 1 of 4 selectable markers, HIS3, TRP 1, LEU2, and URA3 (Stratagene, La Jolla, Calif.). These vectors allow controlled expression from the same plasmid of up to 2 different genes or cloned sequences containing either Flag™ or Myc epitope tags in the same yeast cell. This system is useful to confirm protein-protein interactions of 213P1F11. In addition, expression in yeast yields similar post-translational modifications, such as glycosylations and phosphorylations, that are found when expressed in eukaryotic cells.
[0476] pESP Constructs: To express 213P1F11 in the yeast species Saccharomyces pombe, all or parts of the 213P1F11 cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 213P1F11 protein sequence that is fused at either the amino terminus or at the carboxyl terminus to GST which aids purification of the recombinant protein. A Flag™ epitope tag allows detection of the recombinant protein with anti-Flag™ antibody.
Example 8 Production of Recombinant 213P1F11 in Eukaryotic Systems A. Mammalian Constructs:[0477] To express recombinant 213P1F11 in eukaryotic cells, the full or partial length 213P1F11 cDNA sequences can be cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 213P1F11 are expressed in these constructs, amino acids 1 to 242, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 213P1F11, variants, or analogs thereof. In certain embodiments a region of a specific variant of 213P1F11 is expressed that encodes an amino acid at a specific position which differs from the amino acid of any other variant found at that position. In other embodiments, a region of a variant of 213P1F11 is expressed that lies partly or entirely within a sequence that is unique to that variant.
[0478] The constructs can be transfected into any one of a wide variety of mammalian cells such as 293T cells. Transfected 293T cell lysates can be probed with the anti-213P1F11 polyclonal serum, described herein.
[0479] pcDNA4/HisMax Constructs: To express 213P1F11 in mammalian cells, a 213P1F11 ORF, or portions thereof, of 213P1F11 are cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter and the SP 16 translational enhancer. The recombinant protein has Xpress™ and six histidine (6×His) epitopes fused to the amino-terminus. The pcDNA4/HisMax vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli.
[0480] pcDNA3.1/MycHis Constructs: To express 213P1F11 in mammalian cells, a 213P1F11 ORF, or portions thereof, of 213P1F11 with a consensus Kozak translation initiation site are cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and 6×His epitope fused to the carboxyl-terminus. The pcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability, along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene can be used, as it allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli.
[0481] pcDNA3.1/CT-GFP-TOPO Construct: To express 213P1F11 in mammalian cells and to allow detection of the recombinant proteins using fluorescence, a 213P1F11 ORF, or portions thereof, with a consensus Kozak translation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the Green Fluorescent Protein (GFP) fused to the carboxyl-terminus facilitating non-invasive, in vivo detection and cell biology studies. The pcDNA3.1 CT-GFP-TOPO vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. Additional constructs with an amino-terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning the entire length of a 213P1F11 protein.
[0482] PAPtag: A 213P1F11 ORF, or portions thereof, is cloned into pAPtag-5 (GenHunter Corp. Nashville, Tenn.). This construct generates an alkaline phosphatase fusion at the carboxyl-terminus of a 213P1F11 protein while fusing the IgG&kgr; signal sequence to the amino-terminus. Constructs are also generated in which alkaline phosphatase with an amino-terminal IgG&kgr; signal sequence is fused to the amino-terminus of a 213P1F11 protein. The resulting recombinant 213P1F11 proteins are optimized for secretion into the media of transfected mammalian cells and can be used to identify proteins such as ligands or receptors that interact with 213P1F11 proteins. Protein expression is driven from the CMV promoter and the recombinant proteins also contain myc and 6×His epitopes fused at the carboxyl-terminus that facilitates detection and purification. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the recombinant protein and the ampicillin resistance gene permits selection of the plasmid in E. coli.
[0483] ptag5: A 213P1F11 ORF, or portions thereof, is cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generates 213P1F11 protein with an amino-terminal IgGK signal sequence and myc and 6×His epitope tags at the carboxyl-terminus that facilitate detection and affinity purification. The resulting recombinant 213P1F11 protein is optimized for secretion into the media of transfected mammalian cells, and is used as immunogen or ligand to identify proteins such as ligands or receptors that interact with the 213P1F11 proteins. Protein expression is driven from the CMV promoter. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.
[0484] PsecFc: A 213P1F11 ORF, or portions thereof, is also cloned into psecFc. The psecFc vector was assembled by cloning the human immunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, Calif.). This construct generates an IgG1 Fc fusion at the carboxyl-terminus of the 213P1F11 proteins, while fusing the IgGK signal sequence to N-terminus. 213P1F11 fusions utilizing the murine IgG1 Fc region are also used. The resulting recombinant 213P1F11 proteins are optimized for secretion into the media of transfected mammalian cells, and can be used as immunogens or to identify proteins such as ligands or receptors that interact with 213P1F11 protein. Protein expression is driven from the CMV promoter. The hygromycin resistance gene present in the vector allows for selection of mammalian cells that express the recombinant protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.
[0485] pSR&agr;Constructs: To generate mammalian cell lines that express 213P1F11 constitutively, 213P1F11 ORF, or portions thereof, of 213P1F11 are cloned into pSR&agr; constructs. Amphotropic and ecotropic retroviruses are generated by transfection of pSR&agr; constructs into the 293T-10A1 packaging line or co-transfection of pSR&agr; and a helper plasmid (containing deleted packaging sequences) into the 293 cells, respectively. The retrovirus is used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, 213P1F11, into the host cell-lines. Protein expression is driven from a long terminal repeat (LTR). The Neomycin resistance gene present in the vector allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE 1 origin permit selection and maintenance of the plasmid in E. coli. The retroviral vectors can thereafter be used for infection and generation of various cell lines using, for example, PC3, NIH 3T3, TsuPr1, 293 or rat-i cells.
[0486] Additional pSRc constructs are made that fuse an epitope tag such as the FLAG™ tag to the carboxyl-terminus of 213P1F11 sequences to allow detection using anti-Flag antibodies. For example, the FLAG™ sequence 5′ gat tac aag gat gac gac gat aag 3′ (SEQ ID NO:______) is added to cloning primer at the 3′ end of the ORF. Additional pSRox constructs are made to produce both amino-terminal and carboxyl-terminal GFP and myc/6×His fusion proteins of the full-length 213P1F11 proteins.
[0487] Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of 213P1F11. High virus titer leading to high level expression of 213P1F11 is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. A 213P1F11 coding sequences or fragments thereof are amplified by PCR and subcloned into the AdEasy shuttle vector (Stratagene). Recombination and virus packaging are performed according to the manufacturer's instructions to generate adenoviral vectors. Alternatively, 213P1F11 coding sequences or fragments thereof are cloned into the HSV-1 vector (Imgenex) to generate herpes viral vectors. The viral vectors are thereafter used for infection of various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
[0488] Regulated Expression Systems: To control expression of 213P1F11 in mammalian cells, coding sequences of 213P1F11, or portions thereof, are cloned into regulated mammalian expression systems such as the T-Rex System (Invitrogen), the GeneSwitch System (Invitrogen) and the tightly-regulated Ecdysone System (Sratagene). These systems allow the study of the temporal and concentration dependent effects of recombinant 213P1F11. These vectors are thereafter used to control expression of 213P1F11 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
[0489] B. Baculovirus Expression Systems
[0490] To generate recombinant 213P1F11 proteins in a baculovirus expression system, 213P1F11 ORF, or portions thereof, are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminus. Specifically, pBlueBac-213P1F11 is co-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details). Baculovirus is then collected from cell supernatant and purified by plaque assay.
[0491] Recombinant 213P1F11 protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant 213P1F11 protein can be detected using anti-213P1F11 or anti-His-tag antibody. 213P1F11 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 213P1F11.
Example 9 Antigenicity Profiles and Secondary Structure[0492] FIGS. 5A-D, FIGS. 6A-D, FIGS. 7A-D, FIGS. 8A-D, and FIGS. 9A-D depict graphically five amino acid profiles of the 213P1F11 variants 1 through 4 respectively, each assessment available by accessing the ProtScale website (URL www.expasy.ch/cgi-bin/protscale.pl) on the ExPasy molecular biology server.
[0493] These profiles: FIG. 5, Hydrophilicity, (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 6, Hydropathicity, (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132); FIG. 7, Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); FIG. 8, Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255); FIG. 9, Beta-turn (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294); and optionally others available in the art, such as on the ProtScale website, were used to identify antigenic regions of the 213P1F11 protein. Each of the above amino acid profiles of 213P1F11 were generated using the following ProtScale parameters for analysis: 1) A window size of 9; 2) 100% weight of the window edges compared to the window center; and, 3) amino acid profile values normalized to lie between 0 and 1.
[0494] Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and Percentage Accessible Residues (FIG. 7) profiles were used to determine stretches of hydrophilic amino acids (i.e., values greater than 0.5 on the Hydrophilicity and Percentage Accessible Residues profile, and values less than 0.5 on the Hydropathicity profile). Such regions are likely to be exposed to the aqueous environment, be present on the surface of the protein, and thus available for immune recognition, such as by antibodies.
[0495] Average Flexibility (FIG. 8) and Beta-turn (FIG. 9) profiles determine stretches of amino acids (i.e., values greater than 0.5 on the Beta-turn profile and the Average Flexibility profile) that are not constrained in secondary structures such as beta sheets and alpha helices. Such regions are also more likely to be exposed on the protein and thus accessible to immune recognition, such as by antibodies.
[0496] Antigenic sequences of the 213P1F11 protein and of the variant proteins indicated, e.g., by the profiles set forth in FIGS. 5A-D, FIGS. 6A-D, FIGS. 7A-D, FIGS. 8A-D, and/or FIGS. 9A-D are used to prepare immunogens, either peptides or nucleic acids that encode them, to generate therapeutic and diagnostic anti-213P1F11 antibodies. The immunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50 contiguous amino acids, or the corresponding nucleic acids that encode them, from the 213P1F11 protein variants listed in FIGS. 2 and 3. In particular, peptide immunogens of the invention can comprise, a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment up to the full length of the respective variant's sequence that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profiles of FIGS. 5A-D; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment up to the full length of the respective variant's sequence that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIGS. 6A-D; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment up to the full length of the respective variant's sequence that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profiles of FIGS. 7A-D; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment up to the full length of the respective variant's sequence that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profiles on FIGS. 8A-D; and, a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment up to the full length of the respective variant's sequence that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIGS. 9A-D. Peptide immunogens of the invention can also comprise nucleic acids that encode any of the forgoing.
[0497] All immunogens of the invention, peptide or nucleic acid, can be embodied in human unit dose form, or comprised by a composition that includes a pharmaceutical excipient compatible with human physiology.
[0498] The secondary structures of 213P1F11 variants 1 through 4, namely the predicted presence and location of alpha helices, extended strands, and random coils, are predicted from the primary amino acid sequence using the HNN—Hierarchical Neural Network method (Guermeur, 1997, http://pbil.ibcp.fr/cgi-bin/npsa_automat.p1?pagemnpsa_nn.html), accessed from the ExPasy molecular biology server (http://www.expasy.ch/tools/). The analysis indicates that 213P1F11 variant 1 is composed 47.93% alpha helix, 11.57% extended strand, and 40.50% random coil (FIG. 12A), variant 2 is composed of 38.70% alpha helix, 9.57% extended strand, and 51.74% random coil (FIG. 12B), variant 3 is composed of 50.68% alpha helix, 6.85% extended strand, and 42.47% random coil (FIG. 12C), and variant 4 is composed of 39.25% alpha helix, 12.15% extended strand, and 48.60% random coil (FIG. 12D).
[0499] Analysis for the potential presence of transmembrane domains in 213P1F11 variant 1 was carried out using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server (http://www.expasy.ch/tools/) (Table XXII). The programs do not predict the presence of transmembrane domains in any of the 213P1F11 variants, suggesting that each is a soluble protein.
Example 10 Generation of 213P1F11 Polyclonal Antibodies[0500] Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with the full length 213P1F11 protein, computer algorithms are employed in design of immunogens that, based on amino acid sequence analysis contain characteristics of being antigenic and available for recognition by the immune system of the immunized host (see the Example entitled “Antigenicity Profiles and Secondary Structure”). Such regions would be predicted to be hydrophilic, flexible, in beta-turn conformations, and be exposed on the surface of the protein (see, e.g., FIGS. 5A-D, FIGS. 6A-D, FIGS. 7A-D, FIGS. 8A-D, or FIGS. 9A-D for amino acid profiles that indicate such regions of 213P1F11 and variants).
[0501] For example, 213P1F11 recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turn regions of 213P1F11 variant proteins are used as antigens to generate polyclonal antibodies in New Zealand White rabbits. For example, such regions include, but are not limited to, amino acids 1-17, amino acids 25-80, amino acids 88-108, amino acids 131-147, and 207-242 of 213P1F11 variant 1. It is useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. In one embodiment, a peptide encoding amino acids 1-17 of 213P1F11 variant 1 is conjugated to KLH and used to immunize the rabbit. Alternatively the immunizing agent may include all or portions of the 213P1F11 variant proteins, analogs or fusion proteins thereof. For example, the 213P1F11 variant 1 amino acid sequence can be fused using recombinant DNA techniques to any one of a variety of fusion protein partners that are well known in the art, such as glutathione-S-transferase (GST) and HIS tagged fusion proteins. Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix.
[0502] In one embodiment, a GST-fusion protein encoding amino acids 1-147, encompassing several predicted antigenic regions, is produced and purified and used as immunogen. Other recombinant bacterial fusion proteins that may be employed include maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see the section entitled “Production of 213P1F11 in Prokaryotic Systems” and Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P. S., Brady, W., Umes, M., Grosmaire, L., Damle, N., and Ledbetter, L. (1991) J. Exp. Med. 174, 561-566).
[0503] In addition to bacterial derived fusion proteins, mammalian expressed protein antigens are also used. These antigens are expressed from mammalian expression vectors such as the Tag5 and Fc-fusion vectors (see the section entitled “Production of Recombinant 213P1F11 in Eukaryotic Systems”), and retain post-translational modifications such as glycosylations found in native protein. In one embodiment, the full length sequence of variant 1, amino acids 1-242, is cloned into the Tag5 mammalian secretion vector. The recombinant protein is purified by metal chelate chromatography from tissue culture supernatants of 293T cells stably expressing the recombinant vector. The purified Tag5 213P1F11 protein is then used as immunogen.
[0504] During the immunization protocol, it is useful to mix or emulsify the antigen in adjuvants that enhance the immune response of the host animal. Examples of adjuvants include, but are not limited to, complete Freund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
[0505] In a typical protocol, rabbits are initially immunized subcutaneously with up to 200 &mgr;g, typically 100-200 &mgr;g, of fusion protein or peptide conjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits are then injected subcutaneously every two weeks with up to 200 &mgr;g, typically 100-200 &mgr;g, of the immunogen in incomplete Freund's adjuvant (IFA). Test bleeds are taken approximately 7-10 days following each immunization and used to monitor the titer of the antiserum by ELISA.
[0506] To test reactivity and specificity of immune serum, such as the rabbit serum derived from immunization with a KLH-conjugated peptide encoding amino acids 1-17 of variant 1, the full-length 213P11 variant 1 cDNA is cloned into pcDNA 3.1 myc-his expression vector (Invitrogen, see the Example entitled “Production of Recombinant 213P1F11 in Eukaryotic Systems”). After transfection of the constructs into 293T cells, cell lysates are probed with the anti-213P1P11 serum and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.) to determine specific reactivity to denatured 213P1F11 protein using the Western blot technique. The immune serum is then tested by the Western blot technique against 293T-213P1F11 cells. In addition, the immune serum is tested by fluorescence microscopy, flow cytometry and immunoprecipitation against 293T and other recombinant 213P1F11-expressing cells to determine specific recognition of native protein. Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometric techniques using cells that endogenously express 213P1F11 are also carried out to test reactivity and specificity.
[0507] Anti-serum from rabbits immunized with 213P1F11 variant fusion proteins, such as GST and MBP fusion proteins, are purified by depletion of antibodies reactive to the fusion partner sequence by passage over an affinity column containing the fusion partner either alone or in the context of an irrelevant fusion protein. For example, antiserum derived from a GST-213P1F11 fusion protein encoding amino acids 1-147 is first purified by passage over a column of GST protein covalently coupled to AffiGel matrix (BioRad, Hercules, Calif.). The antiserum is then affinity purified by passage over a column composed of a MBP-fusion protein also encoding amino acids 1-147 covalently coupled to Affigel matrix. The serum is then further purified by protein G affinity chromatography to isolate the IgG fraction. Sera from other His-tagged antigens and peptide immunized rabbits as well as fusion partner depleted sera are affinity purified by passage over a column matrix composed of the original protein immunogen or free peptide.
Example 11 Generation of 213P1F11 Monoclonal Antibodies (mAbs)[0508] In one embodiment, therapeutic mAbs to 213P1F11 variants comprise those that react with epitopes specific for each variant protein or specific to sequences in common between the variants that would disrupt or modulate the biological function of the 213P1F11 variants, for example those that would disrupt the interaction with ligands and binding partners. Immunogens for generation of such mAbs include those designed to encode or contain the entire 213P1F11 protein variant sequence, regions of the 213P1F11 protein variants predicted to be antigenic from computer analysis of the amino acid sequence (see, e.g., FIGS. 5A-D, FIGS. 6A-D, FIGS. 7A-D, FIGS. 8A-D, or FIGS. 9A-D, and the Example entitled “Antigenicity Profiles and Secondary Structure”). Immunogens include peptides, recombinant bacterial proteins, and mammalian expressed Tag 5 proteins and human and murine IgG FC fusion proteins. In addition, cells engineered to express high levels of a respective 213P1F11 variant, such as 293T-213P1F11 variant 1 or 300.19-213P1F11 variant 1 murine Pre-B cells, are used to immunize mice.
[0509] To generate mAbs to a 213P1F11 variant, mice are first immunized intraperitoneally (IP) with, typically, 10-50 &mgr;g of protein immunogen or 107 213P1F11-expressing cells mixed in complete Freund's adjuvant. Mice are then subsequently immunized IP every 2-4 weeks with, typically, 10-50 &mgr;g of protein immunogen or 107 cells mixed in incomplete Freund's adjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. In addition to the above protein and cell-based immunization strategies, a DNA-based immunization protocol is employed in which a mammalian expression vector encoding a 213P1F11 variant sequence is used to immunize mice by direct injection of the plasmid DNA. For example, the full length variant I sequence, encoding amino acids 1-242, is cloned into the Tag5 mammalian secretion vector and the recombinant vector is used as immunogen. In another example the same amino acids are cloned into an Fc-fusion secretion vector in which the 213P1F11 variant I sequence is fused at the amino-terminus to an IgK leader sequence and at the carboxyl-terminus to the coding sequence of the human or murine IgG Fc region. This recombinant vector is then used as immunogen. The plasmid immunization protocols are used in combination with purified proteins expressed from the same vector and with cells expressing the respective 213P1F11 variant.
[0510] During the immunization protocol, test bleeds are taken 7-10 days following an injection to monitor titer and specificity of the immune response. Once appropriate reactivity and specificity is obtained as determined by ELISA, Western blotting, immunoprecipitation, fluorescence microscopy, and flow cytometric analyses, fusion and hybridoma generation is then carried out with established procedures well known in the art (see, e.g., Harlow and Lane, 1988).
[0511] In one embodiment for generating 213P1F11 monoclonal antibodies, a Tag5-213P1F11 variant 1 antigen encoding amino acids 1-242, is expressed and purified from stably transfected 293T cells. Balb C mice are initially immunized intraperitoneally with 25 &mgr;g of the Tag5-213P1F11 variant 1 protein mixed in complete Freund's adjuvant. Mice are subsequently immunized every two weeks with 25 &mgr;g of the antigen mixed in incomplete Freund's adjuvant for a total of three immunizations. ELISA using the Tag5 antigen determines the titer of serum from immunized mice. Reactivity and specificity of serum to full length 213P1F11 variant protein is monitored by Western blotting, immunoprecipitation and flow cytometry using 293T cells transfected with an expression vector encoding the 213P1F11 variant 1 cDNA (see e.g., the Example entitled “Production of Recombinant 213P1F11 in Eukaryotic Systems”). Other recombinant 213P1F11 variant 1-expressing cells or cells endogenously expressing 213P1F11 variant 1 are also used. Mice showing the strongest reactivity are rested and given a final injection of Tag5 antigen in PBS and then sacrificed four days later. The spleens of the sacrificed mice are harvested and fused to SPO/2 myeloma cells using standard procedures (Harlow and Lane, 1988). Supernatants from HAT selected growth wells are screened by ELISA, Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometry to identify 213P1F11 specific antibody-producing clones.
[0512] Monoclonal antibodies are also derived that react only with specific 213P1F11 variants. To this end, immunogens are designed to encode amino acid regions specific to the respective variant. For example, a Tag5 immunogen encoding amino acids 175-230 of variant 2 is produced, purified, and used to immunize mice to generate hybridomas. In another example, a KLH-coupled peptide encoding amino acids 135-146 of variant 3 is produced and used as immunogen. In another example amino acids 1-86 of variant 4 is fused to GST and used as immunogen. Monoclonal antibodies raised to these immunogens are then screened for reactivity to cells expressing the respective variants but not to other 213P1F11 variants. These strategies for raising 213P1F11 variant specific monoclonal antibodies are also applied to polyclonal reagents described in the Example entitled “Generation of 213P1F11 Polyclonal Antibodies.”
[0513] The binding affinity of a 213P1F11 monoclonal antibody is determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 213P1F11 monoclonal antibodies preferred for diagnostic or therapeutic use, as appreciated by one of skill in the art. The BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity. The BIAcore system uses surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23: 1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants.
Example 12 HLA Class I and Class II Binding Assays[0514] HLA class I and class II binding assays using purified HLA molecules are performed in accordance with disclosed protocols (e.g., PCT publications WO 94/20127 and WO 94/03205; Sidney et al, Current Protocols in Immunology 18.3.1 (1998); Sidney, et al, J. Immunol. 154:247 (1995); Sette, et al, Mol Immunol 31:813 (1994)). Briefly, purified MHC molecules (5 to 500 nM) are incubated with various unlabeled peptide inhibitors and 1-10 nM 125I-radiolabeled probe peptides as described. Following incubation, MHC-peptide complexes are separated from free peptide by gel filtration and the fraction of peptide bound is determined. Typically, in preliminary experiments, each MHC preparation is titered in the presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind 10-20% of the total radioactivity. All subsequent inhibition and direct binding assays are performed using these HLA concentrations.
[0515] Since under these conditions [label]<[HLA] and IC50≧[HLA], the measured IC50 values are reasonable approximations of the true KD values. Peptide inhibitors are typically tested at concentrations ranging from 120 &mgr;g/ml to 1.2 ng/ml, and are tested in two to four completely independent experiments. To allow comparison of the data obtained in different experiments, a relative binding figure is calculated for each peptide by dividing the IC50 of a positive control for inhibition by the IC50 for each tested peptide (typically unlabeled versions of the radiolabeled probe peptide). For database purposes, and inter-experiment comparisons, relative binding values are compiled. These values can subsequently be converted back into IC50 nM values by dividing the IC50 nM of the positive controls for inhibition by the relative binding of the peptide of interest. This method of data compilation is accurate and consistent for comparing peptides that have been tested on different days, or with different lots of purified MHC.
[0516] Binding assays as outlined above may be used to analyze HLA supermotif and/or HLA motif-bearing peptides.
Example 13 Identification of HLA Supermotif- and Motif-Bearing CTL Candidate Epitopes[0517] HLA vaccine compositions of the invention can include multiple epitopes. The multiple epitopes can comprise multiple HLA supermotifs or motifs to achieve broad population coverage. This example illustrates the identification and confirmation of supermotif- and motif-bearing epitopes for the inclusion in such a vaccine composition. Calculation of population coverage is performed using the strategy described below.
[0518] Computer Searches and Algorithms for Identification of Supermotif and/or Motif-Bearing Epitopes
[0519] The searches performed to identify the motif-bearing peptide sequences in the Example entitled “Antigenicity Profiles and Secondary Structure” and Tables V-XIX employ the protein sequence data from the gene product of 213P1F11 set forth in FIGS. 2 and 3. ***THIS IS A PLACE WHERE WE CAN REFERENCE NEW SEARCH FRAGMENT TABLE***,
[0520] Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs are performed as follows. All translated 213P1F11 protein sequences are analyzed using a text string search software program to identify potential peptide sequences containing appropriate HLA binding motifs; such programs are readily produced in accordance with information in the art in view of known motif/supermotif disclosures. Furthermore, such calculations can be made mentally.
[0521] Identified A2-, A3-, and DR-supermotif sequences are scored using polynomial algorithms to predict their capacity to bind to specific HLA-Class I or Class II molecules. These polynomial algorithms account for the impact of different amino acids at different positions, and are essentially based on the premise that the overall affinity (or &Dgr;G) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type:
[0522] “&Dgr;G”=a1t×a2t×a3t . . . ×ant
[0523] where ajt is a coefficient which represents the effect of the presence of a given amino acid (U) at a given position (i) along the sequence of a peptide of n amino acids. The crucial assumption of this method is that the effects at each position are essentially independent of each other (i.e., independent binding of individual side-chains). When residue j occurs at position i in the peptide, it is assumed to contribute a constant amount j, to the free energy of binding of the peptide irrespective of the sequence of the rest of the peptide.
[0524] The method of derivation of specific algorithm coefficients has been described in Gulukota et al., J. Mol. Biol. 267:1258-126, 1997; (see also Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al, J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions, anchor and non-anchor alike, the geometric mean of the average relative binding (ARB) of all peptides carrying j is calculated relative to the remainder of the group, and used as the estimate of jt. For Class II peptides, if multiple alignments are possible, only the highest scoring alignment is utilized, following an iterative procedure. To calculate an algorithm score of a given peptide in a test set, the ARB values corresponding to the sequence of the peptide are multiplied. If this product exceeds a chosen threshold, the peptide is predicted to bind. Appropriate thresholds are chosen as a function of the degree of stringency of prediction desired.
[0525] Selection of HLA-A2 Supertype Cross-Reactive Peptides
[0526] Protein sequences from 213P1F11 are scanned utilizing motif identification software, to identify 8-, 9-10- and 11-mer sequences containing the HLA-A2-supermotif main anchor specificity. Typically, these sequences are then scored using the protocol described above and the peptides corresponding to the positive-scoring sequences are synthesized and tested for their capacity to bind purified HLA-A*0201 molecules in vitro (HLA-A*0201 is considered a prototype A2 supertype molecule).
[0527] These peptides are then tested for the capacity to bind to additional A2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). Peptides that bind to at least three of the five A2-supertype alleles tested are typically deemed A2-supertype cross-reactive binders. Preferred peptides bind at an affinity equal to or less than 500 nM to three or more HLA-A2 supertype molecules.
[0528] Selection of HLA-A3 Supermotif-Bearing Epitopes
[0529] The 213P1F11 protein sequence(s) scanned above is also examined for the presence of peptides with the HLA-A3-supermotif primary anchors. Peptides corresponding to the HLA A3 supermotif-bearing sequences are then synthesized and tested for binding to HLA-A*0301 and HLA-A* 1101 molecules, the molecules encoded by the two most prevalent A3-supertype alleles. The peptides that bind at least one of the two alleles with binding affinities of ≦500 nM, often ≦200 nM, are then tested for binding cross-reactivity to the other common A3-supertype alleles (e.g., A*3101, A*3301, and A*6801) to identify those that can bind at least three of the five HLA-A3-supertype molecules tested.
[0530] Selection of HLA-B7 Supermotif Bearing Epitopes
[0531] The 213P1F11 protein(s) scanned above is also analyzed for the presence of 8-, 9-10-, or 11-mer peptides with the HLA-B7-supermotif. Corresponding peptides are synthesized and tested for binding to HLA-B*0702, the molecule encoded by the most common B7-supertype allele (i.e., the prototype B7 supertype allele). Peptides binding B*0702 with IC50 of ≦500 nM are identified using standard methods. These peptides are then tested for binding to other common B7-supertype molecules (e.g., B*3501, B*5101, B*5301, and B*5401). Peptides capable of binding to three or more of the five B7-supertype alleles tested are thereby identified.
[0532] Selection of A1 and A24 Motif-Bearing Epitopes
[0533] To further increase population coverage, HLA-A1 and -A24 epitopes can also be incorporated into vaccine compositions. An analysis of the 213P1F11 protein can also be performed to identify HLA-A1- and A24-motif-containing sequences.
[0534] High affinity and/or cross-reactive binding epitopes that bear other motif and/or supermotifs are identified using analogous methodology.
Example 14 Confirmation of Immunogenicity[0535] Cross-reactive candidate CTL A2-supermotif-bearing peptides that are identified as described herein are selected to confirm in vitro immunogenicity. Confirmation is performed using the following methodology:
[0536] Target Cell Lines for Cellular Screening:
[0537] The 0.221A2.1 cell line, produced by transferring the HLA-A2.1 gene into the HLA-A, -B, -C null mutant human B-lymphoblastoid cell line 721.221, is used as the peptide-loaded target to measure activity of HLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 medium supplemented with antibiotics, sodium pyruvate, nonessential amino acids and 10% (v/v) heat inactivated FCS. Cells that express an antigen of interest, or transfectants comprising the gene encoding the antigen of interest, can be used as target cells to confirm the ability of peptide-specific CTLs to recognize endogenous antigen.
[0538] Primary CTL Induction Cultures:
[0539] Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with 30 &mgr;g/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640 plus 5% AB human serum, non-essential amino acids, sodium pyruvate, L-glutamine and penicillin/streptomycin). The monocytes are purified by plating 10×106 PBMC/well in a 6-well plate. After 2 hours at 37° C., the non-adherent cells are removed by gently shaking the plates and aspirating the supernatants. The wells are washed a total of three times with 3 ml RPMI to remove most of the non-adherent and loosely adherent cells. Three ml of complete medium containing 50 ng/ml of GM-CSF and 1,000 U/ml of IL-4 are then added to each well. TNF&agr; is added to the DCs on day 6 at 75 ng/1 ml and the cells are used for CTL induction cultures on day 7.
[0540] Induction of CTL with DC and Peptide: CD8+ T-cells are isolated by positive selection with Dynal immunomagnetic beads (Dynabeads® M-450) and the detacha-bead® reagent. Typically about 200-250×106 PBMC are processed to obtain 24×106 CD8+ T-cells (enough for a 48-well plate culture). Briefly, the PBMCs are thawed in RPMI with 30 &mgr;g/ml DNAse, washed once with PBS containing 1% human AB serum and resuspended in PBS/1% AB serum at a concentration of 20×106cells/ml. The magnetic beads are washed 3 times with PBS/AB serum, added to the cells (140 &mgr;l beads/20×106 cells) and incubated for 1 hour at 4° C. with continuous mixing. The beads and cells are washed 4×with PBS/AB serum to remove the nonadherent cells and resuspended at 100×106 cells/mil (based on the original cell number) in PBS/AB serum containing 100 &mgr;l/ml detacha-bead® reagent and 30 &mgr;g/ml DNAse. The mixture is incubated for 1 hour at room temperature with continuous mixing. The beads are washed again with PBS/AB/DNAse to collect the CD8+T-cells. The DC are collected and centrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1% BSA, counted and pulsed with 40 &mgr;g/ml of peptide at a cell concentration of 1-2×106/ml in the presence of 3 &mgr;g/ml &bgr;2-microglobulin for 4 hours at 20° C. The DC are then irradiated (4,200 rads), washed 1 time with medium and counted again.
[0541] Setting up induction cultures: 0.25 ml cytokine-generated DC (at 1×105 cells/ml) are co-cultured with 0.25 ml of CD8+ T-cells (at 2×106 cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml of IL-7. Recombinant human IL-10 is added the next day at a final concentration of 10 ng/ml and rhuman IL-2 is added 48 hours later at 10 IU/ml.
[0542] Restimulation of the induction cultures with peptide-pulsed adherent cells: Seven and fourteen days after the primary induction, the cells are restimulated with peptide-pulsed adherent cells. The PBMCs are thawed and washed twice with RPMI and DNAse. The cells are resuspended at 5×106 cells/ml and irradiated at ˜4200 rads. The PBMCs are plated at 2×106 in 0.5 ml complete medium per well and incubated for 2 hours at 37° C. The plates are washed twice with RPMI by tapping the plate gently to remove the nonadherent cells and the adherent cells pulsed with 10 &mgr;g/ml of peptide in the presence of 3 &mgr;g/ml B2 microglobulin in 0.25 ml RPMI/5%AB per well for 2 hours at 37° C. Peptide solution from each well is aspirated and the wells are washed once with RPMI. Most of the media is aspirated from the induction cultures (CD8+ cells) and brought to 0.5 ml with fresh media. The cells are then transferred to the wells containing the peptide-pulsed adherent cells. Twenty four hours later recombinant human IL-10 is added at a final concentration of 10 ng/ml and recombinant human IL2 is added the next day and again 2-3 days later at 501U/ml (Tsai et al., Critical Reviews in Immunology 18(1-2):65-75, 1998). Seven days later, the cultures are assayed for CTL activity in a 51Cr release assay. In some experiments the cultures are assayed for peptide-specific recognition in the in situ IFN&ggr; ELISA at the time of the second restimulation followed by assay of endogenous recognition 7 days later. After expansion, activity is measured in both assays for a side-by-side comparison.
[0543] Measurement of CTL Lytic Activity by 51Cr Release.
[0544] Seven days after the second restimulation, cytotoxicity is determined in a standard (5 hr) 51Cr release assay by assaying individual wells at a single E:T. Peptide-pulsed targets are prepared by incubating the cells with 10 &mgr;g/ml peptide overnight at 37° C.
[0545] Adherent target cells are removed from culture flasks with trypsin-EDTA. Target cells are labeled with 200 &mgr;Ci of 51Cr sodium chromate (Dupont, Wilmington, Del.) for 1 hour at 37° C. Labeled target cells are resuspended at 106 per ml and diluted 1:10 with K562 cells at a concentration of 3.3×106/ml (an NK-sensitive erythroblastoma cell line used to reduce non-specific lysis). Target cells (100 &mgr;l) and effectors (100 &mgr;l) are plated in 96 well round-bottom plates and incubated for 5 hours at 37° C. At that time, 100 &mgr;l of supernatant are collected from each well and percent lysis is determined according to the formula:
[(cpm of the test sample−cpm of the spontaneous 51Cr release sample)/(cpm of the maximal 51Cr release sample−cpm of the spontaneous 51Cr release sample)]×100.
[0546] Maximum and spontaneous release are determined by incubating the labeled targets with 1% Triton X-100 and media alone, respectively. A positive culture is defined as one in which the specific lysis (sample-background) is 10% or higher in the case of individual wells and is 15% or more at the two highest E:T ratios when expanded cultures are assayed.
[0547] In Situ Measurement of Human IFN&ggr; Production as an Indicator of Peptide-Specific and Endogenous Recognition
[0548] Immulon 2 plates are coated with mouse anti-human IFN&ggr; monoclonal antibody (4 &mgr;g/ml 0.1 M NaHCO3, pH 8.2) overnight at 4° C. The plates are washed with Ca2+, Mg2+-free PBS/0.05% Tween 20 and blocked with PBS/10% FCS for two hours, after which the CTLs (100 &mgr;l/well) and targets (100 &mgr;l/well) are added to each well, leaving empty wells for the standards and blanks (which received media only). The target cells, either peptide-pulsed or endogenous targets, are used at a concentration of 1×106 cells/ml. The plates are incubated for 48 hours at 37° C. with 5% CO2.
[0549] Recombinant human IFN-ganma is added to the standard wells starting at 400 pg or 1200 pg/100 microliter/well and the plate incubated for two hours at 37° C. The plates are washed and 100 &mgr;l of biotinylated mouse anti-human IFN-gamma monoclonal antibody (2 microgram/ml in PBS/3%FCS/0.05% Tween 20) are added and incubated for 2 hours at room temperature. After washing again, 100 microliter HRP-streptavidin (1:4000) are added and the plates incubated for one hour at room temperature. The plates are then washed 6× with wash buffer, 100 microliter/well developing solution (TMB 1:1) are added, and the plates allowed to develop for 5-15 minutes. The reaction is stopped with 50 microliter/well 1M H3PO4 and read at OD450. A culture is considered positive if it measured at least 50 pg of IFN-gamma/well above background and is twice the background level of expression.
[0550] CTL Expansion.
[0551] Those cultures that demonstrate specific lytic activity against peptide-pulsed targets and/or tumor targets are expanded over a two week period with anti-CD3. Briefly, 5×104 CD8+ cells are added to a T25 flask containing the following: 1×106 irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2×105 irradiated (8,000 rad) EBV—transformed cells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640 containing 10% (v/v) human AB serum, non-essential amino acids, sodium pyruvate, 25 &mgr;M 2-mercaptoethanol, L-glutamine and penicillin/streptomycin. Recombinant human IL2 is added 24 hours later at a final concentration of 200 IU/ml and every three days thereafter with fresh media at 50 IU/ml. The cells are split if the cell concentration exceeds 1×106/ml and the cultures are assayed between days 13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the 51Cr release assay or at 1×106/ml in the in situ IFN&ggr; assay using the same targets as before the expansion.
[0552] Cultures are expanded in the absence of anti-CD3+ as follows. Those cultures that demonstrate specific lytic activity against peptide and endogenous targets are selected and 5×104 CD8+ cells are added to a T25 flask containing the following: 1×106 autologous PBMC per ml which have been peptide-pulsed with 10 &mgr;g/ml peptide for two hours at 37° C. and irradiated (4,200 rad); 2×105 irradiated (8,000 rad) EBV-transformed cells per ml RPMI-1640 containing 10%(v/v) human AB serum, non-essential AA, sodium pyruvate, 25 mM 2-ME, L-glutamine and gentamicin.
[0553] Immunogenicity of A2 Supermotif-Bearing Peptides
[0554] A2-supermotif cross-reactive binding peptides are tested in the cellular assay for the ability to induce peptide-specific CTL in normal individuals. In this analysis, a peptide is typically considered to be an epitope if it induces peptide-specific CTLs in at least individuals, and preferably, also recognizes the endogenously expressed peptide.
[0555] Immunogenicity can also be confirmed using PBMCs isolated from patients bearing a tumor that expresses 213P11. Briefly, PBMCs are isolated from patients, re-stimulated with peptide-pulsed monocytes and assayed for the ability to recognize peptide-pulsed target cells as well as transfected cells endogenously expressing the antigen.
[0556] Evaluation of A*03/A11 Immunogenicity
[0557] HLA-A3 supermotif-bearing cross-reactive binding peptides are also evaluated for immunogenicity using methodology analogous for that used to evaluate the immunogenicity of the HLA-A2 supermotif peptides.
[0558] Evaluation of B7 Immunogenicity
[0559] Immunogenicity screening of the B7-supertype cross-reactive binding peptides identified as set forth herein are confirmed in a manner analogous to the confirmation of A2-and A3-supermotif-bearing peptides.
[0560] Peptides bearing other supermotifs/motifs, e.g., HLA-A 1, HLA-A24 etc. are also confirmed using similar methodology
Example 15 Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creating Analogs[0561] HLA motifs and supermotifs (comprising primary and/or secondary residues) are useful in the identification and preparation of highly cross-reactive native peptides, as demonstrated herein. Moreover, the definition of HLA motifs and supermotifs also allows one to engineer highly cross-reactive epitopes by identifying residues within a native peptide sequence which can be analoged to confer upon the peptide certain characteristics, e.g. greater cross-reactivity within the group of HLA molecules that comprise a supertype, and/or greater binding affinity for some or all of those HLA molecules. Examples of analoging peptides to exhibit modulated binding affinity are set forth in this example.
[0562] Analoging at Primary Anchor Residues
[0563] Peptide engineering strategies are implemented to further increase the cross-reactivity of the epitopes. For example, the main anchors of A2-supermotif-bearing peptides are altered, for example, to introduce a preferred L, I, V, or M at position 2, and I or V at the C-terminus.
[0564] To analyze the cross-reactivity of the analog peptides, each engineered analog is initially tested for binding to the prototype A2 supertype allele A*0201, then, if A*0201 binding capacity is maintained, for A2-supertype cross-reactivity.
[0565] Alternatively, a peptide is confirmed as binding one or all supertype members and then analoged to modulate binding affinity to any one (or more) of the supertype members to add population coverage.
[0566] The selection of analogs for inmmunogenicity in a cellular screening analysis is typically further restricted by the capacity of the parent wild type (WT) peptide to bind at least weakly, i.e., bind at an IC50 of 5000 nM or less, to three of more A2 supertype alleles. The rationale for this requirement is that the WT peptides must be present endogenously in sufficient quantity to be biologically relevant. Analoged peptides have been shown to have increased immunogenicity and cross-reactivity by T cells specific for the parent epitope (see, e.g., Parkhurst et al, J. Immunol. 157:2539, 1996; and Pogue et al., Proc. Natl. Acad. Sci. USA 92:8166, 1995).
[0567] In the cellular screening of these peptide analogs, it is important to confirm that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, target cells that endogenously express the epitope.
[0568] Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides
[0569] Analogs of HLA-A3 supermotif-bearing epitopes are generated using strategies similar to those employed in analoging HLA-A2 supermotif-bearing peptides. For example, peptides binding to 3/5 of the A3-supertype molecules are engineered at primary anchor residues to possess a preferred residue (V, S, M, or A) at position 2.
[0570] The analog peptides are then tested for the ability to bind A*03 and A*11 (prototype A3 supertype alleles). Those peptides that demonstrate <500 nM binding capacity are then confirmed as having A3-supertype cross-reactivity.
[0571] Similarly to the A2- and A3-motif bearing peptides, peptides binding 3 or more B7-supertype alleles can be improved, where possible, to achieve increased cross-reactive binding or greater binding affinity or binding half life. B7 supermotif-bearing peptides are, for example, engineered to possess a preferred residue (V, I, L, or F) at the C-terminal primary anchor position, as demonstrated by Sidney et al. (J. Immunol. 157:3480-3490, 1996).
[0572] Analoging at primary anchor residues of other motif and/or supermotif-bearing epitopes is performed in a like manner.
[0573] The analog peptides are then be confirmed for immunogenicity, typically in a cellular screening assay. Again, it is generally important to demonstrate that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, targets that endogenously express the epitope.
[0574] Analoging at Secondary Anchor Residues
[0575] Moreover, HLA supermotifs are of value in engineering highly cross-reactive peptides and/or peptides that bind HLA molecules with increased affinity by identifying particular residues at secondary anchor positions that are associated with such properties. For example, the binding capacity of a B7 supermotif-bearing peptide with an F residue at position 1 is analyzed. The peptide is then analoged to, for example, substitute L for F at position 1. The analoged peptide is evaluated for increased binding affinity, binding half life and/or increased cross-reactivity. Such a procedure identifies analoged peptides with enhanced properties.
[0576] Engineered analogs with sufficiently improved binding capacity or cross-reactivity can also be tested for immunogenicity in HLA-B7-transgenic mice, following for example, IFA immunization or lipopeptide immunization. Analoged peptides are additionally tested for the ability to stimulate a recall response using PBMC from patients with 213P1F11-expressing tumors.
[0577] Other Analoging Strategies
[0578] Another form of peptide analoging, unrelated to anchor positions, involves the substitution of a cysteine with &agr;-amino butyric acid. Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity. Substitution of &agr;-amino butyric acid for cysteine not only alleviates this problem, but has been shown to improve binding and crossbinding capabilities in some instances (see, e.g., the review by Sette et al., In: Persistent Viral Infections, Eds. R. Ahlned and I. Chen, John Wiley & Sons, England, 1999).
[0579] Thus, by the use of single amino acid substitutions, the binding properties and/or cross-reactivity of peptide ligands for HLA supertype molecules can be modulated.
Example 16 Identification and Confirmation of 213P1F11-Derived Sequences with HLA-DR Binding Motifs[0580] Peptide epitopes bearing an HLA class II supermotif or motif are identified and confirmed as outlined below using methodology similar to that described for HLA Class I peptides.
[0581] Selection of HLA-DR-Supermotif-Bearing Epitopes.
[0582] To identify 213P1F11-derived, HLA class II HTL epitopes, a 213P1F11 antigen is analyzed for the presence of sequences bearing an HLA-DR-motif or supermotif. Specifically, 15-mer sequences are selected comprising a DR-supermotif, comprising a 9-mer core, and three-residue N- and C-terminal flanking regions (15 amino acids total).
[0583] Protocols for predicting peptide binding to DR molecules have been developed (Southwood et al., J. Immunol. 160:3363-3373, 1998). These protocols, specific for individual DR molecules, allow the scoring, and ranking, of 9-mer core regions. Each protocol not only scores peptide sequences for the presence of DR-supermotif primary anchors (i.e., at position 1 and position 6) within a 9-mer core, but additionally evaluates sequences for the presence of secondary anchors. Using allele-specific selection tables (see, e.g., Southwood et al., ibid.), it has been found that these protocols efficiently select peptide sequences with a high probability of binding a particular DR molecule. Additionally, it has been found that performing these protocols in tandem, specifically those for DR1, DR4w4, and DR7, can efficiently select DR cross-reactive peptides.
[0584] The 213P1F11-derived peptides identified above are tested for their binding capacity for various common HLA-DR molecules. All peptides are initially tested for binding to the DR molecules in the primary panel: DR1, DR4w4, and DR7. Peptides binding at least two of these three DR molecules are then tested for binding to DR2w2 &bgr;1, DR2w2 &bgr;2, DR6w19, and DR9 molecules in secondary assays. Finally, peptides binding at least two of the four secondary panel DR molecules, and thus cumulatively at least four of seven different DR molecules, are screened for binding to DR4w15, DR5w11, and DR8w2 molecules in tertiary assays. Peptides binding at least seven of the ten DR molecules comprising the primary, secondary, and tertiary screening assays are considered cross-reactive DR binders. 213P1F11-derived peptides found to bind common HLA-DR alleles are of particular interest.
[0585] Selection of DR3 Motif Peptides
[0586] Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, and Hispanic populations, DR3 binding capacity is a relevant criterion in the selection of HTL epitopes. Thus, peptides shown to be candidates may also be assayed for their DR3 binding capacity. However, in view of the binding specificity of the DR3 motif, peptides binding only to DR3 can also be considered as candidates for inclusion in a vaccine formulation.
[0587] To efficiently identify peptides that bind DR3, target 213P1F11 antigens are analyzed for sequences carrying one of the two DR3-specific binding motifs reported by Geluk et al. (J. Immunol. 152:5742-5748, 1994). The corresponding peptides are then synthesized and confirmed as having the ability to bind DR3 with an affinity of 1 &mgr;M or better, i.e., less than 1 &mgr;M. Peptides are found that meet this binding criterion and qualify as HLA class II high affinity binders.
[0588] DR3 binding epitopes identified in this manner are included in vaccine compositions with DR supermotif-bearing peptide epitopes.
[0589] Similarly to the case of HLA class I motif-bearing peptides, the class II motif-bearing peptides are analoged to improve affinity or cross-reactivity. For example, aspartic acid at position 4 of the 9-mer core sequence is an optimal residue for DR3 binding, and substitution for that residue often improves DR 3 binding.
Example 17 Immunogenicity of 213P1F11-Derived HTL Epitopes[0590] This example determines immunogenic DR supermotif- and DR3 motif-bearing epitopes among those identified using the methodology set forth herein.
[0591] Immunogenicity of HTL epitopes are confirmed in a manner analogous to the determination of immunogenicity of CTL epitopes, by assessing the ability to stimulate HTL responses and/or by using appropriate transgenic mouse models. Immunogenicity is determined by screening for: 1.) in vitro primary induction using normal PBMC or 2.) recall responses from patients who have 213P1F11-expressing tumors.
Example 18 Calculation of Phenotypic Frequencies of HLA-Supertypes in Various Ethnic Backgrounds to Determine Breadth of Population Coverage[0592] This example illustrates the assessment of the breadth of population coverage of a vaccine composition comprised of multiple epitopes comprising multiple supermotifs and/or motifs.
[0593] In order to analyze population coverage, gene frequencies of HLA alleles are determined. Gene frequencies for each HLA allele are calculated from antigen or allele frequencies utilizing the binomial distribution formulae gf=1−(SQRT(1−af)) (see, e.g., Sidney et al., Human Immunol. 45:79-93, 1996). To obtain overall phenotypic frequencies, cumulative gene frequencies are calculated, and the cumulative antigen frequencies derived by the use of the inverse formula [af=1−(1−Cgf)2].
[0594] Where frequency data is not available at the level of DNA typing, correspondence to the serologically defined antigen frequencies is assumed. To obtain total potential supertype population coverage no linkage disequilibrium is assumed, and only alleles confirmed to belong to each of the supertypes are included (minimal estimates). Estimates of total potential coverage achieved by inter-loci combinations are made by adding to the A coverage the proportion of the non-A covered population that could be expected to be covered by the B alleles considered (e.g., total=A+B*(1−A)). Confirmed members of the A3-like supertype are A3, A11, A31, A*3301, and A*6801. Although the A3-like supertype may also include A34, A66, and A*7401, these alleles were not included in overall frequency calculations. Likewise, confirmed members of the A2-like supertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmed alleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601, B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, and B*5602).
[0595] Population coverage achieved by combining the A2-, A3- and B7-supertypes is approximately 86% in five major ethnic groups. Coverage may be extended by including peptides bearing the A1 and A24 motifs. On average, A1 is present in 12% and A24 in 29% of the population across five different major ethnic groups (Caucasian, North American Black, Chinese, Japanese, and Hispanic). Together, these alleles are represented with an average frequency of 39% in these same ethnic populations. The total coverage across the major ethnicities when A1 and A24 are combined with the coverage of the A2-, A3- and B7-supertype alleles is >95%. An analogous approach can be used to estimate population coverage achieved with combinations of class II motif-bearing epitopes.
[0596] Immunogenicity studies in humans (e.g., Bertoni et al., J. Clin. Invest. 100:503, 1997; Doolan et al., Immunity 7:97, 1997; and Threlkeld et al., J. Immunol 159:1648, 1997) have shown that highly cross-reactive binding peptides are almost always recognized as epitopes. The use of highly cross-reactive binding peptides is an important selection criterion in identifying candidate epitopes for inclusion in a vaccine that is immunogenic in a diverse population.
[0597] With a sufficient number of epitopes (as disclosed herein and from the art), an average population coverage is predicted to be greater than 95% in each of five major ethnic populations. The game theory Monte Carlo simulation analysis, which is known in the art (see e.g., Osborne, M. J. and Rubinstein, A. “A course in game theory” MIT Press, 1994), can be used to estimate what percentage of the individuals in a population comprised of the Caucasian, North American Black, Japanese, Chinese, and Hispanic ethnic groups would recognize the vaccine epitopes described herein. A preferred percentage is 90%. A more preferred percentage is 95%.
Example 19 CTL Recognition of Endogenously Processed Antigens After Priming[0598] This example confirms that CTL induced by native or analoged peptide epitopes identified and selected as described herein recognize endogenously synthesized, i.e., native antigens.
[0599] Effector cells isolated from transgenic mice that are immunized with peptide epitopes, for example HLA-A2 supermotif-bearing epitopes, are re-stimulated in vitro using peptide-coated stimulator cells. Six days later, effector cells are assayed for cytotoxicity and the cell lines that contain peptide-specific cytotoxic activity are further re-stimulated. An additional six days later, these cell lines are tested for cytotoxic activity on 51Cr labeled Jurkat-A2.1/Kb target cells in the absence or presence of peptide, and also tested on 51Cr labeled target cells bearing the endogenously synthesized antigen, i.e. cells that are stably transfected with 213P1F11 expression vectors.
[0600] The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized 213P1F11 antigen. The choice of transgenic mouse model to be used for such an analysis depends upon the epitope(s) that are being evaluated. In addition to HLA-A*0201/Kb transgenic mice, several other transgenic mouse models including mice with human A11, which may also be used to evaluate A3 epitopes, and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-A 1 and A24) are being developed. HLA-DR1 and HLA-DR3 mouse models have also been developed, which may be used to evaluate HTL epitopes.
Example 20 Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice[0601] This example illustrates the induction of CTLs and HTLs in transgenic mice, by use of a 213P1F11-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a 213P1F11-expressing tumor. The peptide composition can comprise multiple CTL and/or HTL epitopes. The epitopes are identified using methodology as described herein. This example also illustrates that enhanced immunogenicity can be achieved by inclusion of one or more HTL epitopes in a CTL vaccine composition; such a peptide composition can comprise an HTL epitope conjugated to a CTL epitope. The CTL epitope can be one that binds to multiple HLA family members at an affinity of 500 nM or less, or analogs of that epitope. The peptides may be lipidated, if desired.
[0602] Immunization procedures: Immunization of transgenic mice is performed as described (Alexander et al., J. Immunol. 159:4753-4761, 1997). For example, A2/Kb mice, which are transgenic for the human HLA A2.1 allele and are used to confirm the immunogenicity of HLA-A*0201 motif- or HLA-A2 supermotif-bearing epitopes, and are primed subcutaneously (base of the tail) with a 0.1 ml of peptide in Incomplete Freund's Adjuvant, or if the peptide composition is a lipidated CTL/HTL conjugate, in DMSO/saline, or if the peptide composition is a polypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days after priming, splenocytes obtained from these animals are restimulated with syngenic irradiated LPS-activated lymphoblasts coated with peptide.
[0603] Cell lines: Target cells for peptide-specific cytotoxicity assays are Jurkat cells transfected with the HLA-A2.1/Kb chimeric gene (e.g., Vitiello et al., J. Exp. Med. 173:1007, 1991) In vitro CTL activation. One week after priming, spleen cells (30×106 cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10×106 cells/flask) in 10 ml of culture medium/T25 flask. After six days, effector cells are harvested and assayed for cytotoxic activity.
[0604] Assay for cytotoxic activity: Target cells (1.0 to 1.5×106) are incubated at 37° C. in the presence of 200 &mgr;l of 51Cr. After 60 minutes, cells are washed three times and resuspended in R10 medium. Peptide is added where required at a concentration of 1 &mgr;g/ml. For the assay, 104 51Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 &mgr;l) in U-bottom 96-well plates. After a six hour incubation period at 37° C., a 0.1 ml aliquot of supernatant is removed from each well and radioactivity is determined in a Micromedic automatic gamma counter. The percent specific lysis is determined by the formula: percent specific release=100×(experimental release−spontaneous release)/(maximum release−spontaneous release). To facilitate comparison between separate CTL assays run under the same conditions, % 51Cr release data is expressed as lytic units/106 cells. One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a six hour 51Cr release assay. To obtain specific lytic units/106, the lytic units/106 obtained in the absence of peptide is subtracted from the lytic units/106 obtained in the presence of peptide. For example, if 30% 51Cr release is obtained at the effector (E): target (T) ratio of 50:1 (i.e., 5×105 effector cells for 10,000 targets) in the absence of peptide and 5:1 (i.e., 5×104 effector cells for 10,000 targets) in the presence of peptide, the specific lytic units would be: [(1/50,000)−(1/500,000)]×106=18 LU.
[0605] The results are analyzed to assess the magnitude of the CTL responses of animals injected with the immunogenic CTL/HTL conjugate vaccine preparation and are compared to the magnitude of the CTL response achieved using, for example, CTL epitopes as outlined above in the Example entitled “Confirmation of Immunogenicity.” Analyses similar to this may be performed to confirm the immunogenicity of peptide conjugates containing multiple CTL epitopes and/or multiple HTL epitopes. In accordance with these procedures, it is found that a CTL response is induced, and concomitantly that an HTL response is induced upon administration of such compositions.
Example 21 Selection of CTL and HTL Epitopes for Inclusion in a 213P1F11-Specific Vaccine[0606] This example illustrates a procedure for selecting peptide epitopes for vaccine compositions of the invention. The peptides in the composition can be in the form of a nucleic acid sequence, either single or one or more sequences (i.e., minigene) that encodes peptide(s), or can be single and/or polyepitopic peptides.
[0607] The following principles are utilized when selecting a plurality of epitopes for inclusion in a vaccine composition. Each of the following principles is balanced in order to make the selection.
[0608] Epitopes are selected which, upon administration, mimic immune responses that are correlated with 213P1F11 clearance. The number of epitopes used depends on observations of patients who spontaneously clear 213P1F11. For example, if it has been observed that patients who spontaneously clear 213P1F11-expressing cells generate an immune response to at least three (3) from 213P F11 antigen, then at least three epitopes should be included for HLA class I. A similar rationale is used to determine HLA class II epitopes.
[0609] Epitopes are often selected that have a binding affinity of an IC50 of 500 nM or less for an HLA class I molecule, or for class II, an IC50 of 1000 nM or less; or HLA Class I peptides with high binding scores from the BIMAS web site, at URL bimas.dcrt.nih.gov/.
[0610] In order to achieve broad coverage of the vaccine through out a diverse population, sufficient supermotif bearing peptides, or a sufficient array of allele-specific motif bearing peptides, are selected to give broad population coverage. In one embodiment, epitopes are selected to provide at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess breadth, or redundancy, of population coverage.
[0611] When creating polyepitopic compositions, or a minigene that encodes same, it is typically desirable to generate the smallest peptide possible that encompasses the epitopes of interest. The principles employed are similar, if not the same, as those employed when selecting a peptide comprising nested epitopes. For example, a protein sequence for the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes. Epitopes may be nested or overlapping (i.e., frame shifted relative to one another). For example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Each epitope can be exposed and bound by an HLA molecule upon administration of such a peptide. A multi-epitopic, peptide can be generated synthetically, recombinantly, or via cleavage from the native source. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes. This embodiment provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup that is presently unknown. Furthermore, this embodiment (absent the creating of any analogs) directs the immune response to multiple peptide sequences that are actually present in 213P1F11, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing nucleic acid vaccine compositions. Related to this embodiment, computer programs can be derived in accordance with principles in the art, which identify in a target sequence, the greatest number of epitopes per sequence length.
[0612] A vaccine composition comprised of selected peptides, when administered, is safe, efficacious, and elicits an immune response similar in magnitude to an immune response that controls or clears cells that bear or overexpress 213P1F11.
Example 22 Construction of “Minigene” Multi-Epitope DNA Plasmids[0613] This example discusses the construction of a minigene expression plasmid. Minigene plasmids may, of course, contain various configurations of B cell, CTL and/or HTL epitopes or epitope analogs as described herein.
[0614] A minigene expression plasmid typically includes multiple CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearing peptide epitopes are used in conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearing peptide epitopes derived 213P1F11, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from 213P1F11 to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct. The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.
[0615] Such a construct may additionally include sequences that direct the HTL epitopes to the endoplasmic reticulum. For example, the Ii protein may be fused to one or more HTL epitopes as described in the art, wherein the CLIP sequence of the Ii protein is removed and replaced with an HLA class II epitope sequence so that HLA class II epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA class II molecules.
[0616] This example illustrates the methods to be used for construction of a minigene-bearing expression plasmid. Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art.
[0617] The minigene DNA plasmid of this example contains a consensus Kozak sequence and a consensus murine kappa Ig-light chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disclosed herein. The sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.
[0618] Overlapping oligonucleotides that can, for example, average about 70 nucleotides in length with 15 nucleotide overlaps, are synthesized and HPLC-purified. The oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides, Kozak sequence, and signal sequence. The final multiepitope minigene is assembled by extending the overlapping oligonucleotides in three sets of reactions using PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95° C. for 15 sec, annealing temperature (5° below the lowest calculated Tm of each primer pair) for 30 sec, and 72° C. for 1 min.
[0619] For example, a minigene is prepared as follows. For a first PCR reaction, 5 &mgr;g of each of two oligonucleotides are annealed and extended: In an example using eight oligonucleotides, i.e., four pairs of primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100 &mgr;l reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM (NH4)2SO4, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO4, 0.1% Triton X-100, 100 &mgr;g/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. The full-length dimer products are gel-purified, and two reactions containing the product of 1+2 and 3+4, and the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the two reactions are then mixed, and 5 cycles of annealing and extension carried out before flanking primers are added to amplify the full length product. The full-length product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing.
Example 23 The Plasmid Construct and the Degree to Which it Induces Immunogenicity[0620] The degree to which a plasmid construct, for example a plasmid constructed in accordance with the previous Example, is able to induce immunogenicity is confirmed in vitro by determining epitope presentation by APC following transduction or transfection of the APC with an epitope-expressing nucleic acid construct. Such a study determines “antigenicity” and allows the use of human APC. The assay determines the ability of the epitope to be presented by the APC in a context that is recognized by a T cell by quantifying the density of epitope-HLA class I complexes on the cell surface. Quantitation can be performed by directly measuring the amount of peptide eluted from the APC (see, e.g., Sijts et al., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684, 1989); or the number of peptide-HLA class I complexes can be estimated by measuring the amount of lysis or lymphokine release induced by diseased or transfected target cells, and then determining the concentration of peptide necessary to obtain equivalent levels of lysis or lymphokine release (see, e.g., Kageyama et al., J. Immunol. 154:567-576, 1995).
[0621] Alternatively, immunogenicity is confirmed through in vivo injections into mice and subsequent in vitro assessment of CTL and HTL activity, which are analyzed using cytotoxicity and proliferation assays, respectively, as detailed e.g., in Alexander et al., Immunity 1:751-761, 1994.
[0622] For example, to confirm the capacity of a DNA minigene construct containing at least one HLA-A2 supermotif peptide to induce CTLs in vivo, HLA-A2.11 Kb transgenic mice, for example, are immunized intramuscularly with 100 &mgr;g of naked cDNA. As a means of comparing the level of CTLs induced by cDNA immunization, a control group of animals is also immunized with an actual peptide composition that comprises multiple epitopes synthesized as a single polypeptide as they would be encoded by the minigene.
[0623] Splenocytes from immunized animals are stimulated twice with each of the respective compositions (peptide epitopes encoded in the minigene or the polyepitopic peptide), then assayed for peptide-specific cytotoxic activity in a 51Cr release assay. The results indicate the magnitude of the CTL response directed against the A2-restricted epitope, thus indicating the in vivo immunogenicity of the minigene vaccine and polyepitopic vaccine.
[0624] It is, therefore, found that the minigene elicits immune responses directed toward the HLA-A2 supermotif peptide epitopes as does the polyepitopic peptide vaccine. A similar analysis is also performed using other HLA-A3 and HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby it is also found that the minigene elicits appropriate immune responses directed toward the provided epitopes.
[0625] To confirm the capacity of a class II epitope-encoding minigene to induce HTLs in vivo, DR transgenic mice, or for those epitopes that cross react with the appropriate mouse MHC molecule, I-Ab-restricted mice, for example, are immunized intramuscularly with 100 &mgr;g of plasmid DNA. As a means of comparing the level of HTLs induced by DNA immunization, a group of control animals is also immunized with an actual peptide composition emulsified in complete Freund's adjuvant. CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunized animals and stimulated with each of the respective compositions (peptides encoded in the minigene). The HTL response is measured using a 3H-thymidine incorporation proliferation assay, (see, e.g., Alexander et al. Immunity 1:751-761, 1994). The results indicate the magnitude of the HTL response, thus demonstrating the in vivo immunogenicity of the minigene.
[0626] DNA minigenes, constructed as described in the previous Example, can also be confirmed as a vaccine in combination with a boosting agent using a prime boost protocol. The boosting agent can consist of recombinant protein (e.g., Barnett et al., Aids Res. and Human Retroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia, for example, expressing a minigene or DNA encoding the complete protein of interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegah et al., Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177-181, 1999; and Robinson et al, Nature Med. 5:526-34, 1999).
[0627] For example, the efficacy of the DNA minigene used in a prime boost protocol is initially evaluated in transgenic mice. In this example, A2.1/Kb transgenic mice are immunized IM with 100 &mgr;g of a DNA minigene encoding the immunogenic peptides including at least one HLA-A2 supermotif-bearing peptide. After an incubation period (ranging from 3-9 weeks), the mice are boosted IP with 107 pfu/mouse of a recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are immunized with 100 &mgr;g of DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding the minigene, but without the vaccinia boost. After an additional incubation period of two weeks, splenocytes from the mice are immediately assayed for peptide-specific activity in an ELISPOT assay. Additionally, splenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vaccinia, then assayed for peptide-specific activity in an alpha, beta and/or gamma IFN ELISA.
[0628] It is found that the minigene utilized in a prime-boost protocol elicits greater immune responses toward the HLA-A2 supermotif peptides than with DNA alone. Such an analysis can also be performed using HLA-A 11 or HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 or HLA-B7 motif or supermotif epitopes. The use of prime boost protocols in humans is described below in the Example entitled “Induction of CTL Responses Using a Prime Boost Protocol.”
Example 24 Peptide Compositions for Prophylactic Uses[0629] Vaccine compositions of the present invention can be used to prevent 213P1F11 expression in persons who are at risk for tumors that bear this antigen. For example, a polyepitopic peptide epitope composition (or a nucleic acid comprising the same) containing multiple CTL and HTL epitopes such as those selected in the above Examples, which are also selected to target greater than 80% of the population, is administered to individuals at risk for a 213P1F11-associated tumor.
[0630] For example, a peptide-based composition is provided as a single polypeptide that encompasses multiple epitopes. The vaccine is typically administered in a physiological solution that comprises an adjuvant, such as Incomplete Freunds Adjuvant. The dose of peptide for the initial immunization is from about 1 to about 50,000 &mgr;g, generally 100-5,000 &mgr;g, for a 70 kg patient. The initial administration of vaccine is followed by booster dosages at 4 weeks followed by evaluation of the magnitude of the immune response in the patient, by techniques that determine the presence of epitope-specific CTL populations in a PBMC sample. Additional booster doses are administered as required. The composition is found to be both safe and efficacious as a prophylaxis against 213P1F11-associated disease.
[0631] Alternatively, a composition typically comprising transfecting agents is used for the administration of a nucleic acid-based vaccine in accordance with methodologies known in the art and disclosed herein.
Example 25 Polyepitopic Vaccine Compositions Derived from Native 213P1F11 Sequences[0632] A native 213P1F11 polyprotein sequence is analyzed, preferably using computer algorithms defined for each class I and/or class II supermotif or motif, to identify “relatively short” regions of the polyprotein that comprise multiple epitopes. The “relatively short” regions are preferably less in length than an entire native antigen. This relatively short sequence that contains multiple distinct or overlapping, “nested” epitopes is selected; it can be used to generate a minigene construct. The construct is engineered to express the peptide, which corresponds to the native protein sequence. The “relatively short” peptide is generally less than 250 amino acids in length, often less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than 50 amino acids in length. The protein sequence of the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes. As noted herein, epitope motifs may be nested or overlapping (i.e., frame shifted relative to one another). For example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes.
[0633] The vaccine composition will include, for example, multiple CTL epitopes from 213P1F11 antigen and at least one HTL epitope. This polyepitopic native sequence is administered either as a peptide or as a nucleic acid sequence which encodes the peptide. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide.
[0634] The embodiment of this example provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup(s) that is presently unknown. Furthermore, this embodiment (excluding an analoged embodiment) directs the immune response to multiple peptide sequences that are actually present in native 213P1F11, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing peptide or nucleic acid vaccine compositions.
[0635] Related to this embodiment, computer programs are available in the art which can be used to identify in a target sequence, the greatest number of epitopes per sequence length.
Example 26 Polyepitopic Vaccine Compositions from Multiple Antigens[0636] The 213P1F11 peptide epitopes of the present invention are used in conjunction with epitopes from other target tumor-associated antigens, to create a vaccine composition that is useful for the prevention or treatment of cancer that expresses 213P1F11 and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from 213P1F11 as well as tumor-associated antigens that are often expressed with a target cancer associated with 213P1F11 expression, or can be administered as a composition comprising a cocktail of one or more discrete epitopes. Alternatively, the vaccine can be administered as a minigene construct or as dendritic cells which have been loaded with the peptide epitopes in vitro.
Example 27 Use of Peptides to Evaluate an Immune Response[0637] Peptides of the invention may be used to analyze an immune response for the presence of specific antibodies, CTL or HTL directed to 213P1F11. Such an analysis can be performed in a manner described by Ogg et al., Science 279:2103-2106, 1998. In this Example, peptides in accordance with the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.
[0638] In this example highly sensitive human leukocyte antigen tetrameric complexes (“tetramers”) are used for a cross-sectional analysis of, for example, 213P1F11 HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of disease or following immunization comprising a 213P1F11 peptide containing an A*0201 motif. Tetrameric complexes are synthesized as described (Musey et al., N. Engl J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201 in this example) and P2-microglobulin are synthesized by means of a prokaryotic expression system. The heavy chain is modified by deletion of the transmembrane-cytosolic tail and COOH-terminal addition of a sequence containing a BirA enzymatic biotinylation site. The heavy chain, &bgr;2-microglobulin, and peptide are refolded by dilution. The 45-kD refolded product is isolated by fast protein liquid chromatography and then biotinylated by BirA in the presence of biotin (Sigma, St. Louis, Mo.), adenosine 5′ triphosphate and magnesium. Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, and the tetrameric product is concentrated to 1 mg/ml. The resulting product is referred to as tetramer-phycoerythrin.
[0639] For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 &mgr;l of cold phosphate-buffered saline. Tri-color analysis is performed with the tetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. The PBMCs are incubated with tetramer and antibodies on ice for 30 to 60 min and then washed twice before formaldehyde fixation. Gates are applied to contain >99.98% of control samples. Controls for the tetramers include both A*0201-negative individuals and A*0201-positive non-diseased donors. The percentage of cells stained with the tetramer is then determined by flow cytometry. The results indicate the number of cells in the PBMC sample that contain epitope-restricted CTLs, thereby readily indicating the extent of immune response to the 213P1F11 epitope, and thus the status of exposure to 213P1F11, or exposure to a vaccine that elicits a protective or therapeutic response.
Example 28 Use of Peptide Epitopes to Evaluate Recall Responses[0640] The peptide epitopes of the invention are used as reagents to evaluate T cell responses, such as acute or recall responses, in patients. Such an analysis may be performed on patients who have recovered from 213P1F11-associated disease or who have been vaccinated with a 213P1F11 vaccine.
[0641] For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any 213P1F11 vaccine. PBMC are collected from vaccinated individuals and HLA typed. Appropriate peptide epitopes of the invention that, optimally, bear supermotifs to provide cross-reactivity with multiple HLA supertype family members, are then used for analysis of samples derived from individuals who bear that HLA type.
[0642] PBMC from vaccinated individuals are separated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis, Mo.), washed three times in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2 mM), penicillin (50U/ml), streptomycin (50 &mgr;g/ml), and Hepes (10 mM) containing 10% heat-inactivated human AB serum (complete RPMI) and plated using microculture formats. A synthetic peptide comprising an epitope of the invention is added at 10 &mgr;g/ml to each well and HBV core 128-140 epitope is added at 1 &mgr;g/ml to each well as a source of T cell help during the first week of stimulation.
[0643] In the microculture format, 4×105 PBMC are stimulated with peptide in 8 replicate cultures in 96-well round bottom plate in 100 &mgr;l/well of complete RPMI. On days 3 and 10, 100 &mgr;l of complete RPMI and 20 U/ml final concentration of rIL-2 are added to each well. On day 7 the cultures are transferred into a 96-well flat-bottom plate and restimulated with peptide, rIL-2 and 105 irradiated (3,000 rad) autologous feeder cells. The cultures are tested for cytotoxic activity on day 14. A positive CTL response requires two or more of the eight replicate cultures to display greater than 10% specific 51Cr release, based on comparison with non-diseased control subjects as previously described (Rehermann, et al, Nature Med. 2:1104,1108, 1996; Rehermann et al, J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al J. Clin. Invest. 98:1432-1440, 1996).
[0644] Target cell lines are autologous and allogeneic EBV-transformed B-LCL that are either purchased from the American Society for Histocompatibility and Immunogenetics (ASHI, Boston, Mass.) or established from the pool of patients as described (Guilhot, et al J. Virol. 66:2670-2678, 1992).
[0645] Cytotoxicity assays are performed in the following manner. Target cells consist of either allogeneic HLA-matched or autologous EBV-transformed B lymphoblastoid cell line that are incubated overnight with the synthetic peptide epitope of the invention at 10 &mgr;M, and labeled with 100 &mgr;Ci of 51Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after which they are washed four times with HBSS.
[0646] Cytolytic activity is determined in a standard 4-h, split well 51Cr release assay using U-bottomed 96 well plates containing 3,000 targets/well. Stimulated PBMC are tested at effector/target (E/T) ratios of 20-50:1 on day 14. Percent cytotoxicity is determined from the formula: 100×[(experimental release-spontaneous release)/maximum release-spontaneous release)]. Maximum release is determined by lysis of targets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis, Mo.). Spontaneous release is <25% of maximum release for all experiments.
[0647] The results of such an analysis indicate the extent to which HLA-restricted CTL populations have been stimulated by previous exposure to 213P1F11 or a 213P1F11 vaccine.
[0648] Similarly, Class II restricted HTL responses may also be analyzed. Purified PBMC are cultured in a 96-well flat bottom plate at a density of 1.5×105 cells/well and are stimulated with 10 &mgr;g/ml synthetic peptide of the invention, whole 213P1F11 antigen, or PHA. Cells are routinely plated in replicates of 4-6 wells for each condition. After seven days of culture, the medium is removed and replaced with fresh medium containing 10U/ml IL-2. Two days later, 1 &mgr;Ci 3H-thymidine is added to each well and incubation is continued for an additional 18 hours. Cellular DNA is then harvested on glass fiber mats and analyzed for 3H-thymidine incorporation. Antigen-specific T cell proliferation is calculated as the ratio of 3H-thymidine incorporation in the presence of antigen divided by the 3H-thymidine incorporation in the absence of antigen.
Example 29 Induction of Specific CTL Response in Humans[0649] A human clinical trial for an immunogenic composition comprising CTL and HTL epitopes of the invention is set up as an IND Phase I, dose escalation study and carried out as a randomized, double-blind, placebo-controlled trial. Such a trial is designed, for example, as follows:
[0650] A total of about 27 individuals are enrolled and divided into 3 groups:
[0651] Group I: 3 subjects are injected with placebo and 6 subjects are injected with 5 &mgr;g of peptide composition;
[0652] Group II: 3 subjects are injected with placebo and 6 subjects are injected with 50 &mgr;g peptide composition;
[0653] Group III: 3 subjects are injected with placebo and 6 subjects are injected with 500 &mgr;g of peptide composition.
[0654] After 4 weeks following the first injection, all subjects receive a booster inoculation at the same dosage.
[0655] The endpoints measured in this study relate to the safety and tolerability of the peptide composition as well as its immunogenicity. Cellular immune responses to the peptide composition are an index of the intrinsic activity of this the peptide composition, and can therefore be viewed as a measure of biological efficacy. The following summarize the clinical and laboratory data that relate to safety and efficacy endpoints.
[0656] Safety: The incidence of adverse events is monitored in the placebo and drug treatment group and assessed in terms of degree and reversibility.
[0657] Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy, subjects are bled before and after injection. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.
[0658] The vaccine is found to be both safe and efficacious.
Example 30 Phase II Trials in Patients Expressing 213P1F11[0659] Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses 213P1F11. The main objectives of the trial are to determine an effective dose and regimen for inducing CTLs in cancer patients that express 213P1F11, to establish the safety of inducing a CTL and HTL response in these patients, and to see to what extent activation of CTLs improves the clinical picture of these patients, as manifested, e.g., by the reduction and/or shrinking of lesions. Such a study is designed, for example, as follows:
[0660] The studies are performed in multiple centers. The trial design is an open-label, uncontrolled, dose escalation protocol wherein the peptide composition is administered as a single dose followed six weeks later by a single booster shot of the same dose. The dosages are 50, 500 and 5,000 micrograms per injection. Drug-associated adverse effects (severity and reversibility) are recorded.
[0661] There are three patient groupings. The first group is injected with 50 micrograms of the peptide composition and the second and third groups with 500 and 5,000 micrograms of peptide composition, respectively. The patients within each group range in age from 21-65 and represent diverse ethnic backgrounds. All of them have a tumor that expresses 213P1F11.
[0662] Clinical manifestations or antigen-specific T-cell responses are monitored to assess the effects of administering the peptide compositions. The vaccine composition is found to be both safe and efficacious in the treatment of 213P1F11-associated disease.
Example 31 Induction of CTL Responses Using a Prime Boost Protocol[0663] A prime boost protocol similar in its underlying principle to that used to confirm the efficacy of a DNA vaccine in transgenic mice, such as described above in the Example entitled “The Plasmid Construct and the Degree to Which It Induces Immunogenicity,” can also be used for the administration of the vaccine to humans. Such a vaccine regimen can include an initial administration of, for example, naked DNA followed by a boost using recombinant virus encoding the vaccine, or recombinant protein/polypeptide or a peptide mixture administered in an adjuvant.
[0664] For example, the initial immunization may be performed using an expression vector, such as that constructed in the Example entitled “Construction of “Minigene” Multi-Epitope DNA Plasmids” in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 &mgr;g) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-107 to 5×109 pfu. An alternative recombinant virus, such as an MVA, canarypox, adenovirus, or adeno-associated virus, can also be used for the booster, or the polyepitopic protein or a mixture of the peptides can be administered. For evaluation of vaccine efficacy, patient blood samples are obtained before immunization as well as at intervals following administration of the initial vaccine and booster doses of the vaccine. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.
[0665] Analysis of the results indicates that a magnitude of response sufficient to achieve a therapeutic or protective immunity against 213P1F11 is generated.
Example 32 Administration of Vaccine Compositions Using Dendritic Cells (DC)[0666] Vaccines comprising peptide epitopes of the invention can be administered using APCs, or “professional” APCs such as DC. In this example, peptide-pulsed DC are administered to a patient to stimulate a CTL response in vivo. In this method, dendritic cells are isolated, expanded, and pulsed with a vaccine comprising peptide CTL and HTL epitopes of the invention. The dendritic cells are infused back into the patient to elicit CTL and HTL responses in vivo. The induced CTL and HTL then destroy or facilitate destruction, respectively, of the target cells that bear the 213P1F11 protein from which the epitopes in the vaccine are derived.
[0667] For example, a cocktail of epitope-comprising peptides is administered ex vivo to PBMC, or isolated DC therefrom. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin™ (Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC with peptides, and prior to reinfusion into patients, the DC are washed to remove unbound peptides.
[0668] As appreciated clinically, and readily determined by one of skill based on clinical outcomes, the number of DC reinfused into the patient can vary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 and Prostate 32:272, 1997). Although 2-50×106 DC per patient are typically administered, larger number of DC, such as 107 or 108 can also be provided. Such cell populations typically contain between 50-90% DC.
[0669] In some embodiments, peptide-loaded PBMC are injected into patients without purification of the DC. For example, PBMC generated after treatment with an agent such as Progenipoietin™ are injected into patients without purification of the DC. The total number of PBMC that are administered often ranges from 108 to 1010. Generally, the cell doses injected into patients is based on the percentage of DC in the blood of each patient, as determined, for example, by immunofluorescence analysis with specific anti-DC antibodies. Thus, for example, if Progenipoietin™ mobilizes 2% DC in the peripheral blood of a given patient, and that patient is to receive 5×106 DC, then the patient will be injected with a total of 2.5×108 peptide-loaded PBMC. The percent DC mobilized by an agent such as Progenipoietin™ is typically estimated to be between 2-10%, but can vary as appreciated by one of skill in the art.
[0670] Ex vivo activation of CTL/HTL responses Alternatively, ex vivo CTL or HTL responses to 213P1F11 antigens can be induced by incubating, in tissue culture, the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of APC, such as DC, and immunogenic peptides. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cells, i.e., tumor cells.
Example 33 An Alternative Method of Identifying and Confirming Motif-Bearing Peptides[0671] Another method of identifying and confirming motif-bearing peptides is to elute them from cells bearing defined MHC molecules. For example, EBV transformed B cell lines used for tissue typing have been extensively characterized to determine which HLA molecules they express. In certain cases these cells express only a single type of HLA molecule. These cells can be transfected with nucleic acids that express the antigen of interest, e.g. 213P1F11. Peptides produced by endogenous antigen processing of peptides produced as a result of transfection will then bind to HLA molecules within the cell and be transported and displayed on the cell's surface. Peptides are then eluted from the HLA molecules by exposure to mild acid conditions and their amino acid sequence determined, e.g., by mass spectral analysis (e.g., Kubo et al., J. Immunol. 152:3913, 1994). Because the majority of peptides that bind a particular HLA molecule are motif-bearing, this is an alternative modality for obtaining the motif-bearing peptides correlated with the particular HLA molecule expressed on the cell.
[0672] Alternatively, cell lines that do not express endogenous HLA molecules can be transfected with an expression construct encoding a single HLA allele. These cells can then be used as described, i.e., they can then be transfected with nucleic acids that encode 213P1F11 to isolate peptides corresponding to 213P1F11 that have been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that correspond to binding to the single HLA allele that is expressed in the cell.
[0673] As appreciated by one in the art, one can perform a similar analysis on a cell bearing more than one HLA allele and subsequently determine peptides specific for each HLA allele expressed. Moreover, one of skill would also recognize that means other than transfection, such as loading with a protein antigen, can be used to provide a source of antigen to the cell.
Example 34 Complementary Polynucleotides[0674] Sequences complementary to the 213P1F11-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring 213P1F11. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06 software (National Biosciences) and the coding sequence of 213P1F11. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5′ sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to a 213P1F11-encoding transcript.
Example 35 Purification of Naturally-Occurring or Recombinant 213P1F11 Using 213P1F11-Specific Antibodies[0675] Naturally occurring or recombinant 213P1F11 is substantially purified by immunoaffinity chromatography using antibodies specific for 213P1F11. An immunoaffinity column is constructed by covalently coupling anti-213P1F11 antibody to an activated chromatographic resin, such as CNBr-activated SEPFIAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
[0676] Media containing 213P1F11 are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of 213P1F11 (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/213P1F11 binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and GCR.P is collected.
Example 36 Identification of Molecules Which Interact with 213P1F11[0677] 213P1F11, or biologically active fragments thereof, are labeled with 121 1 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled 213P1F11, washed, and any wells with labeled 213P1F11 complex are assayed. Data obtained using different concentrations of 213P1F11 are used to calculate values for the number, affinity, and association of 213P1F11 with the candidate molecules.
Example 37 In Vivo Assay for 213P1F11 Tumor Growth Promotion[0678] The effect of the 213P1F11 protein on tumor cell growth is evaluated in vivo by evaluating tumor development and growth of cells expressing or lacking 213P1F11. For example, SCID mice are injected subcutaneously on each flank with 1×106 of either prostate, bladder or breast cancer cell lines (such as PC3, DU145, UM-UC3, J82, MCF7) or N1H-3T3 cells containing tkNeo empty vector or 213P1F11. At least two strategies may be used: (1) Constitutive 213P1F11 expression under regulation of a promoter such as a constitutive promoter obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published Jul. 5, 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, provided such promoters are compatible with the host cell systems, and (2) Regulated expression under control of an inducible vector system, such as ecdysone, tet, etc., provided such promoters are compatible with the host cell systems. Tumor volume is then monitored at the appearance of palpable tumors and followed over time to determine if 213P1F11-expressing cells grow at a faster rate and whether tumors produced by 213P1F11-expressing cells demonstrate characteristics of altered aggressiveness (e.g. enhanced metastasis, vascularization, reduced responsiveness to chemotherapeutic drugs).
[0679] Additionally, mice can be implanted with 1×105 of the same cells orthotopically to determine if 213P1F11 has an effect on local growth in the prostate or on the ability of the cells to metastasize, specifically to lungs, lymph nodes, and bone marrow.
[0680] The assay is also useful to determine the 213P1F11 inhibitory effect of candidate therapeutic compositions, such as for example, 213P1F11 intrabodies, 213P1F11 antisense molecules and ribozymes.
Example 38 213P1F11 Monoclonal Antibody-mediated Inhibition of Tumors In Vivo[0681] The significant expression of 213P1F11 in cancer tissues, together with its restricted expression in normal tissues, makes 213P1F11 an excellent target for antibody therapy. In cases where the monoclonal antibody target is a cell surface protein, antibodies have been shown to be efficacious at inhibiting tumor growth (See, e.g., (Saffran, D., et al., PNAS 10: 1073-1078 or www.pnas.org/cgi/doi/10.1073/pnas.051624698). In cases where the target is not on the cell surface, such as for 213P1F11 and including PSA and PAP in prostate cancer, antibodies have still been shown to recognize and inhibit growth of cells expressing those proteins (Saffran, D. C., et al., Cancer and Metastasis Reviews, 1999. 18: p. 437-449). As with any cellular protein with a restricted expression profile, 213P1F11 is a target for T cell-based immunotherapy.
[0682] Accordingly, the therapeutic efficacy of anti-213P1F11 mAbs in human prostate, bladder and breast cancer mouse models is investigated using in 213P1F11-expressing prostate and bladder cancer xenografts as well as prostate, bladder and breast cancer cell lines, such as those described in the Example entitled “In Vivo Assay for 213P1F11 Tumor Growth Promotion,” that have been engineered to express 213P1F11.
[0683] Antibody efficacy on tumor growth and metastasis formation is confirmed, e.g., in a mouse orthotopic prostate or bladder cancer xenograft models, as well as SCID mice injected with prostate, bladder and breast cancer cell lines, such as those described in the Example entitled “In Vivo Assay for 213P1F11 Tumor Growth Promotion,” designed to express or lack 213P1F11. Therapeutic efficacy of anti-213P1F11 mAbs in prostate cancer is also evaluated in human prostate xenograft mouse models such as the LAPC-9 xenografts (Craft, N., et al.,. Cancer Res, 1999. 59(19): p. 5030-6). The antibodies can be unconjugated, as discussed in this Example, or can be conjugated to a therapeutic modality, as appreciated in the art. It is confirmed that anti-213P1F11 mabs inhibit formation of 213P1F11-expressing tumors. Anti-213P1F11 mAbs inhibit formation of the androgen-independent LAPC-9-AI tumor xenografts, as well as PC3-213P1F11, MCF7-213P1F11 and UM-UC3-213P1F11 tumors. Anti-213P1F11 mAbs also retard the growth of established orthotopic tumors and prolong survival of tumor-bearing mice. These results indicate the utility of anti-213P1F11 mAbs in the treatment of local and advanced stages of prostate, bladder or breast cancer. (See, e.g., Saffran, D., et al., PNAS 10:1073-1078 or www.pnas.org/cgi/doi/10. 1073/pnas.051624698)
[0684] Administration of anti-213P1F11 mAbs retard established orthotopic tumor growth and inhibit metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies indicate that 213P1F11 is an attractive target for immunotherapy and demonstrate the therapeutic potential of anti-213P1F11 mAbs for the treatment of local and metastatic bladder cancer.
[0685] This example demonstrates that unconjugated 213P1F11 monoclonal antibodies effectively to inhibit the growth of human prostate, bladder and breast tumors grown in SCID mice; accordingly a combination of such efficacious monoclonal antibodies is also effective.
[0686] Tumor Inhibition Using Multiple Unconjugated 213P1F11 mAbs
[0687] Materials and Methods
[0688] 213P1F11 Monoclonal Antibodies:
[0689] Monoclonal antibodies are raised against 213P1F11 as described in the Example entitled “Generation of 213P1F11 Monoclonal Antibodies (mAbs).” The antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation, in accordance with techniques known in the art, for their capacity to bind 213P1F11. Epitope mapping data for the anti-213P1F11 mAbs, as determined by ELISA and Western analysis, recognize epitopes on the 213P1F11 protein. Immunohistochemical analysis of bladder cancer tissues and cells with these antibodies is performed.
[0690] The monoclonal antibodies are purified from ascites or hybridoma tissue culture supernatants by Protein-G Sepharose chromatography, dialyzed against PBS, filter sterilized, and stored at −20° C. Protein determinations are performed by a Bradford assay (Bio-Rad, Hercules, Calif.). A therapeutic monoclonal antibody or a cocktail comprising a mixture of individual monoclonal antibodies is prepared and used for the treatment of mice receiving subcutaneous or orthotopic injections of bladder tumor xenografts.
[0691] Cancer Xenograft and Cell Lines
[0692] The LAPC-9 xenograft, which expresses a wild-type androgen receptor and produces prostate-specific antigen (PSA), is passaged in 6- to 8-week-old male ICR-severe combined immunodeficient (SCID) mice (Taconic Farns) by s.c. trocar implant (Craft, N., et al., supra). Prostate, bladder or breast cancer cell lines (such as PC3, DU145, UM-UC3, J82, MCF7) expressing 213P1F11 are generated by retroviral gene transfer as described in Hubert, R. S., et al., STEAP: a prostate-specific cell-surface antigen highly expressed in human prostate tumors. Proc Natl Acad Sci USA, 1999. 96(25):14523-8. Anti-213P1F11 staining is detected by using an FITC-conjugated goat anti-mouse antibody (Southern Biotechnology Associates) followed by analysis on a Coulter Epics-XL f low cytometer.
[0693] In Vivo Mouse Models.
[0694] Subcutaneous (s.c.) tumors are generated by injection of 1×106 213P1F11-expressing cancer cells mixed at a 1:1 dilution with Matrigel (Collaborative Research) in the right flank of male SCID mice. To test antibody efficacy on tumor formation, i.p. antibody injections are started on the same day as tumor-cell injections. As a control, mice are injected with either purified mouse IgG (ICN) or PBS; or a purified monoclonal antibody that recognizes an irrelevant antigen not expressed in human cells. In preliminary studies, no difference is found between mouse IgG or PBS on tumor growth. Tumor sizes are determined by vernier caliper measurements, and the tumor volume is calculated as length×width×height. Mice with s.c. tumors greater than 1.5 cm in diameter are sacrificed. Circulating levels of anti-213P1F11 Abs are determined by a capture ELISA kit (Bethyl Laboratories, Montgomery, Tex.). (See, e.g., (Saffran, D., et al., PNAS 10:1073-1078)
[0695] Orthotopic injections are performed, for example, in two alternative embodiments, under anesthesia by, for example, use of ketamine/xylazine. In a first embodiment, an intravesicular injection of bladder cancer cells is administered directly through the urethra and into the bladder (Peralta, E. A., et al., J. Urol., 1999. 162:1806-1811). In a second embodiment, an incision is made through the abdominal wall, the bladder is exposed, and bladder tumor tissue pieces (1-2 mm in size) derived from a s.c. tumor are surgically glued onto the exterior wall of the bladder, termed “onplantation” (Fu, X., et al., Int. J. Cancer, 1991. 49: 938-939; Chang, S., et al., Anticancer Res., 1997. 17: p.3239-3242). For prostate orthotopic studies, an incision is made through the abdominal muscles to expose the bladder and seminal vesicles, which then are delivered through the incision to the exposed the dorsal prostate. Antibodies can be administered to groups of mice at the time of tumor injection or onplantation, or after 1-2 weeks to allow tumor establishment.
[0696] Anti-213P1F11 mAbs Inhibit Growth of 213P1F11-Expressing Cancer Tumors
[0697] In one embodiment, the effect of anti-213P1F11 mAbs on tumor formation is tested by using the prostate and bladder orthotopic models. As compared with the s.c. tumor model, the orthotopic model, which requires surgical attachment of tumor tissue directly on the prostate or bladder, results in a local tumor growth, development of metastasis in distal sites, and subsequent death (Fu, X., et al., Int. J. Cancer, 1991. 49: p. 938-939; Chang, S., et al., Anticancer Res., 1997. 17: p. 3239-3242). This feature make the orthotopic model more representative of human disease progression and allows one to follow the therapeutic effect of mAbs, as well as other therapeutic modalities, on clinically relevant end points.
[0698] Accordingly, 213P1F11-expressing tumor cells are implanted orthotopically, and 2 days later, the mice are segregated into two groups and treated with either: a) 50-2000 &mgr;g, usually 200-500 &mgr;g, of anti-213P1F11 Ab, or b) PBS, three times per week for two to five weeks. Mice are monitored weekly for indications of tumor growth.
[0699] As noted, a major advantage of the orthotopic prostate and bladder cancer models is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is studied by histological analysis of tissue sections, including lung and lymph nodes (Fu, X., et al., Int. J. Cancer, 1991. 49:938-939; Chang, S., et al., Anticancer Res., 1997. 17:3239-3242). Additionally, 1HC analysis using anti-213P1F11 antibodies can be performed on the tissue sections.
[0700] Mice bearing established orthotopic 213P1F11-expressing tumors are administered 1000 &mgr;g injections of either anti-213P1F11 mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden (1-2 weeks growth), to ensure a high frequency of metastasis formation in mouse lungs and lymph nodes. Mice are then sacrificed and their local bladder tumor and lung and lymph node tissue are analyzed for the presence of tumor cells by histology and IHC analysis.
[0701] These studies demonstrate a broad anti-tumor efficacy of anti-213P1F11 antibodies on initiation and progression of bladder cancer in mouse models. Anti-213P1F11 antibodies inhibit tumor formation and retard the growth of already established tumors and prolong the survival of treated mice. Moreover, anti-213P1F11 mAbs demonstrate a dramatic inhibitory effect on the spread of local prostate, bladder and breast tumors to distal sites, even in the presence of a large tumor burden. Thus, anti-213P1F11 mAbs are efficacious on major clinically relevant end points including lessened tumor growth, lessened metastasis, and prolongation of survival.
Example 39 Therapeutic and Diagnostic use of Anti-213P1F11 Antibodies in Humans[0702] Anti-213P1F11 monoclonal antibodies are safely and effectively used for diagnostic, prophylactic, prognostic and/or therapeutic purposes in humans. Western blot and immunohistochemical analysis of cancer tissues and cancer xenografts with anti-213P1F11 mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. Detection of 213P1F11 in carcinoma and in metastatic disease demonstrates the usefulness of the mAb as a diagnostic and/or prognostic indicator. Anti-213P1F11 antibodies are therefore used in diagnostic applications such as immunohistochemistry of kidney biopsy specimens to detect cancer from suspect patients.
[0703] As determined by flow cytometry, anti-213P1F11 mAb specifically binds to carcinoma cells. Thus, anti-213P1F11 antibodies are used in diagnostic whole body imaging applications, such as radioinmrunoscintigraphy and radioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res 20(2A):925-948 (2000)) for the detection of localized and metastatic cancers that exhibit expression of 213P1F11. Shedding or release of an extracellular domain of 213P1F11 into the extracellular milieu, such as that seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology 27:563-568 (1998)), allows diagnostic detection of 213P1F11 by anti-213P1F11 antibodies in serum and/or urine samples from suspect patients.
[0704] Anti-213P1F11 antibodies that specifically bind 213P1F11 are used in therapeutic applications for the treatment of cancers that express 213P1F11. Anti-213P1F11 antibodies are used as an unconjugated modality and as conjugated form in which the antibodies are attached to one of various therapeutic or imaging modalities well known in the art, such as a prodrugs, enzymes or radioisotopes. In preclinical studies, unconjugated and conjugated anti-213P1F11 antibodies are tested for efficacy of tumor prevention and growth inhibition in the SCID mouse cancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6, (see, e.g., the Example entitled “213P1F11 Monoclonal Antibody-mediated Inhibition of Bladder and Lung Tumors In Vivo”). Conjugated and unconjugated anti-213P1F11 antibodies are used as a therapeutic modality in human clinical trials either alone or in combination with other treatments as described in following Examples.
Example 40 Human Clinical Trials for the Treatment and Diagnosis of Human Carcinomas through use of Human Anti-213P1F11 Antibodies In Vivo[0705] Antibodies are used in accordance with the present invention which recognize an epitope on 213P1F11, and are used in the treatment of certain tumors such as those listed in Table I. Based upon a number of factors, including 213P1F11 expression levels, tumors such as those listed in Table I are presently preferred indications. In connection with each of these indications, three clinical approaches are successfully pursued.
[0706] I.) Adjunctive therapy: In adjunctive therapy, patients are treated with anti-213P1F11 antibodies in combination with a chemotherapeutic or antineoplastic agent and/or radiation therapy. Primary cancer targets, such as those listed in Table I, are treated under standard protocols by the addition anti-213P1F11 antibodies to standard first and second line therapy. Protocol designs address effectiveness as assessed by reduction in tumor mass as well as the ability to reduce usual doses of standard chemotherapy. These dosage reductions allow additional and/or prolonged therapy by reducing dose-related toxicity of the chemotherapeutic agent. Anti-213P1F11 antibodies are utilized in several adjunctive clinical trials in combination with the chemotherapeutic or antineoplastic agents adriamycin (advanced prostrate carcinoma), cisplatin (advanced head and neck and lung carcinomas), taxol (breast cancer), and doxorubicin (preclinical).
[0707] II.) Monotherapy: In connection with the use of the anti-213P1F11 antibodies in monotherapy of tumors, the antibodies are administered to patients without a chemotherapeutic or antineoplastic agent. In one embodiment, monotherapy is conducted clinically in end stage cancer patients with extensive metastatic disease. Patients show some disease stabilization. Trials demonstrate an effect in refractory patients with cancerous tumors.
[0708] III.) Imaging Agent: Through binding a radionuclide (e.g., iodine or yttrium (I131, Y90) to anti-213P1F11 antibodies, the radiolabeled antibodies are utilized as a diagnostic and/or imaging agent. In such a role, the labeled antibodies localize to both solid tumors, as well as, metastatic lesions of cells expressing 213P1F11. In connection with the use of the anti-213P1F11 antibodies as imaging agents, the antibodies are used as an adjunct to surgical treatment of solid tumors, as both a pre-surgical screen as well as a post-operative follow-up to determine what tumor remains and/or returns. In one embodiment, a (111In)-213P1F11 antibody is used as an imaging agent in a Phase I human clinical trial in patients having a carcinoma that expresses 213P1F11 (by analogy see, e.g., Divgi et al. J. Natl Cancer Inst. 83:97-104 (1991)). Patients are followed with standard anterior and posterior gamma camera. The results indicate that primary lesions and metastatic lesions are identified
[0709] Dose and Route of Administration
[0710] As appreciated by those of ordinary skill in the art, dosing considerations can be determined through comparison with the analogous products that are in the clinic. Thus, anti-213P1F11 antibodies can be administered with doses in the range of 5 to 400 mg/m2, with the lower doses used, e.g., in connection with safety studies. The affinity of anti-213P1F11 antibodies relative to the affinity of a known antibody for its target is one parameter used by those of skill in the art for determining analogous dose regimens. Further, anti-213P1F11 antibodies that are fully human antibodies, as compared to the chimeric antibody, have slower clearance; accordingly, dosing in patients with such fully human anti-213P1F11 antibodies can be lower, perhaps in the range of 50 to 300 mg/m 2, and still remain efficacious. Dosing in mg/m2, as opposed to the conventional measurement of dose in mg/kg, is a measurement based on surface area and is a convenient dosing measurement that is designed to include patients of all sizes from infants to adults.
[0711] Three distinct delivery approaches are useful for delivery of anti-213P1F11 antibodies. Conventional intravenous delivery is one standard delivery technique for many tumors. However, in connection with tumors in the peritoneal cavity, such as tumors of the ovaries, biliary duct, other ducts, and the like, intraperitoneal administration may prove favorable for obtaining high dose of antibody at the tumor and to also minimize antibody clearance. In a similar manner, certain solid tumors possess vasculature that is appropriate for regional perfusion. Regional perfusion allows for a high dose of antibody at the site of a tumor and minimizes short term clearance of the antibody.
[0712] Clinical Development Plan (CDP)
[0713] Overview: The CDP follows and develops treatments of anti-213P1F11 antibodies in connection with adjunctive therapy, monotherapy, and as an imaging agent. Trials initially demonstrate safety and thereafter confirm efficacy in repeat doses. Trails are open label comparing standard chemotherapy with standard therapy plus anti-213P1F11 antibodies. As will be appreciated, one criteria that can be utilized in connection with enrollment of patients is 213P1F11 expression levels in their tumors as determined by biopsy.
[0714] As with any protein or antibody infusion-based therapeutic, safety concerns are related primarily to (i) cytokine release syndrome, i.e., hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the antibody therapeutic, or HAMA response); and, (iii) toxicity to normal cells that express 213P1F11. Standard tests and follow-up are utilized to monitor each of these safety concerns. Anti-213P1F11 antibodies are found to be safe upon human administration.
Example 41 Human Clinical Trial Adjunctive Therapy with Human Anti-213P1F11 Antibody and Chemotherapeutic Agent[0715] A phase I human clinical trial is initiated to assess the safety of six intravenous doses of a human anti-213P1F11 antibody in connection with the treatment of a solid tumor, e.g., a cancer of a tissue listed in Table I. In the study, the safety of single doses of anti-213P1F11 antibodies when utilized as an adjunctive therapy to an antineoplastic or chemotherapeutic agent, such as cisplatin, topotecan, doxorubicin, adriamycin, taxol, or the like, is assessed. The trial design includes delivery of six single doses of an anti-213P1F11 antibody with dosage of antibody escalating from approximately about 25 mg/m2 to about 275 mg/2 over the course of the treatment in accordance with the following schedule: 3 Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 mAb Dose 25 75 125 175 225 275 mg/m2 mg/m2 mg/m2 mg/m2 mg/m2 mg/m2 Chemotherapy + + + + + + (standard dose)
[0716] Patients are closely followed for one-week following each administration of antibody and chemotherapy. In particular, patients are assessed for the safety concerns mentioned above: (i) cytokine release syndrome, i.e., hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the human antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express 213P1F11. Standard tests and follow-up are utilized to monitor each of these safety concerns. Patients are also assessed for clinical outcome, and particularly reduction in tumor mass as evidenced by MRI or other imaging.
[0717] The anti-213P1F11 antibodies are demonstrated to be safe and efficacious, Phase II trials confirm the efficacy and refine optimum dosing.
Example 42 Human Clinical Trial: Monotherapy with Human Anti-213P1F11 Antibody[0718] Anti-213P11F11 antibodies are safe in connection with the above-discussed adjunctive trial, a Phase II human clinical trial confirms the efficacy and optimum dosing for monotherapy. Such trial is accomplished, and entails the same safety and outcome analyses, to the above-described adjunctive trial with the exception being that patients do not receive chemotherapy concurrently with the receipt of doses of anti-213P1F11 antibodies.
Example 43 Human Clinical Trial: Diagnostic Imaging with Anti-213P1F11 Antibody[0719] Once again, as the adjunctive therapy discussed above is safe within the safety criteria discussed above, a human clinical trial is conducted concerning the use of anti-213P1F11 antibodies as a diagnostic imaging agent. The protocol is designed in a substantially similar manner to those described in the art, such as in Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991). The antibodies are found to be both safe and efficacious when used as a diagnostic modality.
Example 44 Homology Comparison of 213P1F11 to Known Sequences[0720] The 213P1F11 gene is homologous to a previously cloned gene, namely the human caspase 14 precursor (gi 6912286) (Hu S et al, J. Biol. Chem. 1998, 273:29648), also known as mini-ICE (MICE). The 213P1F11 gene resulted in several protein variants, which share several characteristics (Table XXII), including homology to ICE family of cysteine proteases. Several variants of 213P1F11, namely 213P1F11-v.2, -v.3 and -v.4, are novel proteins that maintain some homology to the published caspase 14 precursor (gi 6912286). For example, 213P1F11-v.2 shows 100% identity to the human caspase 14 precursor (gi 6912286) over the first 174 aa of the protein (FIG. 4D), while differing from the published caspase 14 precursor protein by 56 amino acids at its C-terminus, thus resulting in 76% overall identity to caspase 14 precursor. 213P1F11-v.2 also maintains homology to the mouse caspase 14, and shows 83% homology and 72% identity to that protein (gi 6753280) (FIG. 4F). The 213P1F11-v.3 variant protein show 100% identity to the human caspase 14 precursor (gi 6912286) over 134 amino acids, while differing from that protein by 12 aa at its C-terminus. Similarly, 213P1F11-v.4 shows 97% identity with the human caspase 14 precursor over 235 amino acids, while differing from the human caspase 14 precursor (gi 6912286) by 86 aa at its N-terminus (FIG. 4G). 213P1F11-v.1 consists of 242 amino acids, with calculated molecular weight of 28.0 kDa, and pI of 5.4. 213P1F11-v.1 is an intercellular protein, located in the cytosol with potential localization to the nucleus (Table XXII). Similar localization patterns are observed for 213P1F11 protein variants 1, 3, and 4 (Table XXII). Bioinformatic analysis indicates that 213P1F11-v.2 may also localize to the mitochondria (Table XXII).
[0721] Caspases are a family of cyteine proteases that function as effectors of apoptosis or programmed cell death (Salvesen G S, Dixit V, Cell. 1997, 91:443; Thomberry N, Lazebnik Y, Science. 281: 1312). These proteases cleave different cellular substrates in an aspartate-specific manner. Cleavage may result in activation or inactivation of the cleaved cellular proteins, but not in protein degradation (Nunez et al, Oncogene. 1998, 17:3237; Stennicke H R, Salvesen G S, Cell Death Differ 1999, 6:1054). Caspases traditionally exist as precursor proteins also known as single polypeptide zymogens consisting of a pro-domain, and 2 catalytic subunits, p20 and p1O and contain a conserved QACXG active site (Stennicke H R, Salvesen G S, Cell Death Differ 1999, 6:1054; Cohen M. Biochem J 1997, 326:1). Similar to other members of the caspase family, 213P1F11 contains two catalytic subunits, p20 and p10, in addition to the conserved penta-peptide active site. In 213P1F11 v.1, p20 spans aa 16-139 and p10 spans aa 155-241, while the active site is located at aa 129. Similarly, 213P1F11 v.4 carries both p20 and p10 subunits, while 213P1F11 v.2 and v.3 contain the p20 subunit only, indicating that all 4 variants of 213P1F11 can function in a similar manner. Caspases are activated by proteolytic cleavage of their internal aspartate by an upstream enzyme, often another caspase. However, unlike other caspases with short pro-domains, caspase 14 is not reported to associate with known caspases (Hu S et al, J. Biol. Chem. 1998, 273:29648). Caspase 14 has been shown to be processed by caspase 8 and caspase 10 as well as granzyme B, resulting in two catalytic subunits, p20 and p10 (Ahmad M et al, Cancer Res. 1998, 58:5201). These 2 cleavage products are detected in human epidermis and in vitro during keratinocyte differentiation (Eckhart L et al, J. Invest. Drmatol. 2000, 115:1148). Overexpression of caspase 14 in breast carcinoma cells MCF7 resulted in the apoptosis of these cells, suggesting that caspase 14 participates in the process of programmed cell death (Hu S et al, J. Biol. Chem. 1998, 273:29648).
[0722] Our findings that 213P1F11 is highly expressed in several cancers while showing a restricted expression pattern in normal tissues suggests that the 213P1F11 gene plays an important role in various cancers, including cancers of the prostate, bladder and breast. Based on its similarity to caspase 14 213P1F11 has the ability to control tumor growth, apoptosis, survival, differentiation and progression. Accordingly, when 213P1F11 functions as a regulator of cell growth and apoptosis, or expression, 213P1F11 is used for therapeutic, diagnostic, prognostic or preventative purposes.
[0723] Our findings that 213P1F11 is highly expressed in several cancers while showing a restricted expression pattern in normal tissues suggests that the 213P1F11 gene plays an important role in various cancers, including cancers of the prostate, bladder and breast. Based on its similarity to caspase 14 213P1F11 has the ability to control tumor growth, apoptosis, survival, differentiation and progression. Accordingly, when 213P1F11 functions as a regulator of cell growth and apoptosis, or expression, 213P1F11 is used for therapeutic, diagnostic, prognostic or preventative purposes.
Example 45 Identification and Confirmation of Signal Transduction Pathways[0724] Many mammalian proteins have been reported to interact with signaling molecules and to participate in regulating signaling pathways. (J. Neurochem. 2001; 76:217-223). Caspases participate in signal transduction processes by getting recruited to signaling complexes and cleaving specific cellular substrates including other caspases and structural proteins, ultimately resulting in morphologic changes that represent the hallmark of apoptosis (Cohen G M. Biochem J. 1997, 326:1). Recent studies have demonstrated that caspases also cleave signaling molecules, such as the guanine nucleotide exchange factor TIAM1, leading to the inactivation of TIAMI and thereby the Rae cascade (Qi H et al, Cell Growth Differ. 2001, 12:603). Using iimmunoprecipitation and Western blotting techniques, proteins are identified that associate with 213P1F11 and mediate signaling events. Several pathways known to play a role in cancer biology can be regulated by 213P1F11, including phospholipid pathways such as P13K, survival pathways such as AKT, NFkB, etc, adhesion and migration pathways, including FAK, Rho, Rac-1, etc, as well as mitogenic/survival cascades such as ERK, p38, etc (Cell Growth Differ. 2000,11:279; J. Biol. Chem. 1999, 274:801; Oncogene. 2000, 19:3003, J. Cell Biol. 1997, 138:913.). Bioinformatic analysis revealed that 213P1F11 can become phosphorylated by serine/threonine as well as tyrosine kinases. Thus, the phosphorylation of 213P1F11 is provided by the present invention to lead to activation of the above listed pathways.
[0725] Using, e.g., Western blotting techniques the ability of 213P F11 to regulate these pathways is confirmed. Cells expressing or lacking 213P1F11 are either left untreated or stimulated with cytokines, hormones and anti-integrin antibodies. Cell lysates are analyzed using anti-phospho-specific antibodies (Cell Signaling, Santa Cruz Biotechnology) in order to detect phosphorylation and regulation of ERK, p38, AKT, P13K, PLC and other signaling molecules. When 213P1F11 plays a role in the regulation of signaling pathways, whether individually or communally, it is used as a target for diagnostic, prognostic, preventative and therapeutic purposes.
[0726] To confirm that 213P1F11 directly or indirectly activates known signal transduction pathways in cells, luciferase (luc) based transcriptional reporter assays are carried out in cells expressing individual genes. These transcriptional reporters contain consensus-binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways. The reporters and examples of these associated transcription factors, signal transduction pathways, and activation stimuli are listed below.
[0727] NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress
[0728] SRE-luc, SRF/TCF/ELKI; MAPK/SAPK; growth/differentiation
[0729] AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress
[0730] ARE-luc, androgen receptor; steroids/MAPK; growth/differentiation/apoptosis
[0731] p53-luc, p53; SAPK; growth/differentiation/apoptosis
[0732] CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress
[0733] Gene-mediated effects can be assayed in cells showing mRNA expression. Luciferase reporter plasmids can be introduced by lipid-mediated transfection (TFX-50, Promega). Luciferase activity, an indicator of relative transcriptional activity, is measured by incubation of cell extracts with luciferin substrate and luminescence of the reaction is monitored in a luminometer.
[0734] Signaling pathways activated by 213P1F11 are mapped and used for the identification and validation of therapeutic targets. When 213P1F11 is involved in cell signaling, it is used as target for diagnostic, prognostic, preventative and therapeutic purposes.
Example 46 Involvement in Tumor Progression[0735] Some apoptosis intermediates, such as DcRI, FLICE and TRAIL-R3, function as cellular inhibitors of apoptosis by acting as decoys and interfering with normal function of the apoptotic machinery (Sheikh M S et al, Oncogene. 1999, 18:4153; Ashkenazi A, Dixit V M. Curr Opin Cell Biol. 1999, 11:255). When 213P1F11 functions as a decoy, it can contribute to the growth of cancer cells. The role of 213P1F11 in tumor growth is confirmed in a variety of primary and transfected cell lines including prostate, bladder and breast cell lines as well as NIH 3T3 cells engineered to stably express 213P1F11. Parental cells lacking 213P1F11 and cells expressing 213P1F11 are evaluated for cell growth using a well-documented proliferation assay (Fraser S P, Grimes J A, Djamgoz M B. Prostate. 2000;44:61, Johnson D E, Ochieng J, Evans S L. Anticancer Drugs. 1996, 7:288).
[0736] To confirm the role of 213P1F11 in the transformation process, its effect in colony forming assays is investigated. Parental NIH3T3 cells lacking 213P1F11 are compared to NHI-3T3 cells expressing 213P1F11, using a soft agar assay under stringent and more permissive conditions (Song Z. et al. Cancer Res. 2000, 60:6730).
[0737] To confirm the role of 213P1F11 in invasion and metastasis of cancer cells, a well-established assay is used, e.g., a Transwell Insert System assay (Becton Dickinson) (Cancer Res. 1999, 59:6010). Control cells. including prostate, colon, bladder and kidney cell lines lacking 213P1F11 are compared to cells expressing 213P1F11. Cells are loaded with the fluorescent dye, calcein, and plated in the top well of the Transwell insert coated with a basement membrane analog. Invasion is determined by fluorescence of cells in the lower chamber relative to the fluorescence of the entire cell population.
[0738] 213P1F11 can also play a role in cell cycle and apoptosis. Parental cells and cells expressing 213P1F11 are compared for differences in cell cycle regulation using a well-established BrdU assay (Abdel-Malek ZA. J Cell Physiol. 1988, 136:247). In short, cells are grown under both optimal (full serum) and limiting (low serum) conditions are labeled with BrdU and stained with anti-BrdU Ab and propidium iodide. Cells are analyzed for entry into the G1, S, and G2M phases of the cell cycle. Alternatively, the effect of stress on apoptosis is evaluated in control parental cells and cells expressing 213P1F11, including normal and tumor bladder cells. Engineered and parental cells are treated with various chemotherapeutic agents, such as paclitaxel, gemcitabine, etc, and protein synthesis inhibitors, such as cycloheximide. Cells are stained with annexin V-FITC and cell death is measured by FACS analysis. The modulation of cell death by 213P1F11 can play a critical role in regulating tumor progression and tumor load.
[0739] When 213P1F11 plays a role in cell growth, transformation, invasion or apoptosis, it is used as a target for diagnostic, prognostic, preventative and therapeutic purposes.
Example 47 Involvement in Angiogenesis[0740] Angiogenesis or new capillary blood vessel formation is necessary for tumor growth (Hanahan D, Folkman J. Cell. 1996, 86:353; Folkman J. Endocrinology. 1998 139:441). Several assays have been developed to measure angiogenesis in vitro and in vivo, such as the tissue culture assays, endothelial cell tube formation, and endothelial cell proliferation. Using these assays as well as in vitro neo-vascularization, the effect of 213P1F11 on angiogenesis is confirmed. For example, endothelial cells engineered to express 213P1F11 are evaluated using tube formation and proliferation assays. The effect of 213P1F11 is also confirmed in animal models in vivo. For example, cells either expressing or lacking 213P1F11 are implanted subcutaneously in immunocompromised mice. Endothelial cell migration and angiogenesis are evaluated 5-15 days later using immunohistochemistry techniques. When 213P1F11 affects angiogenesis, it is used as a target for diagnostic, prognostic, preventative and therapeutic purposes
Example 48 Regulation of Transcription[0741] The localization of 213P1F11 to the cytoplasm with potential nuclear localization (Table XXII), support the present invention use of 213P1F11 based on its role in the transcriptional regulation of eukaryotic genes. Regulation of gene expression is confirmed, e.g., by studying gene expression in cells expressing or lacking 213P1F11. For this purpose, two types of experiments are performed.
[0742] In the first set of experiments, RNA from parental and 213P1F11-expressing cells are extracted and hybridized to commercially available gene arrays (Clontech) (Smid-Koopman E et al. Br J Cancer. 2000. 83:246). Resting cells as well as cells treated with FBS or androgen are compared. Differentially expressed genes are identified in accordance with procedures known in the art. The differentially expressed genes are then mapped to biological pathways (Chen K et al., Thyroid. 2001. 11:41.).
[0743] In the second set of experiments, specific transcriptional pathway activation is evaluated using commercially available (Stratagene) luciferase reporter constructs including: NFkB-luc, SRE-luc, ELK1-luc, ARE-luc, p53-luc, and CRE-luc. These transcriptional reporters contain consensus binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways, and represent a good tool to ascertain pathway activation and screen for positive and negative modulators of pathway activation.
[0744] When 213P1F11 plays a role in gene regulation, it is used as a target for diagnostic, prognostic, preventative and therapeutic purposes.
Example 49 Subcellular Localization of 213P1F11[0745] The cellular location of 213P1F11 can be assessed using subcellular fractionation techniques widely used in cellular biology (Storrie B, et al. Methods Enzymol. 1990;182:203-25). A variety of cell lines, including prostate, bladder and breast cell lines as well as cell lines engineered to express 213P1F11 are separated into nuclear, cytosolic and membrane fractions. Gene expression and location in nuclei, heavy membranes (lysosomes, peroxisomes, and mitochondria), light membranes (plasma membrane and endoplasmic reticulum), and soluble protein fractions are tested using Western blotting techniques.
[0746] Alternatively, 293T cells can be transfected with an expression vector encoding individual genes, HIS-tagged (PcDNA 3.1 MYC/HIS, Invitrogen) and the subcellular localization of these genes is determined as described above. In short, the transfected cells can be harvested and subjected to a differential subcellular fractionation protocol (Pemberton, P. A. et al, 1997, J of Histochemistry and Cytochemistry, 45:1697-1706.). Location of the HIS-tagged genes is followed by Western blotting.
[0747] Using 213P1F11 antibodies, it is possible to demonstrate cellular localization by irumunofluorescence and immunohistochemistry. For example, cells expressing or lacking 213P1F11 are adhered to a microscope slide and stained with anti-213P1F11 specific Ab. Cells are incubated with an FITC-coupled secondary anti-species Ab, and analyzed by fluorescent microscopy.
[0748] When 213P1F11 is localized to specific cell compartments, it is used as a target for diagnostic, preventative and therapeutic purposes.
Example 50 213P1F11 Proteolytic Activity[0749] The similarity of 213P1F11 to casapase cysteine proteases supports the use of 213P1F11 as a protease. Protease activity can be confirmed using on in vitro protease assay coupled to detection of protein fragments by western blotting (Hu S et al, above; Slee E et al, J. Biol. Chem. 2001, 276:7320). In one embodiment, recombinant 213P1F11 protein is incubated with apoptotic substrates, including other caspases known to associate with caspase 14, namely caspase 2 and caspase 4, as well as recombinant RIP and poly(ADP-ribose) polymerase (i.e. PARP) (Slee E et al, J. Biol. Chem. 2001, 276:7320; Hayakawa et al, Apoptosis. 2002, 7:107). Proteins are separated by SDS-Page and analyzed by western blotting with substrate specific antibodies. In another embodiment, 213P1F11 activity is compared in control cells lacking 213P1F11 and cells expressing 213P1F11. Cell lysates from control and 213P1F11 expressing cells are incubated in the presence of the recombinant substrates listed above. Whole proteins are analyzed by western blotting with antibodies directed to the apoptotic substrates.
[0750] When 213P1F11 functions as a protease, it is used as a target for diagnostic, preventative and therapeutic purposes Throughout this application, various website data content, publications, patent applications and patents are referenced. (Websites are referenced by their Uniform Resource Locator, or URL, addresses on the World Wide Web.) The disclosures of each of these references are hereby incorporated by reference herein in their entireties.
[0751] The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention. 4 TABLE I Tissues that Express 213P1F11 When Malignant Bladder Prostate Breast
[0752] 5 TABLE II Amino Acid Abbreviations SINGLE LETTER THREE LETTER FULL NAME F Phe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cys cysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamine R Arg arginine I Ile isoleucine M Met methiomne T Thr threonine N Asn asparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid E Glu glutamic acid G Gly glycine
[0753] 6 TABLE III AMINO ACID SUBSTITUTION MATRIX Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix). The higher the value, the more likely a substitution is found in related, natural proteins. (See URL www.ikp.unibe.ch/manual/blosum62.html) A C D E F G H I K L M N P Q R S T V W Y . 4 0 −2 −1 −2 0 −2 −1 −1 −1 −1 −2 −1 −1 −1 1 0 0 −3 −2 A 9 −3 −4 −2 −3 −3 −1 −3 −1 −1 −3 −3 −3 −3 −1 −1 −1 −2 −2 C 6 2 −3 −1 −1 −3 −1 −4 −3 1 −1 0 −2 0 −1 −3 −4 −3 D 5 −3 −2 0 −3 1 −3 −2 0 −1 2 0 0 −1 −2 −3 −2 E 6 −3 −1 0 −3 0 0 −3 −4 −3 −3 −2 −2 −1 1 3 F 6 −2 −4 −2 −4 −3 0 −2 −2 −2 0 −2 −3 −2 −3 G 8 −3 −1 −3 −2 1 −2 0 0 −1 −2 −3 −2 2 H 4 −3 2 1 −3 −3 −3 −3 −2 −1 3 −3 −1 I 5 −2 −1 0 −1 1 2 0 −1 −2 −3 −2 K 4 2 −3 −3 −2 −2 −2 −1 1 −2 −1 L 5 −2 −2 0 −1 −1 −1 1 −1 −1 M 6 −2 0 0 1 0 −3 −4 −2 N 7 −1 −2 −1 −1 −2 −4 −3 P 5 1 0 −1 −2 −2 −1 Q 5 −1 −1 −3 −3 −2 R 4 1 −2 −3 −2 S 5 0 −2 −2 T 4 −3 −1 V 11 2 W 7 Y
[0754] 7 TABLE IV HLA Class I/II Motifs/Supermotifs TABLE IV (A): HLA Class I Supermotifs/Motifs POSITION POSITION POSITION 2 (Primary Anchor) 3 (Primary Anchor) C Terminus (Primary Anchor) SUPERMOTIFS A1 TILVMS FWY A2 LIVMATQ IVMATL A3 VSMATLI RK A24 YFWIVLMT FIYWLM B7 P VILFMWYA B27 RHK FYLWMIVA B44 ED FWYLIMVA B58 ATS FWVLIVMA B62 QLIVMP FWYMIVLA MOTIFS A1 TSM Y A1 DEAS Y A2.1 LMVQIAT VLIMAT A3 LMVISATFCGD KYRHFA A11 VTMLISAGNCDF KRYH A24 YFWM FLIW A*3101 MVTALIS RK A*3301 MVALFIST RK A*6801 AVTMSLI RK B*0702 P LMFWYAIV B*3501 P LMFWYIVA B51 P LIVFWYAM B*5301 P IMFWYALV B*5401 P ATIVLMFWY Bolded residues are preferred, italicized residues are less preferred: A peptide is considered motif-bearing if it has primary anchors at each primary anchor position for a motif or supermotif as specified in the above table. TABLE IV (B): HLA Class II Supermotif 1 6 9 W, F, Y, V, .I, L A, V, I, L, P, C, S, T A, V, I, L, C, S, T, M, Y TABLE IV (C): HLA Class II Motifs MOTIFS 1° anchor 1 2 3 4 5 1° anchor 6 7 8 9 DR4 preferred FMYLIVW M T I VSTCPALIM MH MH deleterious W R WDE DR1 preferred MFLIVWY PAMQ VMATSPLIC M AVM deleterious C CH FD CWD GDE D DR7 preferred MFLIVWY M W A IVMSACTPL M IV deleterious C G GRD N G DR3 MOTIFS 1° anchor 1 2 3 1° anchor 4 5 1° anchor 6 motif a preferred LIVMFY D motif b preferred LIVMFAY DNQEST KRH DR MFLIVWY VMSTACPLI Supermotif Italicized residues indicate less preferred or “tolerated” residues. TABLE IV (D): HLA Class I Supermotifs POSITION: SUPERMOTIFS 1 2 3 4 5 6 7 8 C-terminus A1 1° Anchor 1° Anchor TILVMS FWY A2 1° Anchor 1° Anchor LIVMATQ LIVMAT A3 preferred 1° Anchor YFW (4/5) YFW (3/5) YFW (4/5) P (4/5) 1° Anchor VSMATLI RK delete- DE (3/5); DE (4/5) rious P (5/5) A24 1° Anchor 1° Anchor YFWIVLMT FIYWLM B7 preferred FWY (5/5) 1° Anchor FWY (4/5) FWY (3/5) 1° Anchor LIVM (3/5) P VILFMWYA delete- DE (3/5); DE (3/5) G (4/5) QN (4/5) DE (4/5) rious P (5/5); G (4/5); A (3/5); QN (3/5) B27 1° Anchor 1° Anchor RHK FYLWMIVA B44 1° Anchor 1° Anchor ED FWYLIMVA B58 1° Anchor 1° Anchor ATS FWYLIVMA B62 1° Anchor 1° Anchor QLIVMP FWYMIVLA TABLE IV (E): HLA Class I Motifs POSITION: C- 1 2 3 4 5 6 7 8 9 terminus or C-terminus A1 preferred GFY 1° Anchor DEA YFW P DEQN YFW 1° Anchor 9-mer W STM Y deleterious DE RHKLIVMP A G A A1 preferred GRHK ASTCLIVM 1° Anchor GSTC ASTC LIVM DE 1° Anchor 9-mer DEAS Y deleterious A RHKDEPY DE PQN RHK PG GP FW A1 preferred YFW 1° Anchor DEAQN A YFWQN PASTC GDE P 1° Anchor 10-mer STM Y deleterious GP RHKGLIVM DE RHK QNA RHKYFW RHK A A1 preferred YFW STCLIVM 1° Anchor A YFW PG G YFW 1° Anchor 10-mer DEAS Y deleterious RHK RHKDEPY P G PRHK QN FW A2.1 preferred YFW 1° Anchor YFW STC YFW A P 1° Anchor 9-mer {overscore (LMIVQAT)} VLIMAT deleterious DEP DERKH RKH DERKH POSITION: C- 1 2 3 4 5 6 7 8 9 Terminus A2.1 preferred AYFW 1° Anchor LVIM G G FYWL 1° Anchor 10-mer LMIVQA VIM VLIMAT T deleterious DEP DE RKHA P RKH DERKH RKH A3 preferred RHK 1° Anchor YFW PRHKYFW A YFW P 1° Anchor LMVISA KYRHFA TFCGD deleterious DEP DE A11 preferred A 1° Anchor YFW YFW A YFW YFW P 1° Anchor VTLMIS KRYH AGNCDF deleterious DEP A G A24 preferred YFWRHK 1° Anchor STC YFW YFW 1° Anchor 9-mer YFWM FLIW deleterious DEG DE G QNP DERH G AQN K A24 preferred 1° Anchor P YFWP P 1° Anchor 10-mer YFWM FLIW deleterious GDE QN RHK DE A QN DEA A3101 preferred RHK 1° Anchor YFW P YFW YFW AP 1° Anchor {overscore (MVTALIS)} RK deleterious DEP DE ADE DE DE DE A3301 preferred 1° Anchor YFW AYFW 1° Anchor MVALFI RK ST deleterious GP DE POSITION: C- 1 2 3 4 5 6 7 8 9 Terminus A6801 preferred YFWSTC 1° Anchor YFWLIV YFW P 1° Anchor {overscore (AVTMSLI)} M RK deleterious GP DEG RHK A B0702 preferred RHKFW 1° Anchor RHK RHK RHK RHK PA 1° Anchor Y P {overscore (LMFWYAIV)} deleterious DEQNP DEP DE DE GDE QN DE B3501 preferred FWYLIV 1° Anchor FWY FWY 1° Anchor M P {overscore (LMFWYIV)} A deleterious AGP G G B51 preferred LIVMFW 1° Anchor FWY STC FWY G FWY 1° Anchor Y P {overscore (LIVFWYAM)} deleterious AGPDER DE G DEQN GDE HKSTC B5301 preferred LIVMFW 1° Anchor FWY STC FWY LIVMFWY FWY 1° Anchor Y P {overscore (IMFWYAL)} V deleterious AGPQN G RHKQN DE B5401 preferred FWY 1° Anchor FWYL LIVM ALIVM FWYAP 1° Anchor P IVM {overscore (ATIVLMF)} WY deleterious GPQNDE GDES RHKDE DE QNDGE DE TC Italicized residues indicate less preferred or “tolerated” residues. The information in this Table is specific for 9-mers unless otherwise specified.
[0755] 8 TABLE V Pos 123456789 Score SeqID v.1-A1-9 mers: 213P1F11 96 KGEDGEMVK 22.500 215 EAELVQEGK 18.000 171 TVEGYIAYR 18.000 191 LVDVFTKRK 10.000 170 STVEGYIAY 6.250 35 DLDALEHMF 5.000 75 REDPVSCAF 2.500 53 KRDPTAEQF 2.500 162 YTDALHVYS 2.500 189 QTLVDVFTK 2.500 228 NPEIQSTLR 2.250 206 LTEVTRRMA 2.250 6 SLEEEKYDM 1.800 145 GDEIVMVIK 1.800 154 DSPQTIPTY 1.500 31 GSEEDLDAL 1.350 99 DGEMVKLEN 1.125 104 KLENLFEAL 0.900 57 TAEQFQEEL 0.900 202 ILELLTEVT 0.900 38 ALEHMFRQL 0.900 232 QSTLRKRLY 0.750 144 GGDEIVMVI 0.625 167 HVYSTVEGY 0.500 71 AIDSREDPV 0.500 19 LALILCVTK 0.400 218 LVQEGKARK 0.400 136 QRDPGETVG 0.250 179 RHDQKGSCF 0.250 157 QTIPTYTDA 0.250 226 KTNPEIQST 0.250 62 QEELEKFQQ 0.225 175 YIAYRHDQK 0.200 125 KVYITQACR 0.200 107 ILFEALNNK 0.200 213 MAEAELVQE 0.180 59 EQFQEELEK 0.150 219 VQEGKARKT 0.135 61 FQEELEKFQ 0.135 33 EEDLDALEH 0.125 217 ELVQEGKAR 0.100 204 ELLTEVTRR 0.100 187 FIQTLVDVF 0.100 21 LILCVTKAR 0.100 190 TLVDVFTKR 0.100 83 FVVLMAHGR 0.100 230 EIQSTLRKR 0.100 64 ELEKFQQAI 0.090 1 MSNPRSLEE 0.075 115 KNCQALRAK 0.050 97 GEDGEMVKL 0.050 86 LMAHGREGF 0.050 142 TVGGDEIVM 0.050 37 DALEHMFRQ 0.050 80 SCAFVVLMA 0.050 119 ALRAKPKVY 0.050 11 KYDMSGARL 0.050 81 CAFVVLMAH 0.050 108 LFEALNNKN 0.045 133 RGEQRDPGE 0.045 74 SREDPVSCA 0.045 7 LEEEKYDMS 0.045 28 AREGSEEDL 0.045 169 YSTVEGYIA 0.030 117 CQALRAKPK 0.030 79 VSCAFVVLM 0.030 14 MSGARLALI 0.030 36 LDALEHMFR 0.025 46 LRFESTMKR 0.025 141 ETVGGDEIV 0.025 212 RMAEAELVQ 0.025 40 EHMFRQLRF 0.025 13 DMSGARLAL 0.025 153 KDSPQTIPT 0.025 161 TYTDALHVY 0.025 8 EEEKYDMSG 0.022 45 QLRFESTMK 0.020 121 PAKPKVYII 0.020 49 ESTMKRDPT 0.015 129 IQACRGEQR 0.015 184 GSCFIQTLV 0.015 199 KGHILELLT 0.013 233 STLRKRLYL 0.013 30 EGSEEDLDA 0.013 77 DPVSCAFVV 0.013 106 ENLFEALNN 0.013 160 PTYTDALHV 0.013 128 IIQACRGEQ 0.010 20 ALILCVTKA 0.010 158 TIPTYTDAL 0.010 18 RLALILCVT 0.010 164 DALHVYSTV 0.010 111 ALNNKNCQA 0.010 193 DVFTKRKGH 0.010 85 VLMAHGREG 0.010 205 LLTEVTRRM 0.010 203 LELLTEVTR 0.010 147 EIVMVIKDS 0.010 110 EALNNKNCQ 0.010 23 LCVTKAREG 0.010 v.2-A1-9 mers: 213P1F11 33 DTSPTDMIR 12.500 12 FQDPLYLPS 3.750 5 TVEGPTPFQ 1.800 34 TSPTDMIRK 1.500 9 PTPFQDPLY 0.250 4 STVEGPTPF 0.250 21 EAPPNPPLW 0.200 36 PTDMIRKAH 0.125 46 LSRPWWMCS 0.075 31 SQDTSPTDM 0.075 8 GPTPFQDPL 0.025 17 YLPSEAPPN 0.020 19 PSEAPPNPP 0.014 22 APPNPPLWN 0.013 10 TPFQDPLYL 0.013 42 KAHALSRPW 0.010 20 SEAPPNPPL 0.010 44 HALSRPWWM 0.010 50 WWMCSRRGK 0.010 47 SRPWWMCSR 0.005 3 YSTVEGPTP 0.003 30 NSQDTSPTD 0.003 40 IRKAHALSR 0.003 24 PNPPLWNSQ 0.003 56 RGKDISWNF 0.003 23 PPNPPLWNS 0.003 14 DPLYLPSEA 0.003 35 SPTDMIRKA 0.003 6 VEGPTPFQD 0.003 48 RPWWMCSRR 0.003 29 WNSQDTSPT 0.003 37 TDMIRKAHA 0.001 52 MCSRRGKDI 0.001 45 ALSRPWWMC 0.001 16 LYLPSEAPP 0.001 1 HVYSTVEGP 0.001 43 AHALSRPWW 0.001 51 WMCSRRGKD 0.001 32 QDTSPTDMI 0.001 38 DMIRKAHAL 0.001 18 LPSEAPPNP 0.001 2 VYSTVEGPT 0.001 54 SRRGKDISW 0.000 25 NPPLWNSQD 0.000 26 PPLWNSQDT 0.000 7 EGPTPFQDP 0.000 39 MIRKAHALS 0.000 53 CSRRGKDIS 0.000 15 PLYLPSEAP 0.000 13 QDPLYLPSE 0.000 27 PLWNSQDTS 0.000 41 RKAHALSRP 0.000 28 LWNSQDTSP 0.000 55 RRGKDTSWN 0.000 11 PFQDPLYLP 0.000 49 PWWMCSRRG 0.000 v.3-A1-9 mers: 213P1F11 9 ATLPSPFPY 62.500 11 LPSPFPYLS 0.050 7 RGATLPSPF 0.025 10 TLPSPFPYL 0.020 1 YIIQACRGA 0.010 2 IIQACRGAT 0.010 12 PSPFPYLSL 0.008 5 ACRGATLPS 0.005 3 IQACRGATL 0.003 8 GATLPSPFP 0.002 4 QACRGATLP 0.000 6 CRGATLPSP 0.000 v.4-A1-9 mers: 213P1F11 85 ASEEEKYDM 2.700 25 NGECGQTFR 2.250 35 KEEQGRAFR 0.900 83 TSASEEEKY 0.750 9 KSLSVQPEK 0.600 82 ETSASEEEK 0.500 34 LKEEQGRAF 0.450 71 GRDLSISFR 0.250 14 QPEKRTGLR 0.225 27 ECGQTFRLK 0.200 80 NSETSASEE 0.135 58 TQEVFGGGV 0.135 4 CQEYDKSLS 0.135 52 VNDPRETQE 0.125 66 VGDIVGRDL 0.125 10 SLSVQPEKR 0.100 12 SVQPEKRTG 0.100 45 SSVHQKLVN 0.075 68 DIVGRDLSI 0.050 64 GGVGDIVGR 0.050 86 SEEEKYDMS 0.045 22 RDENGECGQ 0.045 57 ETQEVFGGG 0.025 24 ENGECGQTF 0.025 18 RTGLRDENG 0.025 21 LRDENGECG 0.025 44 GSSVHQKLV 0.015 11 LSVQPEKRT 0.015 70 VGRDLSISF 0.013 63 GGGVGDIVG 0.013 51 LVNDPRETQ 0.010 3 KCQEYDKSL 0.010 73 DLSISFRNS 0.010 1 MGKCQEYDK 0.010 50 KLVNDPRET 0.010 75 SISFRNSET 0.010 42 FRGSSVHQK 0.010 55 PRETQEVFG 0.009 54 DPRETQEVF 0.003 5 QEYDKSLSV 0.003 56 RETQEVFGG 0.003 72 RDLSISFRN 0.003 6 EYDKSLSVQ 0.003 62 FGGGVGDIV 0.003 30 QTFRLKEEQ 0.003 36 EEQGRAFRG 0.003 26 GECGQTFRL 0.003 43 RGSSVHQKL 0.003 46 SVHQKLVND 0.002 60 EVFGGGVGD 0.002 76 ISFRNSETS 0.002 74 LSISFRNSE 0.002 48 HQKLVNDPR 0.002 13 VQPEKRTGL 0.002 28 CGQTFRLKE 0.001 84 SASEEEKYD 0.001 69 IVGRDLSIS 0.001 20 GLRDENGEC 0.001 33 RLKEEQGPA 0.001 40 RAFRGSSVH 0.001 65 GVGDIVGRD 0.001 32 FRLKEEQGR 0.001 59 QEVFGGGVG 0.001 61 VFGGGVGDT 0.001 78 FRNSETSAS 0.001 67 GDIVGRDLS 0.001 23 DENGECGQT 0.001 79 RNSETSASE 0.001 2 GKCQEYDKS 0.001 39 GPAFRGSSV 0.001 17 KRTGLRDEN 0.001 38 QGRAFRGSS 0.000 37 EQGRAFRGS 0.000 29 GQTFRLKEE 0.000 41 AFRGSSVHQ 0.000 49 QKLVNDPRE 0.000 8 DKSLSVQPE 0.000 81 SETSASEEE 0.000 31 TFRLKEEQG 0.000 77 SFRNSETSA 0.000 53 NDPRETQEV 0.000 19 TGLRDENGE 0.000 7 YDKSLSVQP 0.000 47 VHQKLVNDP 0.000 16 EKRTGLRDE 0.000 15 PEKRTGLRD 0.000 v.5-A1-9 mers: 213P1F11 4 LALILRVTK 0.400 1 GARLALILR 0.050 5 ALILRVTKA 0.010 6 LILRVTKAR 0.010 3 RLALILRVT 0.010 2 ARLALILRV 0.003 9 RVTKAREGS 0.001 8 LRVTKAREG 0.001 7 ILRVTKARE 0.000 v.6-A1-9 mers: 213P1F11 1 KLENLFEAM 0.900 4 NLFEANNNK 0.200 5 LFEAMNNKN 0.045 3 ENLFEAMNN 0.013 7 EAMNNKNCQ 0.010 8 AMNNKNCQA 0.005 2 LENLFEAMN 0.001 6 FEAMNNKNC 0.001 9 MNNKNCQAL 0.000
[0756] 9 TABLE VI Pos 1234567890 Score SeqID v.1-A1-10 mers: 213P1F11 35 DLDALEHMFR 25.000 228 NPEIQSTLRK 22.500 202 ILELLTEVTR 18.000 38 ALEHMFRQLR 9.000 144 GGDEIVMVIK 5.000 169 YSTVEGYIAY 3.750 162 YTDALHVYST 2.500 104 KLENLFEALN 1.800 171 TVEGYIAYRH 1.800 206 LTEVTRRMAE 1.125 87 MAHGREGFLK 1.000 71 AIDSREDPVS 1.000 215 EAELVQEGKA 0.900 213 MAEAELVQEG 0.900 6 SLEEEKYDMS 0.900 61 FQEELEKFQQ 0.675 75 REDPVSCAFV 0.500 136 QRDPGETVGG 0.500 153 KDSPQTIPTY 0.500 191 LVDVFTKRKG 0.500 170 STVEGYIAYR 0.500 96 KGEDGEMVKL 0.450 74 SREDPVSCAF 0.450 18 RLALILCVTK 0.400 217 ELVQEGKARK 0.400 31 GSEEDLDALE 0.270 189 QTLVDVFTKR 0.250 226 KTNPEIQSTL 0.250 53 KRDPTAEQFQ 0.250 157 QTIPTYTDAL 0.250 133 RGEQRDPGET 0.225 7 LEEEKYDMSG 0.225 32 SEEDLDALEH 0.225 99 DGEMVKLENL 0.225 116 NCQALRAKPK 0.200 190 TLVDVFTKRK 0.200 188 IQTLVDVFTK 0.150 219 VQEGKARKTN 0.135 141 ETVGGDEIVM 0.125 160 PTYTDALHVY 0.125 152 IKDSPQTIPT 0.125 20 ALILCVTKAP 0.100 128 IIQACRGEQR 0.100 85 VLMAHGREGF 0.100 57 TAEQFQEELE 0.090 232 QSTLRKRLYL 0.075 14 MSGARLALIL 0.075 231 IQSTLRKRLY 0.075 79 VSCAFVVLMA 0.075 121 RAKPKVYIIQ 0.050 118 QALRAKPKVY 0.050 80 SCAFVVLMAH 0.050 106 ENLFEALNNK 0.050 95 LKGEDOEMVK 0.050 58 AEQFQEELEK 0.050 45 QLRFESTMKR 0.050 108 LFEALNNKMC 0.045 62 QEELEKFQQA 0.045 5 RSLEEEKYDM 0.030 49 ESTMKRDPTA 0.030 179 RHDQKGSCFI 0.025 11 KYDMSGARLA 0.025 39 LEHMFRQLRF 0.025 166 LHVYSTVEGY 0.025 33 EEDLDALEHM 0.025 227 TNPEIQSTLR 0.025 139 PGETVGGDEI 0.022 145 GDEIVMVIKD 0.022 142 TVGGDEIVMV 0.020 165 ALHVYSTVEG 0.020 185 SCFIQTLVDV 0.020 81 CAFVVLMAHG 0.020 187 FIQTLVDVFT 0.020 158 TIPTYTDALH 0.020 64 ELEKFQQAID 0.018 59 EQFQEELEKF 0.015 1 MSNPRSLEEE 0.015 154 DSPQTIPTYT 0.015 159 IPTYTDALHV 0.013 3 NPRSLEEEKY 0.013 143 VGGDEIVMVI 0.013 111 ALNNKNCQAL 0.010 19 LALILCVTKA 0.010 70 QAIDSREDPV 0.010 167 HVYSTVEGYI 0.010 125 KVYIIQACRG 0.010 114 NKNCQALRAK 0.010 22 ILCVTKAREG 0.010 149 VMVIKDSPQT 0.010 204 ELLTEVTRRM 0.010 110 EALNNKNCQA 0.010 13 DMSGARLALI 0.010 23 LCVTKAREGS 0.010 175 YIAYRHDQKG 0.010 148 IVMVIKDSPQ 0.010 84 VVLMAHGREG 0.010 230 EIQSTLRKRL 0.010 214 AEAELVQEGK 0.010 201 HILELLTEVT 0.010 193 DVFTKRKGHI 0.010 v.2-A1-10 mers: 213P1F11 34 DTSPTDMIRK 25.000 9 GPTPFQDPLY 2.500 22 EAPPNPPLWM 0.500 6 TVEGPTPFQD 0.450 20 PSEAPPNPPL 0.270 37 PTDMIRKAHA 0.250 47 LSRPWWMCSR 0.150 4 YSTVEGPTPF 0.150 13 FQDPLYLPSE 0.150 32 SQDTSPTDMI 0.075 5 STVEGPTPFQ 0.050 33 QDTSPTDMIR 0.025 43 KAHALSRPWW 0.020 31 NSQDTSPTDM 0.015 35 TSPTDMIRKA 0.015 10 PTPFQDPLYL 0.013 45 HALSRPWWMC 0.010 21 SEAPPNPPLW 0.010 17 LYLPSEAPPN 0.010 2 HVYSTVEGPT 0.010 40 MIRKAHALSR 0.005 52 WMCSRRGKDI 0.005 46 ALSRPWWMCS 0.005 48 SRPWWMCSRR 0.005 8 EGPTPFQDPL 0.003 26 NPPLWNSQDT 0.003 24 PPNPPLWNSQ 0.003 36 SPTDMIRKAH 0.003 23 APPNPPLWNS 0.003 18 YLPSEAPPNP 0.002 39 DMIRKAHALS 0.001 53 MCSRRGKDIS 0.001 54 CSRRGKDISW 0.001 56 RRGKDISWNF 0.001 42 RKAHALSRPW 0.001 44 AHALSRPWWM 0.001 14 QDPLYLPSEA 0.001 11 TPFQDPLYLP 0.001 38 TDMIRKAHAL 0.001 29 LWNSQDTSPT 0.001 7 VEGPTPFQDP 0.001 30 WNSQDTSPTD 0.001 12 PFQDPLYLPS 0.000 27 PPLWNSQDTS 0.000 19 LPSEAPPNPP 0.000 15 DPLYLPSEAP 0.000 25 PNPPLWNSQD 0.000 49 RPWWMCSRRG 0.000 16 PLYLPSEAPP 0.000 50 PWWMCSRRGK 0.000 3 VYSTVEGPTP 0.000 55 SRRGKDISWN 0.000 1 LHVYSTVEGP 0.000 51 WWMCSRRGKD 0.000 28 PLWNSQDTSP 0.000 41 IRKAHALSRP 0.000 v.3-A1-10 mers: 213P1F11 9 CATLPSPFPY 2.500 10 ATLPSPFPYL 0.500 12 LPSPFPYLSL 0.125 11 TLPSPFPYLS 0.020 3 IIQACRGATL 0.020 2 YIIQACRGAT 0.010 7 CRGATLPSPF 0.005 5 QACRGATLPS 0.005 1 VYIIQACRGA 0.001 8 RGATLPSPFP 0.001 6 ACRGATLPSP 0.000 4 IQACRGATLP 0.000 v.4-A1-10 mers: 213P1F11 85 ASEEEKYDMS 1.350 82 ETSASEEEKY 1.250 52 VNDPRETQEV 1.250 25 NGECGQTFRL 1.125 34 LKEEQGRAFR 0.900 4 CQEYDKSLSV 0.675 35 KEEQGRAFRG 0.225 86 SEEEKYDMSG 0.225 9 KSLSVQPEKR 0.150 80 NSETSASEEE 0.135 58 TQEVFGGGVG 0.135 66 VGDIVGRDLS 0.125 71 GRDLSISFRN 0.125 12 SVQPEKRTGL 0.100 44 GSSVHQKLVN 0.075 63 GGGVGDIVGR 0.050 69 IVGRDLSISF 0.050 22 RDENGECGQT 0.045 57 ETQEVFGGGV 0.025 24 ENGECGQTFR 0.025 21 LRDENGECGQ 0.025 55 PRETQEVFGG 0.023 84 SASEEEKYDM 0.020 8 DKSLSVQPEK 0.020 11 LSVQPEKRTG 0.015 13 VQPEKRTGLR 0.015 74 LSISFRNSET 0.015 62 FGGGVGDIVG 0.013 14 QPEKRTGLRD 0.011 10 SLSVQPEKRT 0.010 60 EVFGGGVGDI 0.010 75 SISFRNSETS 0.010 65 GVGDIVGRDL 0.010 3 KCQEYDKSLS 0.010 68 DIVGRDLSIS 0.010 81 SETSASEEEK 0.010 26 GECGQTFRLK 0.010 33 RLKEEQGRAF 0.010 50 KLVNDPRETQ 0.010 23 DENGECGQTF 0.005 27 ECGQTFRLKE 0.005 45 SSVHQKLVND 0.003 67 GDIVGRDLSI 0.003 70 VGRDLSISFR 0.003 30 QTFRLKEEQG 0.003 18 RTGLRDENGE 0.003 43 RGSSVHQKLV 0.003 40 RAFRCSSVHQ 0.002 83 TSASEEEKYD 0.002 76 TSFRNSETSA 0.002 29 GQTFRLKEEQ 0.002 41 AFRGSSVHQK 0.001 20 GLRDENGECG 0.001 73 DLSISFRNSE 0.001 51 LVNDPRETQE 0.001 46 SVHQKLVNDP 0.001 47 VHQKLVNDPR 0.001 31 TFRLKEEQGR 0.001 53 NDPRETQEVF 0.001 49 QKLVNDPRET 0.001 2 GKCQEYDKSL 0.001 56 RETQEVFGGG 0.001 6 EYDKSLSVQP 0.001 54 DPRETQEVFG 0.001 39 GRAFRGSSVH 0.001 42 FRGSSVHQKL 0.001 61 VFGGGVGDIV 0.001 5 QEYDKSLSVQ 0.001 72 RDLSISFRNS 0.001 17 KRTGLRDENG 0.001 36 EEQGRAFRGS 0.001 79 RNSETSASEE 0.000 1 MGKCQEYDKS 0.000 38 QGRAFRGSSV 0.000 19 TGLRDENGEC 0.000 64 GGVGDIVGRD 0.000 28 CGQTFRLKEE 0.000 37 EQGRAFRGSS 0.000 78 FRNSETSASE 0.000 59 QEVFGGGVGD 0.000 77 SFRNSETSAS 0.000 16 EKRTGLRDEN 0.000 32 FRLKEEQGRA 0.000 48 HQKLVNDPRE 0.000 7 YDKSLSVQPE 0.000 15 PEKRTGLRDE 0.000 v.5-A1-10 mers: 213P1F11 4 RLALILRVTK 0.400 6 ALILRVTKAR 0.100 1 SGARLALILR 0.013 5 LALILRVTKA 0.010 2 GARLALILRV 0.005 8 ILRVTKAREG 0.001 9 LRVTKAREGS 0.001 3 ARLALILRVT 0.001 7 LILRVTKARE 0.000 10 RVTKAREGSE 0.000 v.6-A1-10 mers: 213P1F11 2 KLENLFEANN 1.800 4 ENLFEANNNK 0.050 6 LFEAMNNKNC 0.045 8 EAMNNKNCQA 0.010 5 HLFEANNNKN 0.010 10 MNNKNCQALR 0.005 9 AMNNKNCQAL 0.005 3 LENLFEAMNN 0.003 7 FEANNNKNCQ 0.001 1 VKLENLFEAM 0.001
[0757] 10 TABLE VII Pos 123456789 Score SeqID v.1-A2-9 mers: 213P1F11 20 ALILCVTKA 11.426 201 HILELLTEV 11.345 143 VGGDEIVMV 7.278 44 RQLRFESTM 7.082 188 IQTLVDVFT 7.064 18 RLALILCVT 7.027 205 LLTEVTRRM 6.925 233 STLRKRLYL 6.038 111 ALNNKNCQA 4.968 104 KLENLFEAL 4.455 183 KGSCFIQTL 4.266 231 IQSTLRKRL 3.682 118 QALRAKPKV 3.574 103 VKLENLFEA 2.374 71 AIDSREDPV 1.874 100 GEMVKLENL 1.732 158 TIPTYTDAL 1.439 6 SLEEEKYDM 1.304 13 DMSGARLAL 1.157 150 MVIKDSPQT 1.108 226 KTNPEIQST 0.833 227 TNPEIQSTL 0.572 107 NLFEALNNK 0.520 38 ALEHMFRQL 0.520 79 VSCAFVVLM 0.482 184 GSCFIQTLV 0.454 109 FEALNNKNC 0.444 164 DALHVYSTV 0.402 97 GEDGEMVKL 0.382 87 MAHGREGFL 0.361 151 VIKDSPQTI 0.350 95 LKGEDGEMV 0.330 155 SPQTIPTYT 0.268 14 MSGARLALI 0.266 50 STMKRDPTA 0.255 112 LNNKNCQAL 0.237 63 EELEKFQQA 0.209 64 ELEKFQQAI 0.190 142 TVGGDEIVM 0.178 199 KGHILELLT 0.170 202 ILELLTEVT 0.163 190 TLVDVFTKR 0.116 186 CFIQTLVDV 0.111 144 GGDEIVMVT 0.105 94 FLKGEDGEM 0.104 157 QTIPTYTDA 0.104 140 GETVGGDEI 0.099 85 VLMAHGREG 0.094 17 ARLALILCV 0.082 77 DPVSCAFVV 0.081 135 EQRDPGETV 0.081 208 EVTRRMAEA 0.075 80 SCAFVVLMA 0.075 160 PTYTDALHV 0.068 169 YSTVEGYIA 0.061 15 SGARLALIL 0.057 216 AELVQEGKA 0.046 86 LMAHGREGF 0.045 31 GSEEDLDAL 0.041 76 EDPVSCAFV 0.040 223 KARKTNPEI 0.039 153 KDSPQTIPT 0.036 57 TAEQFQEEL 0.035 198 RKGHILELL 0.033 78 PVSCAFVVL 0.032 22 ILCVTKARE 0.025 163 TDALHVYST 0.024 12 YDMSGARLA 0.023 187 FIQTLVDVF 0.022 182 QKGSCFIQT 0.020 195 FTKRKGHIL 0.020 134 GEQRDPGET 0.019 149 VMVIKDSPQ 0.018 212 RMAEAELVQ 0.018 211 RRMAEAELV 0.018 219 VQEGKARKT 0.016 125 KVYIIQACR 0.015 141 ETVGGDEIV 0.015 214 AEAELVQEG 0.014 120 LRAKPKVYI 0.014 21 LILCVTKAR 0.013 16 GARLALILC 0.012 162 YTDALHVYS 0.010 189 QTLVDVFTK 0.010 61 FQEELEKFQ 0.010 30 EGSEEDLDA 0.010 204 ELLTEVTRR 0.010 105 LENLFEALN 0.009 123 KPKVYIIQA 0.009 218 LVQEGKARK 0.009 34 EDLDALEHM 0.009 81 CAFVVLMAH 0.009 5 RSLEEEKYD 0.008 68 FQQAIDSRE 0.007 114 NKNCQALRA 0.007 170 STVEGYIAY 0.006 165 ALHVYSTVE 0.006 83 FVVLMAHGR 0.006 176 IAYRHDQKG 0.006 121 RAKPKVYII 0.005 v.2-A2-9 mers: 213P1F11 45 ALSRPWWMC 63.342 10 TPFQDPLYL 2.838 38 DMIRKAHAL 1.157 20 SEAPPNPPL 0.415 44 HALSRPWWM 0.358 17 YLPSEAPPN 0.343 8 GPTPFQDPL 0.260 29 WNSQDTSPT 0.224 31 SQDTSPTDM 0.201 52 MCSRRGKDI 0.116 35 SPTDMIRKA 0.061 37 TDMIRKAHA 0.026 12 FQDPLYLPS 0.021 14 DPLYLPSEA 0.009 32 QDTSPTDMI 0.007 27 PLWNSQDTS 0.007 51 WMCSRRGKD 0.006 4 STVEGPTPF 0.004 26 PPLWNSQDT 0.004 6 VEGPTPFQD 0.003 22 APPNPPLWN 0.003 39 MIRKAHALS 0.001 48 RPWWMCSRR 0.001 42 KAHALSRPW 0.001 18 LPSEAPPNP 0.001 56 RGKDISWNF 0.001 15 PLYLPSEAP 0.001 5 TVEGPTPFQ 0.000 3 YSTVEGPTP 0.000 30 NSQDTSPTD 0.000 43 AHALSRPWW 0.000 2 VYSTVEGPT 0.000 34 TSPTDMIRK 0.000 23 PPNPPLWNS 0.000 1 HVYSTVEGP 0.000 55 RRGKDISWN 0.000 46 LSRPWWMCS 0.000 25 NPPLWNSQD 0.000 21 EAPPNPPLW 0.000 41 RKAHALSRP 0.000 13 QDPLYLPSE 0.000 9 PTPFQDPLY 0.000 7 EGPTPFQDP 0.000 16 LYLPSEAPP 0.000 36 PTDMIRKAH 0.000 53 CSRRGKDIS 0.000 50 WWMCSRRGK 0.000 33 DTSPTDMIR 0.000 47 SRPWWMCSR 0.000 24 PNPPLWNSQ 0.000 28 LWNSQDTSP 0.000 11 PFQDPLYLP 0.000 54 SRRGKDISW 0.000 49 PWWMCSRRG 0.000 19 PSEAPPNPP 0.000 40 IRKAHALSR 0.000 v.3-A2-9 mers: 213P1F11 10 TLPSPFPYL 223.237 3 IQACRGATL 3.682 1 YIIQACRGA 0.628 2 IIQACRGAT 0.226 9 ATLPSPFPY 0.022 12 PSPFPYLSL 0.005 11 LPSPFPYLS 0.003 8 GATLPSPFP 0.001 7 RGATLPSPF 0.000 4 QACRGATLP 0.000 5 ACRGATLPS 0.000 6 CRGATLPSP 0.000 v.4-A2-9 mers: 213P1F11 5 QEYDKSLSV 17.743 13 VQPEKRTGL 15.096 50 KLVNDPRET 5.216 26 GECGQTFRL 2.409 3 KCQEYDKSL 2.001 75 SISFRNSET 1.025 44 GSSVHQKLV 0.454 62 FGGGVGDIV 0.420 58 TQEVFGGGV 0.223 20 GLRDENGEC 0.201 43 RGSSVHQKL 0.139 68 DIVGRDLSI 0.108 53 NDPRETQEV 0.097 33 RLKEEQGRA 0.093 11 LSVQPEKRT 0.083 56 RETQEVFGG 0.019 66 VGDIVGRDL 0.019 69 IVGRDLSIS 0.010 39 GRAFRGSSV 0.010 85 ASEEEKYDM 0.009 10 SLSVQPEKR 0.007 84 SASEEEKYD 0.005 51 LVNDPRETQ 0.004 61 VFGGGVGDI 0.004 29 GQTFRLKEE 0.003 46 SVHQKLVND 0.003 72 RFLSISFRN 0.002 73 DLSISFRNS 0.002 65 GVGDIVGRD 0.002 40 RAFRGSSVH 0.002 76 ISFRNSETS 0.001 23 DENGECGQT 0.001 12 SVQPEKRTG 0.001 9 KSLSVQPEK 0.001 36 EEQGRAFRG 0.001 74 LSISFRNSE 0.001 18 RTGLRDENG 0.001 4 CQEYDKSLS 0.000 79 RNSETSASE 0.000 30 QTFRLKEEQ 0.000 28 CGQTFRLKE 0.000 60 EVFGGGVGD 0.000 19 TGLRDENGE 0.000 35 KEEQGRAFR 0.000 86 SEEEKYDMS 0.000 77 SFRNSETSA 0.000 70 VGRDLSISF 0.000 83 TSASEEEKY 0.000 64 GGVGDIVGR 0.000 37 EQGRAFRGS 0.000 63 GGGVGDIVG 0.000 45 SSVHQKLVN 0.000 24 ENGECGQTF 0.000 81 SETSASEEE 0.000 57 ETQEVFGGG 0.000 59 QEVFGGGVG 0.000 49 QKLVNDPRE 0.000 2 GKCQEYDKS 0.000 52 VNDPRETQE 0.000 67 GDIVGRDLS 0.000 78 FRNSETSAS 0.000 47 VHQLKVNDP 0.000 32 FRLKEEQGR 0.000 25 NGECGQTFR 0.000 42 FRGSSVHQK 0.000 38 QGRAFRGSS 0.000 17 KRTGLRDEN 0.000 21 LRDENGECG 0.000 71 GRDLSISFR 0.000 34 LKEEQGRAF 0.000 82 ETSASEEEK 0.000 80 NSETSASEE 0.000 1 MGKCQEYDK 0.000 8 DKSLSVQPE 0.000 7 YDKSLSVQP 0.000 27 ECGQTFRLK 0.000 54 DPRETQEVF 0.000 31 TFRLKEEQG 0.000 22 RDENGECGQ 0.000 48 HQKLVNDPR 0.000 14 QPEKRTGLR 0.000 41 AFRGSSVHQ 0.000 15 PEKRTGLRD 0.000 55 PRETQEVPG 0.000 6 EYDKSLSVQ 0.000 16 EKRTGLRDE 0.000 v.5-A2-9 mers: 213P1F11 5 ALILRVTKA 11.426 3 RLALILRVT 1.405 2 ARLALILRV 0.082 6 LILRVTKAR 0.013 9 RVTKAREGS 0.003 7 ILRVTKARE 0.002 4 LALILRVTK 0.001 1 GARLALILR 0.000 8 LRVTKAREG 0.000 v.6-A2-9 mers: 213P1F11 8 AMNNKNCQA 3.588 1 KLENLFEAM 1.036 4 NLFEAMNNK 0.520 6 FEAMNNKNC 0.444 9 MNNKNCQAL 0.237 2 LENLFEAMN 0.009 3 ENLFEAMNN 0.000 7 EAMNNKNCQ 0.000 5 LFEAMNNKN 0.000
[0758] 11 TABLE VIII Pos 1234567890 Score SeqID v.1-A2-10mers: 213P1F11 187 FIQTLVDVFT 25.924 111 ALNNKNCQAL 21.362 86 LMAHGREGFL 18.753 142 TVGGDEIVMV 13.997 149 VMVIKDSPQT 9.149 117 CQALRAKPKV 7.052 205 LLTEVTRRMA 6.925 94 FLKGEDGEMV 5.487 119 ALRAKPKVYI 4.361 185 SCFIQTLVDV 3.864 75 REDPVSCAFV 2.975 70 QAIDSREDPV 1.941 183 KGSCFIQTLV 1.589 150 MVIKDSPQTI 1.552 13 DMSGARLALI 1.300 107 NLFEALNNKN 1.130 226 KTNPEIQSTL 1.038 19 LALILCVTKA 0.998 218 LVQEGKARKT 0.909 63 EELEKFQQAI 0.877 159 IPTYTDALHV 0.772 232 QSTLRKRLYL 0.767 103 VKLENLFEAL 0.712 134 GEQRDPGETV 0.663 12 YDMSGARLAL 0.505 5 RSLEEEKYDM 0.492 143 VGGDEIVMVI 0.448 162 YTDALHVYST 0.438 102 MVKLENLFEA 0.345 48 FESTMKRDPT 0.327 96 KGEDGEMVKL 0.295 204 ELLTEVTRRM 0.276 140 GETVGGDEIV 0.272 182 QKGSCFIQTL 0.259 190 TLVDVFTKRK 0.232 85 VLMAHGREGF 0.230 207 TEVTRRMAEA 0.222 230 ETQSTLRKRL 0.220 16 GARLALILCV 0.169 27 KAREGSEEDL 0.159 157 QTIPTYTDAL 0.145 163 TDALHVYSTV 0.145 37 DALEHMFRQL 0.128 79 VSCAFVVLMA 0.127 200 GHILELLTEV 0.111 201 HILELLTEVT 0.106 212 RMAEAELVQE 0.102 14 MSGARLALIL 0.097 29 REGSEEDLDA 0.097 78 PVSCAFVVLM 0.084 15 SGARLALILC 0.075 165 ALHVYSTVEG 0.075 125 KVYIIQACRG 0.073 167 HVYSTVEGYI 0.071 104 KLENLFEALN 0.063 56 PTAEQFQEEL 0.050 30 EGSEEDLDAL 0.048 175 YIAYRHDQKG 0.047 41 HMFRQLRFES 0.039 209 VTRRMAEAEL 0.038 188 IQTLVDVFTK 0.034 193 DVFTKRKGHI 0.033 10 EKYDMSGARL 0.029 22 ILCVTKAREG 0.025 225 RKTNPEIQST 0.024 154 DSPQTIPTYT 0.020 110 EALNNKNCQA 0.019 76 EDPVSCAFVV 0.017 156 PQTIPTYTDA 0.017 179 RHDQKGSCFI 0.016 122 AKPKVYIIQA 0.016 20 ALILCVTKAR 0.015 18 RLALILCVTK 0.015 222 GKARKTNPEI 0.014 6 SLEEEKYDMS 0.014 21 LILCVTKARE 0.013 43 FRQLRFESTM 0.012 62 QEELEKFQQA 0.012 72 IDSREDPVSC 0.012 170 STVEGYIAYR 0.011 61 FQEELEKFQQ 0.011 198 RKGHILELLT 0.010 194 VFTKRKGHLL 0.010 158 TIPTYTDALH 0.010 123 KPKVYIIQAC 0.009 81 CAFVVLMAHG 0.009 148 IVMVIKDSPQ 0.008 84 VVLMAHGREG 0.008 77 DPVSCAFVVL 0.008 152 IKDSPQTIPT 0.007 176 IAYRHDQKGS 0.006 44 RQLRFESTMK 0.006 100 GEMVKLENLF 0.005 196 TKRKGHTLEL 0.005 101 EMVKLENLFE 0.004 203 LELLTEVTRR 0.004 181 DQKGSCFIQT 0.004 38 ALEHMFRQLR 0.004 17 ARLALILCVT 0.004 169 YSTVEGYIAY 0.003 v.2-A2-10 mers: 213P1F11 52 WMCSRRGKDI 34.660 32 SQDTSPTDMI 0.207 44 AHALSRPWWM 0.142 31 NSQDTSPTDM 0.133 46 ALSRPWWMCS 0.127 45 HALSRPWWMC 0.111 38 TDMIRKAHAL 0.110 18 YLPSEAPPNP 0.069 26 NPPLWNSQDT 0.049 10 PTPFQDPLYL 0.036 43 KAHALSRPWW 0.020 8 EGPTPFQDPL 0.019 35 TSPTDMIRKA 0.015 2 HVYSTVEGPT 0.009 23 APPNPPLWNS 0.008 14 QDPLYLPSEA 0.007 13 FQDPLYLPSE 0.006 5 STVEGPTPFQ 0.005 39 DMIRKAHALS 0.004 28 PLWNSQDTSP 0.003 4 YSTVEGPTPF 0.002 36 SPTDMIRKAH 0.002 29 LWNSQDTSPT 0.002 21 SEAPPNPPLW 0.001 16 PLYLPSEAPP 0.001 7 VEGPTPFQDP 0.001 11 TPFQDPLYLP 0.001 49 RPWWMCSRRG 0.001 19 LPSEAPPNPP 0.001 37 PTDMIRKAHA 0.001 9 GPTPFQDPLY 0.000 6 GPTPFQDPLY 0.000 30 WNSQDTSPTD 0.000 22 EAPPNPPLWN 0.000 40 MIRKAHALSR 0.000 20 PSEAPPNPPL 0.000 53 MCSRRGKDIS 0.000 56 RRGKDISWNF 0.000 17 LYLPSEAPPN 0.000 54 CSRRGKDISW 0.000 34 DTSPTDMIRK 0.000 47 LSRPWWMCSR 0.000 42 RKAHALSRPW 0.000 1 LHVYSTVEGP 0.000 27 PPLWNSQDTS 0.000 15 DPLYLPSEAP 0.000 55 SRRGKDISWN 0.000 33 QDTSPTDMIR 0.000 12 PRQDPLYLPS 0.000 51 WWMCSRRGKS 0.000 24 PPNPPLWNSQ 0.000 3 VYSTVEGPTP 0.000 25 PNPPLWNSQD 0.000 48 SRPWWMCSRR 0.000 41 IRKAHALSRP 0.000 50 PWWMCSRRGK 0.000 v.3-A2-10 mers: 213P1F11 10 ATLPSPFPYL 11.472 3 IIQACRGATL 4.993 2 YIIQACRGAT 0.613 12 LPSPFPYLSL 0.356 11 TLPSPFPYLS 0.283 9 GATLPSPFPY 0.006 4 IQACRGATLP 0.003 5 QACRGATLPS 0.001 8 RGATLPSPFP 0.001 1 VYIIQACRGA 0.000 6 ACRGATLPSP 0.000 7 CRGATLPSPF 0.000 v.4-A2-10 mers: 213P1F11 10 SLSVQPEKRT 7.452 12 SVQPEKRTGL 1.869 65 GVGDIVGRDL 1.533 43 RGSSVHQKLV 0.454 4 CQEYDKSLSV 0.451 52 VNDPRETQEV 0.309 84 SASEEEKYDM 0.283 76 ISFRNSETSA 0.204 57 ETQEVFGGGV 0.147 74 LSISFRNSET 0.083 60 EVFGGGVGDI 0.076 25 NGECGQTFRL 0.052 38 QGRAFRGSSV 0.035 2 GKCQEYDKSL 0.030 50 KLVNDPRETQ 0.026 61 VFGGGVGDIV 0.016 19 TGLRDENGEC 0.016 67 GDIVGRDLSI 0.014 42 FRGSSVHQKL 0.014 20 GLRDENGECG 0.011 69 IVGRDLSISF 0.011 51 LVNDPRETQE 0.009 49 QKLVNDPRET 0.008 3 KCQEYDKSLS 0.007 75 SISFRNSETS 0.005 73 DLSISFRNSE 0.004 5 QEYDKSLSVQ 0.004 46 SVHQKLVNDP 0.003 33 RLKEEQGRAF 0.002 35 KEEQGRAFRG 0.002 30 QTFRLKEEQG 0.002 32 FRLKEEQGRA 0.002 13 VQPEKRTGLR 0.001 62 FGGGVGDIVG 0.001 40 RAFRGSSVHQ 0.001 29 GQTFRLKEEQ 0.001 68 DIVGRDLSIS 0.001 70 VGRDLSISFR 0.001 9 KSLSVQPEKR 0.001 83 TSASEEEKYD 0.001 79 RNSETSASEE 0.000 9 KSLSVQPEKR 0.000 83 TSASEEEKYD 0.000 79 RNSETSASEE 0.000 86 SEEEKYDMSG 0.000 56 RETQEVFGGG 0.000 59 QEVFGGGVGD 0.000 37 EQGRAFRGSS 0.000 45 SSVHQKLVND 0.000 28 CGQTFRLKEE 0.000 63 GGGVGDIVGR 0.000 18 RTGLRDENGE 0.000 44 GSSVHQKLVN 0.000 11 LSVQPEKRTG 0.000 24 ENGECGQTFR 0.000 66 VGDIVGRDLS 0.000 72 RDLSISFRNS 0.000 81 SETSASEEEK 0.000 26 GECGQTFRLK 0.000 23 DENGECGQTF 0.000 85 ASEEEKYDMS 0.000 22 RDENGECGQT 0.000 54 DPRETQEVFG 0.000 34 LKEEQGRAFR 0.000 36 EEQGRAFRGS 0.000 82 ETSASEEEKY 0.000 64 GGVGDIVGRD 0.000 27 ECGQTFRLKE 0.000 58 TQEVFGGGVG 0.000 71 GRDLSISFRN 0.000 1 MGKCQEYDKS 0.000 53 NDPRETQEVF 0.000 17 KRTGLRDENG 0.000 78 FRNSETSASE 0.000 47 VHQKLVNDPR 0.000 7 YDKSLSVQPE 0.000 14 QPEKRTGLRD 0.000 21 LRDENGECGQ 0.000 39 GRAFRGSSVH 0.000 77 SFRNSETSAS 0.000 80 NSETSASEEE 0.000 41 AFRGSSVHQK 0.000 48 HQKLVNDPRE 0.000 8 DKSLSVQPEK 0.000 31 TFRLKEEQGR 0.000 16 EKRTGLRDEN 0.000 55 PRETQEVFGG 0.000 15 PEKRTGLFDE 0.000 6 EYDKSLSVQP 0.000 v.5-A2-10 mers: 213P1F11 5 LALILRVTKA 0.998 2 GARLALILRV 0.169 6 ALALRVTKAR 0.015 4 RLALILRVTK 0.015 7 LILRVTKARE 0.013 8 ILRVTKAREG 0.002 3 ARLALILRVT 0.001 1 SGARLALILR 0.000 10 RVTKAREGSE 0.000 9 LRVTKAREGS 0.000 v.6-A2-10mers: 213P1F11 9 AMNNKNCQAL 15.428 5 NLFEAMNNKN 1.130 1 VKLENLFEAM 0.166 2 KLENLFEAMN 0.063 8 EAMNNKNCQA 0.019 3 LENLFEAMNN 0.002 7 FEAMNNKNCQ 0.001 6 LFEAMNNKNC 0.000 10 MNNKNCQALR 0.000 4 ENLFEAMNNK 0.000
[0759] 12 TABLE IX Pos 123456789 Score SeqID v.1-A3-9mers: 213P1F11 107 NLFEALNNK 225.000 190 TLVDVFTKR 27.000 45 QLRFESTMK 20.000 189 QTLVDVFTK 13.500 125 KVYIIQACR 9.000 167 HVYSTVEGY 6.000 204 ELLTEVTRR 5.400 104 KLENLFEAL 5.400 218 LVQEGKARK 3.000 191 LVDVFTKRK 3.000 171 TVEGYIAYR 2.700 119 ALRAKPKVY 2.000 86 LMAHGREGF 2.000 175 YIAYRHDQK 2.000 59 EQFQEELEK 1.800 6 SLEEEKYDM 0.900 217 ELVQEGKAR 0.900 101 EMVKLENLF 0.900 20 ALILCVTKA 0.900 170 STVEGYIAY 0.900 83 FVVLMAHGR 0.600 35 DLDALEHMF 0.600 187 FIQTLVDVF 0.600 64 ELEKFQQAI 0.540 13 DMSGARLAL 0.540 21 LILCVTKAR 0.450 19 LALILCVTK 0.300 117 CQALRAKPK 0.300 18 RLALILCVT 0.225 111 ALNNKNCQA 0.200 3 NPRSLEEEK 0.200 38 ALEHMFRQL 0.180 158 TIPTYTDAL 0.180 145 GDEIVMVIK 0.135 96 KGEDGEMVK 0.120 129 IQACRGEQR 0.120 202 ILELLTEVT 0.100 233 STLRKRLYL 0.090 94 FLKGEDGEM 0.090 234 TLRKRLYLQ 0.090 215 EAELVQEGK 0.090 121 RAKPKVYII 0.081 201 HILELLTEV 0.068 88 AHGREGFLK 0.060 46 LRFESTMKR 0.060 165 ALHVYSTVE 0.060 151 VIKDSPQTT 0.060 212 RMAEAELVQ 0.060 142 TVGGDEIVM 0.060 123 KPKVYIIQA 0.054 205 LLTEVTRRM 0.045 81 CAFVVLMAH 0.045 228 NPEIQSTLR 0.040 39 LEHMFRQLR 0.036 226 KTNPEIQST 0.034 195 FTKRKGHIL 0.030 149 VMVIKDSPQ 0.030 44 RQLRFESTM 0.027 144 GGDEIVMVI 0.024 157 QTIPTYTDA 0.022 31 GSEEDLDAL 0.020 71 AIDSREDPV 0.020 51 TMKRDPTAE 0.020 22 ILCVTKARE 0.020 67 KFQQAIDSR 0.018 115 KNCQALRAK 0.018 57 TAEQFQEEL 0.018 203 LELLTEVTR 0.018 229 PEIQSTLRK 0.018 16 GARLALTLC 0.018 80 SCAFVVLMA 0.018 230 EIQSTLRKR 0.018 223 KARKTNPEI 0.018 78 PVSCAFVVL 0.018 193 DVFTKRKGH 0.015 50 STMKRDPTA 0.015 150 MVIKDSPQT 0.015 41 HMFRQLRFE 0.015 75 REDPVSCAF 0.013 97 GEDGEMVKL 0.012 100 GEMVKLENL 0.012 160 PTYTDALHV 0.010 53 KRDPTAEQF 0.009 79 VSCAFVVLM 0.009 231 IQSTLRKRL 0.009 208 EVTRRMAEA 0.009 154 DSPQTIPTY 0.009 197 KRKGHILEL 0.008 183 KGSCFIQTL 0.008 36 LDALEHMFR 0.008 113 NNKNCQALR 0.008 141 ETVGGDEIV 0.007 161 TYTDALHVY 0.006 140 GETVGGDEI 0.005 60 QEQEELEKE 0.005 188 IQTLVDVFT 0.005 184 GSCFIQTLV 0.005 148 IVMVIKDSP 0.005 14 MSGARLALI 0.005 102 MVKLENLFE 0.004 v.2-A3-9 mers: 213P1F11 45 ALSRPWWMC 0.900 34 TSPTDMIRK 0.300 38 DMIRKAHAL 0.270 4 STVEGPTPF 0.225 48 RPWWMCSRR 0.200 33 DTSPTDMIR 0.180 8 GPTPFQDPL 0.081 10 TPFQDPLYL 0.060 1 HVYSTVEGP 0.030 17 YLPSEAPPN 0.020 27 PLWNSQDTS 0.020 9 PTPFQDPLY 0.020 47 SRPWWMCSR 0.018 15 PLYLPSEAP 0.015 56 RGKDISWNF 0.009 44 HALSRPWWM 0.009 40 IRKAHALSR 0.008 51 WMSCRRGKD 0.006 31 SQDTSPTDM 0.006 5 TVEGPTPFQ 0.005 20 SEAPPNPPL 0.004 39 MIRKAHALS 0.004 12 FQDPLYLPS 0.004 50 WWMCSRRGK 0.003 52 MCSRRGKDI 0.003 46 LSRPWWMCS 0.002 32 QDTSPTDMI 0.001 14 DPLYLPSEA 0.001 21 EAPPNPPLW 0.001 36 PTDMIRKAH 0.001 22 APPNPPLWN 0.001 42 KAHALSRPW 0.001 25 NPPLWNSQD 0.001 54 SRRGKDISW 0.001 23 PPNPPLWNS 0.000 18 LPSEAPPNP 0.000 37 TDMIRKAHA 0.000 35 SPTDMIRKA 0.000 6 VEGPTPFQD 0.000 29 WNSQDTSPT 0.000 43 AHALSRPWW 0.000 53 CSRRGKDIS 0.000 26 PPLWNSQDT 0.000 3 YSTVEGPTP 0.000 30 NSQDTSPTD 0.000 13 QDPLYLPSE 0.000 16 LYLPSEAPP 0.000 2 VYSTVEGPT 0.000 55 RRGKDISWN 0.000 41 RKAHALSRP 0.000 7 EGPTPFQDP 0.000 28 LWNSQDTSP 0.000 19 PSEAPPNPP 0.000 11 PFQDPLYLP 0.000 24 PNPPLWNSQ 0.000 49 PWWMCSRRG 0.000 v.3-A3-9 mers: 213P1F11 10 TLPSPFPYL 2.700 9 ATLPSPFPY 1.350 3 IQACRGATL 0.018 11 LPSPFPYLS 0.005 1 YIIQACRGA 0.003 2 IIQACRGAT 0.003 7 RGATLPSPF 0.002 5 ACRGATLPS 0.001 12 PSPFPYLSL 0.001 8 GATLPSPFP 0.001 4 QACRGATLP 0.000 6 CRGATLPSP 0.000 v.4-A3-9 mers: 213P1F11 10 SLSVQPEKR 4.000 9 KSLSVQPEK 0.675 82 ETSASEEEK 0.300 48 HQKLVNDPR 0.180 20 GLRDENGEC 0.180 33 RLKEEQGRA 0.090 68 DIVGRDLSI 0.081 42 FRGSSVHQK 0.060 1 MGKCQEYDK 0.060 50 KLVNDPRET 0.045 64 GGVGDIVGR 0.041 3 KCQEYDKSL 0.041 35 KEEQGRAFR 0.036 13 VQPEKRTGL 0.027 26 GECGQTFRL 0.024 83 TSASEEEKY 0.020 71 GRDLSISFR 0.018 27 ECGQTFRLK 0.018 14 QPEKRTGLR 0.012 40 RAFRGSSVH 0.010 75 SISFRNSET 0.010 54 DPRETQEVF 0.009 65 GVGDIVGRD 0.008 32 FRLKEEQGR 0.006 69 IVGRDLSIS 0.006 5 QEYDKSLSV 0.006 58 TQEVFGGGV 0.005 30 QTFRLKEEQ 0.005 85 ASEEEKYDM 0.005 60 EVFGGGVGD 0.005 70 VGRDLSISF 0.004 25 NGECGQTFR 0.004 73 DLSISFRNS 0.004 51 LVNDPRETQ 0.003 46 SVHQKLVND 0.003 24 ENGECGQTF 0.002 44 GSSVHQKLV 0.002 29 GQTFRLKEE 0.001 4 CQEYDKSLS 0.001 18 RTGLRDENG 0.001 76 ISFRNSETS 0.001 43 RGSSVHQKL 0.001 61 VFGGGVGDI 0.001 57 ETQEVFGGG 0.001 39 GRAFRGSSV 0.001 11 LSVQPEKRT 0.001 56 RETQEVFGG 0.001 62 FGGGVGDIV 0.000 74 LSISFRNSE 0.000 12 SVQPEKRTG 0.000 34 LKEEQGRAF 0.000 45 SSVHQKLVN 0.000 53 NDPRETQEV 0.000 77 SFRNSETSA 0.000 67 GDIVGRDLS 0.000 86 SEEEKYDMS 0.000 84 SASEEEKYD 0.000 72 RDLSISFRN 0.000 2 GKCQEYDKS 0.000 63 GGGVGDIVG 0.000 28 CGQTFRLKE 0.000 37 EQGRAFRGS 0.000 80 NSETSASEE 0.000 66 VGDIVGRDL 0.000 17 KRTGLRDEN 0.000 36 EEQGRAFRG 0.000 52 VNDPRETQE 0.000 79 RNSETSASE 0.000 47 VHQKLVNDP 0.000 81 SETSASEEE 0.000 23 DENGECGQT 0.000 78 FRNSETSAS 0.000 38 QGRAFRGSS 0.000 21 LRDENGECG 0.000 19 TGLRDENGE 0.000 49 QKLVNDPRE 0.000 41 AFRGSSVHQ 0.000 59 QEVFGGGVG 0.000 31 TFRLKEEQG 0.000 7 YDKSLSVQP 0.000 22 RDENGECGQ 0.000 8 DKSLSVQPE 0.000 15 PEKRTGLRD 0.000 6 EYDKSLSVQ 0.000 55 PRETQEVFG 0.000 16 EKRTGLRDE 0.000 v.5-A3-9 mers: 213P1F11 5 ALILRVTKA 0.900 6 LILRVTKAR 0.450 1 GARLALILR 0.360 4 LALILRVTK 0.300 3 RLALILRVT 0.022 7 ILRVTKARE 0.020 9 RVTKAREGS 0.004 2 ARLALILRV 0.001 8 LRVTKAREG 0.000 v.6-A3-9 mers: 213P1F11 4 NLFEAMNNK 225.000 1 KLENLFEAM 1.800 8 AMNNKNCQA 0.200 9 MNNKNCQAL 0.200 6 FEAMNNKNC 0.000 2 LENLFEAMN 0.000 7 EAMNNKNCQ 0.000 3 ENLFEAMNN 0.000 5 LFEAMNNKN 0.000
[0760] 13 TABLE X Pos 1234567890 Score SeqID v.1-A3-10 mers 190 TLVDVFTKRK 45.000 18 RLALILCVTK 20.000 38 ALEHMFRQLR 12.000 217 ELVQEGKARK 9.000 45 QLRFESTMKR 8.000 188 IQTLVDVFTK 5.400 20 ALILCVTKAR 4.500 202 ILELLTEVTR 4.000 85 VLMAHGREGF 3.000 35 DLDALEHMFR 2.400 170 STVEGYIAYR 2.025 189 QTLVDVFTKR 1.350 87 MAHGREGFLK 0.900 44 RQLRFESTMK 0.900 119 ALRAKPKVYI 0.900 41 HMFRQLRFES 0.600 111 ALNNKNCQAL 0.600 13 DMSGARLALI 0.405 128 IIQACRGEQR 0.400 228 NPEIQSTLRK 0.400 94 FLKGEDGEMV 0.300 144 GGDEIVMVIK 0.203 157 QTIPTYTDAL 0.203 226 KTNPEIQSTL 0.203 86 LMAHGREGFL 0.180 104 KLENLFEALN 0.180 107 NLFEALNNKN 0.150 160 PTYTDALHVY 0.150 149 VMVIKDSPQT 0.150 59 EQFQEELEKF 0.135 214 AEAELVQEGK 0.135 167 HVYSTVEGYI 0.135 171 TVEGYIAYRH 0.135 58 AEQFQEELEK 0.120 116 NCQALRAKPK 0.100 174 GYTAYRHDQK 0.090 150 MVTKDSPQTI 0.090 102 MVKLEHLFEA 0.090 95 LKGEDGEMVK 0.060 6 SLEEEKYDMS 0.060 203 LELLTEVTRR 0.054 162 YTDALHVYST 0.045 142 TVGGDEIVMV 0.045 212 RMAEAELVQE 0.045 169 YSTVEGYIAY 0.040 2 SNPRSLEEEK 0.040 3 NPRSLEEEKY 0.040 118 QALRAKPKVY 0.030 125 KVYIIQACRG 0.030 205 LLTEVTRRMA 0.030 51 TMKRDPTAEQ 0.030 209 VTRRMAEAEL 0.030 193 DVFTKRKGHI 0.027 153 KDSPQTIPTY 0.027 106 ENLFEALNNK 0.027 27 KAREGSEEDL 0.027 100 GEMVKLENLF 0.027 201 HILELLTEVT 0.022 158 TIPTYTDALH 0.020 165 ALHVYSTVEG 0.020 166 LHVYSTVEGY 0.018 16 GARLALILCV 0.018 78 PVSCAFVVLM 0.018 101 EMVKLENIFE 0.018 187 FIQTLVDVFT 0.015 185 SCFIQTLVDV 0.015 123 KPKVYIIQAC 0.013 141 ETVGGDEIVM 0.013 204 ELLTEVTRRM 0.013 56 PTAEQFQEEL 0.013 231 IQSTLRKRLY 0.012 227 TNPEIQSTLR 0.012 39 LEHMFRQLRF 0.012 186 CFIQTLVDVF 0.009 79 VSCAFVVLMA 0.009 66 EKFQQAIDSR 0.009 216 AELVQEGKAR 0.009 19 LALTLCVTKA 0.009 80 SCAFVVLMAH 0.009 230 ETQSTLRKRL 0.009 77 DPVSCAFVVL 0.008 181 DQKGSCFIQT 0.008 112 LNNKNCQALR 0.008 233 STLRKRLYLQ 0.007 5 RSLEEEKYDM 0.007 197 KRKGHILELL 0.006 82 AFVVLMAHGR 0.006 14 MSGARLALIL 0.006 232 QSTLRKRLYL 0.006 117 CQALRAKPKV 0.006 64 ELEKFQQATD 0.006 143 VGGDEIVMVT 0.005 120 LRAKPKVYII 0.005 103 VKLENLFEAL 0.004 159 IPTYTDALHV 0.004 71 AIDSREDPVS 0.004 63 EELEKFQQAI 0.004 74 SREDPVSCAF 0.003 114 NKNCQALRAK 0.003 148 IVMVIKDSPQ 0.003 v.2-A3-10 mers: 213P1F11 34 DTSPTDMIRE 1.350 40 MIRKAHALSR 0.800 52 WMCSRRGKDI 0.300 46 ALSRPWWMCS 0.240 9 GPTPFQDPLY 0.180 47 LSRPWWNCSR 0.135 32 SQDTSPTDMI 0.027 2 HVYSTVEGPT 0.022 18 YLPSEAPPNP 0.020 39 DMIRKAHALS 0.018 45 HALSRPWWMC 0.013 16 PLYLPSEAPP 0.010 4 YSTVEGPTPF 0.010 28 PLWNSQDTSP 0.010 56 RRGKDISWNF 0.009 6 TVEGPTPFQD 0.009 33 QDTSPTDMIR 0.008 11 TPFQDPLYLP 0.007 10 PTPFQDPLYL 0.006 43 KAHALSRPWW 0.006 13 FQDPLYLPSE 0.004 48 SRPWWMCSRR 0.004 5 STVEGPTPFQ 0.003 23 APPNPPLWNS 0.003 54 CSRRGKDISW 0.002 36 SPTDMIRKAH 0.002 50 PWWMCSRRGK 0.001 31 NSQDTSPTDM 0.001 37 PTDMIRKAHA 0.001 26 NPPLWNSQDT 0.001 38 TDMIRKAHAL 0.001 21 SEAPPNPPLW 0.001 44 AHALSRPWWM 0.001 8 EGPTPFQDPL 0.001 20 PSEAPPNPPL 0.000 19 LPSEAPPNPP 0.000 7 VEGPTPFQDP 0.000 53 MCSRRGKDIS 0.000 22 EAPPNPPLWN 0.000 14 QDPLYLPSEA 0.000 35 TSPTDMIRKA 0.000 15 DPLYLPSEAP 0.000 29 LWNSQDTSPT 0.000 49 RPWWMCSRRG 0.000 1 LHVYSTVEGP 0.000 27 PPLWNSQDTS 0.000 55 SRRGKDISWN 0.000 17 LYLPSEAPPN 0.000 30 WNSQDTSPTD 0.000 24 PPNPPLWNSQ 0.000 41 IRKAHALSRP 0.000 3 VYSTVEGPTP 0.000 42 RKAHALSRPW 0.000 25 PNPPLWNSQD 0.000 51 WWMCSRRGKD 0.000 12 PFQDPLYLPS 0.000 v.3-A3-10 mers: 213P1F11 11 TLPSPFPYLS 0.360 9 GATLPSPFPY 0.360 10 ATLPSPFPYL 0.304 3 IIQACRGATL 0.060 12 LPSPFPYLSL 0.027 2 YIIQACRGAT 0.005 7 CRGATLPSPF 0.002 5 QACRGATLSP 0.001 4 IQACRGATLP 0.001 6 ACRGATLPSP 0.000 8 RGATLPSPFP 0.000 1 VYIIQACRGA 0.000 v.4-A3-10 mers: 213P1F11 69 IVGRDLSISF 0.400 33 RLKEEQGRAF 0.300 50 KLVNDPRETQ 0.135 60 EVFGGGVGDI 0.121 41 AFRGSSVHQK 0.090 12 SVQPEKRTGL 0.090 9 KSLSVQPEKR 0.090 26 GECGQTFRLK 0.081 10 SLSVQPEKRT 0.075 20 GLRDENGECG 0.060 81 SETSASEEEK 0.060 82 ETSASEEEKY 0.060 13 VQPEKRTGLR 0.054 65 GVGDIVGRDL 0.027 63 GGGVGDIVGR 0.018 73 DLSISFRNSE 0.018 4 CQEYDKSLSV 0.012 84 SASEEEKYDM 0.009 8 DKSLSVQPEK 0.009 70 VGRDLSISFR 0.006 47 VHQKLVNDPR 0.006 34 LKEEQGRAFR 0.006 46 SVHQKLVNDP 0.006 67 GDIVGRDLSI 0.005 76 ISFRNSETSA 0.005 30 QTFRLKEEQG 0.005 57 ETQEVFGGGV 0.004 68 DIVGRDLSIS 0.004 75 SISFRNSETS 0.004 31 TFRLKEEQGR 0.004 24 ENGECGQTRF 0.004 23 DENGECGQTF 0.003 2 GKCQEYDKSL 0.003 51 LVNDPRETQE 0.003 53 NDPRETQEVG 0.002 25 NGECGQTFRL 0.002 29 GQTFRLKEEQ 0.002 3 KCQEYDKSLS 0.002 40 RAFRGSSVHQ 0.001 18 RTGLRDENGE 0.001 42 FRGSSVHQKL 0.001 74 LSISFRNSET 0.001 44 GSSVHQKLVN 0.001 39 GRAFRGSSVN 0.001 48 HQKLVNDPRE 0.001 52 VNDPRETQEV 0.001 35 KEEQGRAFRG 0.001 86 SEEEKYDMSG 0.001 61 VFGGGVGDIV 0.000 5 QEYDKSLSVQ 0.000 14 QPEKRTGLRD 0.000 27 ECGQTFRLKE 0.000 37 EQGRAFRGSS 0.000 85 ASEEEKYDMS 0.000 71 GRDLSISFRN 0.000 45 SSVHQKLVND 0.000 38 QGRAFRGSSV 0.000 64 GGVGDIVGRD 0.000 58 TQEVFGGGVG 0.000 80 NSETSASEEE 0.000 43 RGSSVHQKLV 0.000 17 KRTGLRDENG 0.000 32 FRLKEEQGRA 0.000 19 TGLRDENGEC 0.000 54 DPRETQEVFG 0.000 59 QEVFGGGVGD 0.000 56 RETQEVFGGG 0.000 79 RNSETSASEE 0.000 62 FGGGVGDIVG 0.000 7 YDKSLSVQPE 0.000 83 TSASEEEKYD 0.000 77 SFRNSETSAS 0.000 66 VGDIVGRDLS 0.000 1 MGKCQEYDKS 0.000 21 LRDENGECGQ 0.000 22 RDENGECGQT 0.000 78 FRNSETSASE 0.000 55 PRETQEVFGG 0.000 72 RDLSISFRNS 0.000 36 EEQGRAFRGS 0.000 28 CGQTFRLKEE 0.000 11 LSVQPEKRTG 0.000 49 QKLVNDPRET 0.000 6 EYDKSLSVQP 0.000 16 EYDKSLSVQP 0.000 15 PEKRTGLRDE 0.000 v.5-A3-10 mers: 213P1F11 4 RLALILRVTK 20.000 6 ALILRVTKAR 4.500 2 GARLALILRV 0.018 1 SGARLALILR 0.012 5 LALILRVTKA 0.009 7 LILRVTKARE 0.003 8 ILRVTKAREG 0.002 10 RVTKAREGSE 0.001 9 LRVTKAREGS 0.000 3 ARLALILRVT 0.000 v.6-A3-10 mers: 213P1F11 9 AMNNKNCQAL 0.600 2 KLENLFEAMN 0.180 5 NLFEAMNNKN 0.150 4 ENLFEAMNNK 0.027 10 MNNKNCQALR 0.008 1 VKLENLFEAM 0.001 8 EAMNNKNCQA 0.001 3 LENLFEAMNN 0.000 6 LFEAMNNKNC 0.000 7 FEAMNNKNCQ 0.000
[0761] 14 TABLE XI Pos 123456789 Score SeqID v.1-A11-9 mers:213P1F11 189 QTLVDVFTK 4.500 125 KVYIIQACR 2.400 218 LVQEGKARK 2.000 191 LVDVFTKRK 1.000 107 NLFEALNNK 0.800 59 EQFQEELEK 0.720 83 FVVLMAHGR 0.600 171 TVEGYIAYR 0.400 45 QLRFESTMK 0.400 175 YIAYRHDQK 0.400 19 LALILCVTK 0.300 117 CQALRAKPK 0.300 3 NPRSLEEEK 0.200 96 KGEDGEMVK 0.120 129 IQACRGEQR 0.120 190 TLVDVFTKR 0.120 67 KFQQAIDSR 0.120 88 AHGREGFLK 0.060 145 GDEIVMVIK 0.060 215 EAELVQEGK 0.060 21 LILCVTKAR 0.060 142 TVGGDEIVM 0.040 228 NPEIQSTLR 0.040 167 HVYSTVEGY 0.040 204 ELLTEVTRR 0.036 233 STLRKRLYL 0.030 170 STVEGYIAY 0.030 44 RQLRFESTM 0.027 50 STMKRDPTA 0.020 217 ELVQEGKAR 0.018 229 PETQSTLRK 0.018 203 LELLTEVTR 0.018 46 LRFESTMKR 0.016 157 QTIPTYTDA 0.015 115 KNCQALRAK 0.012 121 RAKPKVYII 0.012 39 LEIIMFRQLR 0.012 104 KLEHLFEAL 0.012 123 KPKVYIIQA 0.012 11 KYDMSGARL 0.012 195 FTKRKOHIL 0.010 36 LDALEHMFR 0.008 81 CAFVVLMAH 0.008 113 NNKNCQALR 0.008 6 SLEEEKYDM 0.008 201 HILELLTEV 0.006 193 DVFTKRKGH 0.006 20 ALILCVTKA 0.006 223 KARKTNPEI 0.006 208 EVTRRMAEA 0.006 141 ETVGGDEIV 0.005 94 FLKGEDGEM 0.004 80 SCAFVVLMA 0.004 111 ALNNKNCQA 0.004 102 MVKLENLFE 0.004 86 LMAHGREGF 0.004 148 IVMVIKDSP 0.004 160 PTYTDALHV 0.004 187 FIQTLVDVF 0.004 151 VIKDSPQTI 0.004 161 TYTDALHVY 0.004 71 AIDSREDPV 0.004 168 VYSTVEGYI 0.004 158 TIPTYTDAL 0.004 100 GEMVKLENL 0.004 226 KTNPEIQST 0.003 150 MVIKDSPQT 0.003 118 QALPAKPKV 0.003 186 CFIQTLVDV 0.003 84 VVLMAHGRE 0.003 231 IQSTLRKRL 0.003 77 DPVSCAFVV 0.003 212 RMAEAELVQ 0.002 13 DMSGARLAL 0.002 10 EKYDMSGAR 0.002 230 EIQSTLRKR 0.002 57 TAEQFQEEL 0.002 159 IPTYTDALH 0.002 60 QFQEELEKF 0.002 119 ALRAKPKVY 0.002 87 MAHGREGFL 0.002 194 VFTKRKGHI 0.002 78 PVSCAFVVL 0.002 24 CVTKAREGS 0.002 174 GYIAYRHDQ 0.002 135 EQRDPGETV 0.002 75 REDPVSCAF 0.002 101 EMVKLENLF 0.002 140 GETVGGDEI 0.002 97 GEDGEMVKL 0.002 16 GARLALILC 0.001 211 RRMAEAELV 0.001 197 KRKGHILEL 0.001 144 GGDEIVMVI 0.001 18 RLALILCVT 0.001 64 ELEKFQQAI 0.001 172 VEGYIAYRH 0.001 35 DLDALEHMF 0.001 162 YTDALHVYS 0.001 206 LTEVTRRMA 0.001 v.2-A11-9 mers 48 RPWWMCSRR 0.240 33 DTSPTDMIR 0.120 34 TSPTDMIRK 0.040 4 STVEGPTPF 0.015 40 IRKAHALSR 0.008 10 TPFQDPLYL 0.008 31 SQDTSPTDM 0.006 44 HALSRPWWM 0.006 8 GPTPFQDPL 0.006 50 WMMCSRRGK 0.004 1 HVYSTVEGP 0.004 47 SRPWWMCSR 0.004 5 TVEGPTPFQ 0.002 38 DMIRKAHAL 0.002 56 RGKDISWNF 0.001 12 FQDPLYLPS 0.001 52 MCSRRGKDI 0.001 9 PTPFQDPLY 0.001 14 DPLYLPSEA 0.001 45 ALSRPWWMC 0.001 16 LYLPSEAPP 0.001 42 KAHALSRPW 0.001 21 EAPPNPPLW 0.001 20 SEAPPNPPL 0.001 36 PTDMIRKAH 0.001 2 VYSTVEGPT 0.000 22 APPNPPLWN 0.000 17 YLPSEAPPN 0.000 37 TDMIRKAHA 0.000 39 MIRKAHALS 0.000 51 WMCSRRGKD 0.000 54 SRRGKDIWA 0.000 25 NPPLWNSQD 0.000 32 QDTSPTDMI 0.000 18 LPSEAPPNP 0.000 35 SPTDMIRKA 0.000 43 ANALSRPWW 0.000 6 VEGPTPFQD 0.000 15 PLYLPSEAP 0.000 27 PLWNSQDTS 0.000 41 RKAHALSRP 0.000 55 RRGKLISWN 0.000 29 WNSQDTSPT 0.000 11 PFQDPLYLP 0.000 46 LSRPWWMCS 0.000 23 PPNPPLWNS 0.000 26 PPLWNSQDT 0.000 53 CSRRGKDIS 0.000 30 NSQDTSPTD 0.000 13 QDPLYLPSE 0.000 3 YSTVEGPTP 0.000 28 LWNSQDTSP 0.000 7 EGPTPFQDP 0.000 24 PNPPLWNSQ 0.000 19 PSEAPPNPP 0.000 49 PWWMCSRRG 0.000 v.3-A11-9 mers 9 ATLPSPFPY 0.045 3 IQACRGATL 0.006 10 TLPSPFPYL 0.004 8 GATLPSPFP 0.001 1 YIIQACRGA 0.001 7 RGATLPSPF 0.001 11 LPSPFPYLS 0.000 5 ACRGATLPS 0.000 2 IIQACRGAT 0.000 4 QACRGATLP 0.000 12 PSPFPYLSL 0.000 6 CRGATLPSP 0.000 v.4-A11-9 mers: 213P1F11 82 ETSASEEEK 0.300 48 HQKLVNDPR 0.120 9 KSLSVQPEK 0.090 10 SLSVQPEKR 0.080 1 MGKCQEYDK 0.040 14 QPEKRTGLR 0.040 35 KEEQGRAFR 0.036 42 FRGSSVHQK 0.020 64 GGVGDIVGR 0.018 71 GRDLSISFR 0.012 33 RLKEEQGRA 0.012 40 RAFRGSSVH 0.012 32 FRLKEEQGR 0.006 65 GVGDIVGRD 0.006 27 ECGQTFRLK 0.006 58 TQEVFGGGV 0.006 13 VQPEKRTGL 0.006 26 GECGQTFRL 0.005 25 NGECGQTRF 0.004 68 DIVGRDLSI 0.004 3 KCQEYDKSL 0.003 18 RTGLRDENG 0.003 5 QEYDKSLSV 0.002 77 SFRNSETSA 0.002 46 SVHQKLVND 0.002 51 LVNDPRETQ 0.002 30 QTFRLKEEQ 0.002 69 IVGRDLSIS 0.002 61 VFGGGVGDI 0.002 20 GLRDENGEC 0.001 60 EVFGGGVGD 0.001 29 GQTFRLKEE 0.001 43 RGSSVHQKL 0.001 4 CQEYDKSLS 0.001 39 GRAFRGSSV 0.001 54 DPRETQEVF 0.001 56 RETQEVFGG 0.001 75 SISFRNSET 0.000 85 ASEEEKYDM 0.000 70 VGRDLSISF 0.000 44 GSSVHQKLV 0.000 57 ETQEVFGGG 0.000 72 RDLSISFRN 0.000 41 AFRGSSVHQ 0.000 53 NDPRETQEV 0.000 31 TFRLKEEQG 0.000 83 TSASEEEKY 0.000 62 FGGGVGDIV 0.000 12 SVQPEKRTG 0.000 50 KLVNDPRET 0.000 79 RNSETSASE 0.000 63 GGGVGDIVG 0.000 6 EYDKSLSVQ 0.000 24 ENGECGQTF 0.000 84 SASEEEKYL 0.000 67 GDIVGRDLS 0.000 59 QEVFGGGVG 0.000 17 KRTGLRDEN 0.000 45 SSVHQKLVN 0.000 22 RDENGECGQ 0.000 2 GKCQEYDKS 0.000 86 SEEEKYDMS 0.000 81 SETSASEEE 0.000 36 EEQGRAFRG 0.000 52 VNDPRETQE 0.000 28 CGQTFRLKE 0.000 76 ISFRNSETS 0.000 74 LSISFRNSE 0.000 49 QKLVNDPRE 0.000 19 TGLRDENGE 0.000 47 VHQKLVNDP 0.000 66 VGEIVGRDL 0.000 38 QGRAFRGSS 0.000 34 LKEEQGRAF 0.000 80 NSETSASEE 0.000 7 YDKLSLVQP 0.000 78 FRNSETSAS 0.000 21 LRDENGECG 0.000 37 EQGRAFRGS 0.000 23 DENGECGQT 0.000 11 LSVQPEKRT 0.000 15 PEFKRGLRD 0.000 73 DLSISFRNS 0.000 8 DKSLSVQPE 0.000 55 PRETQEVFG 0.000 16 EKRTGLRDE 0.000 v.5-A11-9 mers: 213P1F11 4 LALILRVTK 0.300 1 GARLALILR 0.240 6 LILRVTKAR 0.060 9 RVTKAREGG 0.006 5 ALILRVTKA 0.006 2 ARLALILRV 0.001 7 ILRVTKARE 0.000 3 RLALILRVT 0.000 8 LRVTKAREG 0.000 v.6-A11-9 mers: 213P1F11 4 NLFEAMNNK 0.800 1 KLENLFEAM 0.012 8 AMNNKNCQA 0.004 9 MNNKNCQAL 0.000 5 LFEAMNNKN 0.000 7 EAMNNKNCQ 0.000 2 LENLFEAMN 0.000 3 ENLFEAMNN 0.000 6 FEAMNNKNC 0.000
[0762] 15 TABLE XII Pos 1234567890 Score SeqID v.1-A11-10 mers: 213P1F11 44 RQLRFESTMK 2.700 174 GYIAYRHDQK 1.800 188 IQTLVDVFTK 1.800 18 RLALILCVTK 1.200 87 MAHGREGFLK 0.600 228 NPEIQSTLRK 0.400 190 TLVDVFTKRK 0.300 189 QTLVDVFTKR 0.300 170 STVEGYIAYR 0.300 217 ELVQEGKARK 0.180 45 QLRFESTMKR 0.160 58 AEQEQEELEK 0.120 116 NCQALRAKPK 0.100 38 ALEHMFRQLR 0.080 202 ILELLTEVTR 0.080 128 IIQACRGEQR 0.080 144 GGDETVMVIK 0.060 102 MVKLENLFEA 0.060 214 AEAELVQEGK 0.060 82 AFVVLMAHGR 0.060 20 ALTLCVTKAR 0.060 35 DLDALEHMFR 0.048 171 TVEGYIAYRH 0.040 2 SNPRSLEEEK 0.040 95 LKGEDGEMVK 0.040 167 HVYSTVEGYI 0.040 150 MVIKDSPQTI 0.030 226 KTNPEIQSTL 0.030 142 TVGGDEIVMV 0.020 203 LELLTEVTRR 0.018 106 ENLFEALNNK 0.018 157 QTIPTYTDAL 0.015 193 DVFTKRKGHI 0.012 16 GARLALILCV 0.012 125 KVYIIQACRG 0.012 209 VTRRMAEAEL 0.010 93 GFLKGEDGEM 0.009 216 AELVQEGKAR 0.009 141 ETVGGDEIVM 0.009 168 VYSTVEGYIA 0.008 112 LNNKNCQALR 0.008 85 VLMAHGREGF 0.008 227 TNPEIQSTLR 0.008 27 KAREGSEEDL 0.006 117 CQALRAKPKV 0.006 111 ALNNKNCQAL 0.004 80 SCAFVVLMAH 0.004 185 SCFIQTLVDV 0.004 94 FLKGEDGEMV 0.004 86 LMAHGREGFL 0.004 158 TIPTYTDALH 0.004 119 ALRAKPKVYI 0.004 159 IPTYTDALHV 0.004 148 IVMVIKDSPQ 0.004 59 EQFQEELEKF 0.004 9 EEKYDMSGAR 0.004 29 REGSEEDLDA 0.004 100 GEMVKLENLF 0.004 83 FVVLMAHGRE 0.003 233 STLRKRLYLQ 0.003 19 LALILCVTKA 0.003 186 CFIQTLVDVF 0.003 70 QAIDSREDPV 0.003 41 HMFRQLRFES 0.002 66 EKFQQAIDSR 0.002 114 NKNCQALRAK 0.002 50 STMKRDPTAE 0.002 3 NPRSLEEEKY 0.002 162 YTDALHVYST 0.002 206 LTEVTRRMAE 0.002 195 FTKRKGHILE 0.002 194 VFTKRKGHIL 0.002 24 CVTKAREGSE 0.002 78 PVSCAFVVLM 0.002 160 PTYTDALHVY 0.002 5 RSLEEEKYDM 0.002 61 FQEELEKFQQ 0.002 75 REDPVSCAFV 0.002 140 GETVGGDEIV 0.002 134 GEQRDPGETV 0.002 118 QALRAKPKVY 0.002 212 RMAEAELVQE 0.001 11 KYDMSGARLA 0.001 121 RAKPKVYIIQ 0.001 104 KLENLFEALN 0.001 39 LEHMFRQLRF 0.001 13 DMSGARLALI 0.001 32 SEEDLDALEH 0.001 56 PTAEQFQEEL 0.001 25 VTKAREGSEE 0.001 200 GHILELLTEV 0.001 207 TEVTRRMAEA 0.001 77 DPVSCAFVVL 0.001 110 EALNNKNCQA 0.001 12 YDMSGARLAL 0.001 107 NLFEALNNKN 0.001 113 NNKNCQALRA 0.001 123 KPKVYIIQAC 0.001 179 RHDQKGSCFI 0.001 129 IQACRGEQRD 0.001 v.2-A11-10 mers: 213P1F11 34 DTSPTDMIRK 0.600 40 MIRKAHALSR 0.160 33 QDTSPTDMIR 0.008 43 KAHALSRPWW 0.006 9 GPTPFQDPLY 0.006 6 TVEGPTPFQD 0.006 32 SQDTSPTDMI 0.006 48 SRPWWMCSPR 0.004 47 LSRPWWMCSR 0.004 2 HVYSTVEGPT 0.004 10 PTPFQDPLYL 0.002 52 WMCSRRGKDI 0.002 5 STVEGPTPFQ 0.002 56 RRGKDISWNF 0.001 37 PTDMIRKAHA 0.001 36 SPTDMIRKAH 0.001 46 ALSRPWWMCS 0.001 11 TPFQDPLYLP 0.001 13 FQDPLYLPSE 0.001 45 HALSRPWWMC 0.001 17 LYLPSEAPPN 0.001 21 SEAPPNPPLW 0.001 50 PWWMCSRRGK 0.000 23 APPNPPLWNS 0.000 54 CSRRGKDISW 0.000 38 TDMIRKAHAL 0.000 18 YLPSEAPPNP 0.000 3 VYSTVEGPTP 0.000 44 AHALSRPWWM 0.000 4 YSTVEGPTPF 0.000 26 NPPLWNSQDT 0.000 14 QDPLYLPSEA 0.000 31 NSQDTSPTDM 0.000 19 LPSEAPPNPP 0.000 53 MCSRRGKDIS 0.000 39 DMIRKAHALS 0.000 49 RPWWMCSRRG 0.000 22 EAPPNPPLWN 0.000 15 DPLYLPSEAP 0.000 16 PLYLPSEAPP 0.000 28 PLWNSQDTSP 0.000 42 RKAHALSRPW 0.000 8 EGPTPFQDPL 0.000 7 VEGPTPFQDP 0.000 12 PFQDPLYLPS 0.000 30 WNSQDTSPTD 0.000 51 WWMCSRRGKD 0.000 1 LHVYSTVEGP 0.000 27 PPLWNSQDTS 0.000 41 IRKAHALSRP 0.000 24 PPNPPLWNSQ 0.000 29 LWNSQDTSPT 0.000 55 SRRGKDISWN 0.000 35 TSPTDMIRKA 0.000 20 PSEAPPNPPL 0.000 25 PNPPLWNSQD 0.000 v.3-A11-10 mers: 213P1F11 9 GATLPSPFPY 0.018 10 ATLPSPFPYL 0.015 12 LPSPFPYLSL 0.004 3 IIQACRGATL 0.004 11 TLPSPFPYLS 0.001 1 VYIIQACRGA 0.001 4 IQACRGATLP 0.001 2 UOOQACRGAT 0.001 5 QACRGATLPS 0.000 6 ACRGATLPSP 0.000 7 CRGATLPSPF 0.000 8 RGATLPSPFP 0.000 v.4-A11-10 mers: 213P1F11 41 AFRGSSVHQK 0.200 13 VQPEKRTGLR 0.120 81 SETSASEEEK 0.060 69 IVGRDLSISF 0.040 31 TFRLKEEQGR 0.040 12 SVQPEKRTGL 0.020 9 KSLSVQPEKR 0.018 26 GECGQTFRLK 0.018 63 GGGVGDIVGR 0.012 60 EVFGGGVGDI 0.012 4 CQEYDKSLSV 0.012 65 GVGDIVGRDL 0.006 8 DKSLSVQPEK 0.006 84 SASEEEKYDM 0.004 47 VHQKLVNDPR 0.004 34 LKEEQGRAFR 0.004 70 VGRDLSISFR 0.004 82 ETSASEEEKY 0.003 57 ETQEVFGGGV 0.003 18 RTGLRDENGE 0.003 24 ENGECGQTFR 0.002 30 QTFRLKEEQG 0.002 46 SVHQKLVNDP 0.002 51 LVNDPRETQE 0.002 61 VFGGGVGDIV 0.002 67 GDIVGRDLSI 0.002 50 KLVNDPRETQ 0.002 29 GQTFRLKEEQ 0.002 20 GLRDENGECG 0.001 40 RAFRGSSVHQ 0.001 33 RLKEEQGRAF 0.001 39 GRAFRGSSVH 0.001 3 KCQEYDKSLS 0.001 25 NGECGQTFRL 0.001 58 TQEVFGGGVG 0.001 48 HQKLVNDPRE 0.001 35 KEEQGRAFRG 0.001 76 ISFRNSETSA 0.000 52 VNDPRETQEV 0.000 75 SISFRNSETS 0.000 14 QPEKRTGLRD 0.000 32 FRLKEEQGRA 0.000 43 RGSSVHQKLV 0.000 2 GKCQEYDKSL 0.000 77 SFRNSETSAS 0.000 10 SLSVQPEKRT 0.000 42 FRGSSVHQKL 0.000 53 NDPRETQEVF 0.000 38 QGRAFRGSSV 0.000 71 GRDLSISFRN 0.000 56 RETQEVFGGG 0.000 68 DIVGRDLSIS 0.000 23 DENGECGQTF 0.000 37 EQGRAFRGSS 0.000 79 RNSETSASEE 0.000 44 GSSVHQKLVN 0.000 6 EYDKSLSVQP 0.000 73 DLSISFRNSE 0.000 5 QEYDKSLSVQ 0.000 86 SEEEKYDMSG 0.000 27 ECGQTFRLKE 0.000 64 GGVGDIVGRD 0.000 59 QEVFGGGVGD 0.000 17 KRTGLRDENG 0.000 22 RDENGECGQT 0.000 54 DPRETQEVFG 0.000 62 FGGGVGDIVG 0.000 45 SSVHQKLVND 0.000 19 TGLRDENGEC 0.000 74 LSISFRNSET 0.000 80 NSETSASEEE 0.000 1 MGKCQEYDKS 0.000 21 LRDENGECGQ 0.000 85 ASEEEKYDMS 0.000 66 VGDIVGRDLS 0.000 7 YDKSLSVQPE 0.000 78 FRNSETSASE 0.000 83 TSASEEEKYD 0.000 28 CGQTFRLKEE 0.000 72 RDLSISFRNS 0.000 55 PRETQEVFGG 0.000 16 EKRTGLRDEN 0.000 11 LSVQPEKRTG 0.000 49 QKLVNDPRET 0.000 36 EEQGRAFRGS 0.000 15 PEKRTGLRDE 0.000 v.5-A11-10 mers: 213P1F11 4 RLALILRVTK 1.200 6 ALILRVTKAR 0.060 2 GARLALILRV 0.012 1 SGARLALILR 0.008 10 RVTKAREGSE 0.006 5 LALILRVTKA 0.003 7 LILRVTKARE 0.001 8 ILRVTKAREG 0.001 9 LRVTKAREGS 0.000 3 ARLALILRVT 0.000 v.6-A11-10 mers: 213P1F11 4 ENLFEAMNNK 0.018 10 MNNKNCQALR 0.008 9 AMNNKNCQAL 0.004 2 KLENLFEAMN 0.001 8 EAMNNKNCQA 0.001 5 NLFEAMNNKN 0.001 1 VKLENLFEAM 0.000 3 LENLFEAMNN 0.000 6 LFEAMNNKNC 0.000 7 FEAMNNKNCQ 0.000
[0763] 16 TABLE XIII Pos 123456789 Score SeqID v.1-A24-9 mers: 213P1F11 11 KYDMSGARL 400.000 168 VYSTVEGYI 70.000 60 QFQEELEKF 19.800 104 KLENLFEAL 17.280 227 TNPEIQSTL 10.080 183 KGSCFIQTL 9.600 112 LNNKNCQAL 7.200 31 GSEEDLDAL 7.200 38 ALEHMFRQL 7.200 57 TAEQFQEEL 6.600 233 STLRKRLYL 6.000 158 TIPTYTDAL 6.000 161 TYTDALHVY 6.000 177 AYRHDQKGS 5.000 194 VFTKRKGHI 5.000 15 SGARLALIL 4.800 231 IQSTLRKRL 4.800 101 EMVKLENLF 4.320 87 MAHGREGFL 4.000 195 FTKRKGHIL 4.000 13 DMSGARLAL 4.000 187 FIQTLVDVF 3.600 35 DLDALEHMF 2.400 121 RAKPKVYII 2.400 223 KARKTNPEI 2.200 86 LMAHGREGF 2.000 64 ELEKFQQAI 1.800 144 GGDEIVMVI 1.680 44 RQLRFESTM 1.500 151 VILKDSPQTI 1.440 108 LFEALNNKN 1.188 198 RKGHILELL 1.120 14 MSGARLALI 1.000 6 SLEEEKYDM 0.900 197 KRKGHILEL 0.880 205 LLTEVTRRM 0.840 126 VYIIQACRG 0.750 174 GYIAYRHDQ 0.750 186 CFIQTLVDV 0.750 75 REDPVSCAF 0.672 42 MFRQLRFES 0.660 100 GEMVKLENL 0.600 28 AREGSEEDL 0.600 94 FLKGEDGEM 0.550 79 VSCAFVVLM 0.500 142 TVGGDEIVM 0.500 78 PVSCAFVVL 0.480 53 KRDPTAEQF 0.480 97 GEDGEMVKL 0.440 210 TRRMAEAEL 0.440 226 KTHPEIQST 0.432 179 RHDQKGSCF 0.400 40 EHMFRQLRF 0.300 18 RLALILCVT 0.280 199 KGHILELLT 0.240 201 HILELLTEV 0.238 99 DGEMVKLEM 0.231 155 SPQTIPTYT 0.210 147 EIVMVIKDS 0.210 164 DALHVYSTV 0.210 123 KPKVYIIQA 0.200 157 QTIPTYTDA 0.180 202 ILELLTEVT 0.180 170 STVEGYIAY 0.180 20 ALILCVTKA 0.165 219 VQEGKARKT 0.165 118 QALRAKPKV 0.165 50 STMKRDPTA 0.150 150 MVIKDSPQT 0.150 47 RFESTMKRD 0.150 154 DSPQTIPTY 0.150 206 LTEVTRRMA 0.150 106 ENLFEALNN 0.150 111 ALNNKNCQA 0.150 67 KFQQAIDSR 0.150 141 ETVGGDEIV 0.150 77 DPVSCAFVV 0.150 180 HDQKGSCFI 0.150 80 SCAFVXTLMA 0.140 188 TQTLVDVFT 0.140 184 GSCFIQTLV 0.140 30 EGSEEDLDA 0.120 143 VGGDEIVMV 0.120 162 YTDALHVYS 0.120 73 DSREDPVSC 0.120 135 EQRDPGETV 0.120 208 EVTRRMAEA 0.110 140 GETVGGDEI 0.110 232 QSTLRKRLY 0.100 16 GARLALILC 0.100 119 ALRAKPKVY 0.100 167 HVYSTVEGY 0.100 24 CVTKAREGS 0.100 49 ESTMKRDPT 0.100 71 AIDSREDPV 0.100 120 LRAKPKVYI 0.100 169 YSTVEGYIA 0.100 34 EDLDALEHM 0.090 82 AFVVLMAHG 0.090 93 GFLKGEDGE 0.075 v.2-A24-9 mers: 213P1F11 2 VYSTVEGPT 7.000 56 RGKDISWNF 6.720 38 DMIRKAHAL 6.000 8 GPTPFQDPL 4.800 10 TPFQDPLYL 4.000 4 STVEGPTPF 3.600 52 MCSRRGKDI 1.000 16 LYLPSEAPP 0.900 44 HALSRPWWM 0.750 31 SQDTSPTDM 0.500 20 SEAPPNPPL 0.480 42 KAHALSRPW 0.240 14 DPLYLPSEA 0.198 21 EAPPNPPLW 0.180 22 APPNPPLWN 0.150 17 YLPSEAPPN 0.150 12 FQDPLYLPS 0.150 35 SPTDMIRKA 0.132 46 LSRPWWMCS 0.120 45 ALSRPWWMC 0.100 29 WNSQDTSPT 0.100 32 QDTSPTDMI 0.100 53 CSRRGKDIS 0.100 7 EGPTPFQDP 0.022 48 RPWWMCSRR 0.020 55 RRGKDISWN 0.020 26 PPLWNSQDT 0.018 23 PPNPPLWNS 0.018 30 NSQDTSPTD 0.018 9 PTPFQDPLY 0.015 50 WWMCSRRGK 0.015 25 NPPLWNSQD 0.015 34 TSPTDMIRK 0.015 28 LWNSQDTSD 0.015 37 TDNIRKAHA 0.015 5 TVEGPTPFQ 0.015 18 LPSEAPPNP 0.012 33 DTSPTDMIR 0.012 51 WMCSRRGKD 0.011 54 SRRGKDISW 0.010 43 AHALSRPWW 0.010 27 PLWNSQDTS 0.010 3 YSTVEGPTP 0.010 1 HVYSTVEGP 0.010 11 PFQDPLYLP 0.009 24 PNPPLWNSQ 0.003 41 RKAHALSRP 0.002 19 PSEAPPNPP 0.002 47 SRPWWMCSR 0.002 13 QDPLYLPSE 0.002 36 PTDMIRKAH 0.001 6 VEGPTPFQD 0.001 40 IRKAHALSR 0.001 49 PWWMCSRRG 0.001 15 PLYLPSEAP 0.001 v.3-A24-9 mers: 213P1F11 10 TLPSPFPYL 7.200 7 RGATLPSPF 4.800 3 IQACRGATL 4.000 12 PSPFPYLSL 0.600 9 ATLPSPFPY 0.180 2 IIQACRGAT 0.150 1 YIIQACRGA 0.150 11 LPSPFPYLS 0.120 5 ACRGATLPS 0.100 4 QACRGATLP 0.010 8 GATLPSPFP 0.010 6 CRGATLPSP 0.001 v.4-A24-9 mers: 213P1F11 43 RGSSVHQKL 14.784 3 KCQEYDKSL 14.400 13 VQPEKRTGL 7.200 66 VGDIVGRDL 5.600 61 VFGGGVGDI 5.000 24 ENGECGQTF 2.880 70 VGRDLSISF 2.880 54 DPRETQEVF 2.400 68 DIVGRDLSI 1.500 85 ASEEEKYDM 0.900 77 SFRNSETSA 0.500 6 EYDKSLSVQ 0.500 26 GECGQTFRL 0.400 34 LKEEQGRAF 0.360 50 KLVNDPRET 0.330 33 RLKEEQGRA 0.240 4 CQEYDKSLS 0.150 11 LSVQPEKRT 0.150 58 TQEVFGGGV 0.150 45 SSVHQKLVN 0.150 62 FGGGVGDIV 0.140 20 GLRDENGEC 0.132 37 EQGRAFRGS 0.120 73 DLSISFRNS 0.120 83 TSASEEEKY 0.110 75 SISFRNSET 0.110 44 GSSVHQKLV 0.100 38 QGRAFRGSS 0.100 69 IVGRDLSIS 0.100 76 ISFRNSETS 0.100 41 AFRGSSVHQ 0.050 31 TFRLKEEQG 0.050 9 KSLSVQPEK 0.046 72 RDLSISFRN 0.042 57 ETQEVFGGG 0.030 17 KRTGLRDEN 0.026 79 RNSETSASE 0.024 40 RAFRGSSVH 0.020 18 RTGLRDENG 0.020 53 NDPRETQEV 0.020 86 SEEEKYDMS 0.018 12 SVQPEKRTG 0.018 74 LSISFRNSE 0.018 51 LVNDPRETQ 0.018 19 TGLRDENGE 0.018 65 GVGDIVGRD 0.017 28 CGQTFRLKE 0.017 80 NSETSASEE 0.017 23 DENGECGQT 0.015 25 NGECGQTFR 0.015 64 GGVGDIVGR 0.015 67 GDIVGRDLS 0.015 78 FRNSETSAS 0.015 14 QPEKRTGLR 0.015 48 HQKLVNDPR 0.014 30 QTFRLKEEQ 0.013 5 QEYDKSLSV 0.012 52 VNDPRETQE 0.012 27 ECGQTFRLK 0.012 84 SASEEEKYD 0.012 2 GKCQEYDKS 0.011 10 SLSVQPEKR 0.011 29 GQTFRLKEE 0.011 82 ETSASEEEK 0.011 39 GRAFRGSSV 0.010 46 SVGQKLVND 0.010 60 EVFGGGVGD 0.010 63 GGGVGDIVG 0.010 1 MGKCQEYDE 0.010 35 KEEQGRAFR 0.003 22 RDENGECGQ 0.003 47 VHQKLVNDP 0.002 56 RETQEVFGG 0.002 32 FRLKEEQGR 0.002 59 QEVFGGGVG 0.002 36 EEQGRAFRG 0.002 49 QKLVNDPRE 0.002 8 DKSLSVQPE 0.001 7 YDKSLSVQP 0.001 21 LRDENGECG 0.001 81 SETSASEEE 0.001 42 FRGSSVHQK 0.001 16 EKRTGLRDE 0.001 71 GRDLSISFR 0.001 55 PRETQEVPG 0.000 15 PEKRTGLRD 0.000 v.5-A24-9 mers: 213P1F11 3 RLALILRVT 0.280 9 RVTKAREGS 0.200 5 ALILRVTKA 0.165 6 LILRVTKAR 0.021 2 ARLALILRV 0.018 4 LALILRVTK 0.018 1 GARLALILR 0.010 7 ILRVTKARE 0.010 8 LRVTKAREG 0.002 v.6-A24-9 mers: 213P1F11 9 MNNKNCQAL 7.200 1 KLENLFEAM 2.160 5 LFEAMNNKN 0.990 3 ENLFEAMNN 0.150 8 AMNNKNCQA 0.150 7 EAMNNKNCQ 0.018 2 LENLFEAMN 0.015 4 NLFEAMNNK 0.014 6 FEAMNNKNC 0.010
[0764] 17 TABLE XIV Pos 1234567890 Score SeqID v.1-A24-10 mers: 213P1F11 226 KTNPEIQSTL 20.160 194 VFTKRKGHIL 20.000 186 CFIQTLVDVF 18.000 96 KGEDGEMVKL 15.840 11 KYDMSGARLA 10.000 27 KAREGSEEDL 9.600 37 DALEHMFRQL 8.640 161 TYTDALHVYS 7.200 111 ALNNKNCQAL 7.200 230 EIQSTLRKRL 7.200 77 DPVSCAFVVL 7.200 157 QTIPTYTDAL 7.200 99 DGEMVKLENL 6.000 177 AYRHDQKGSC 5.000 168 VYSTVEGYIA 5.000 14 MSGARLALIL 4.800 30 EGSEEDLDAL 4.800 209 VTRRMAEAEL 4.400 93 GFLKGEDGEM 4.125 232 QSTLRKRLYL 4.000 86 LMAHGREGFL 4.000 85 VLMAHGREGF 3.000 59 EQFQEELEKF 2.200 150 MVIKDSPQTI 1.800 5 RSLEEEKYDM 1.800 143 VGGDEIVMVI 1.680 167 HVYSTVECYI 1.400 197 KRKGHILELL 1.120 204 ELLTEVTRRM 1.050 103 VKLENLFEAL 1.037 119 ALRAKPKVYI 1.000 193 DVFTKRKGHI 1.000 13 DMSGARLALI 1.000 141 ETVGGDEIVM 0.750 108 LFEALNNKNC 0.750 126 VYIIQACRCE 0.750 174 GYIAYRHDQK 0.750 12 YDMSGARLAL 0.600 42 MFRQLRFEST 0.600 56 PTAEQFQEEL 0.528 74 SREDPVSCAF 0.504 10 EKYDMSGARL 0.480 182 QKGSCFIQTL 0.480 196 TKRKGHILEL 0.440 100 GEMVKLENLF 0.432 34 EDLDALEHMF 0.432 123 KPKVYIIQAC 0.336 133 RGEQRDPGET 0.330 104 KLENLFEALN 0.300 183 KGSCFIQTLV 0.280 52 MKRDPTAEQF 0.240 178 YRHDQKGSCF 0.240 63 EELEKFQQAI 0.216 201 HILELLTEVT 0.216 67 KFQQAIDSRE 0.210 187 FIQTLVDVFT 0.210 154 DSPQTIPTYT 0.210 179 RHDQKGSCFI 0.200 39 LEHMFRQLRF 0.200 218 LVQEGKARKT 0.198 107 NLFEALNNKN 0.190 6 SLEEEKYDMS 0.180 70 QAIDSREDPV 0.180 139 PGETVGGDEI 0.165 19 LALILCVTKA 0.165 215 EAELVQEGKA 0.165 47 RFESTMKRDP 0.150 23 LCVTKAREGS 0.150 118 QALRAKPKVY 0.150 149 VMVIKDSPQT 0.150 110 EALNNKNCQA 0.150 219 VQEGKARKTN 0.150 79 VSCAFVVLMA 0.140 41 HMFRQLRFES 0.132 73 DSREDPVSCA 0.120 181 DQKGSCFIQT 0.120 205 LLTEVTRRMA 0.120 16 GARLALILCV 0.120 102 MVKLENLFEA 0.110 3 NPRSLEEEKY 0.110 222 GKARKTNPEI 0.110 117 CQALRAKPKV 0.110 60 QFQEELEKFQ 0.108 169 YSTVEOYIAY 0.100 176 IAYRHDQKGS 0.100 185 SCFIQTLVDV 0.100 120 LRAKPKVYII 0.100 231 IQSTLRKRLY 0.100 113 NNKNCQALRA 0.100 142 TVGGDEIVMV 0.100 94 FLKGEDGEMV 0.100 49 ESTMKRDPTA 0.100 15 SGARLALILC 0.100 162 YTDAILHVYST 0.100 71 AIDSREDPVS 0.100 159 IPTYTDALHV 0.100 82 AFVVLMAHGR 0.075 43 FRQLRFESTM 0.075 78 PVSCAFVVLM 0.050 33 EEDLDALEHM 0.050 v.2-A24-10 mers: 213P1F11 17 LYLPSEAPPN 9.000 8 EGPTPFQDPL 6.000 4 YSTVEGPTPF 2.000 32 SQDTSPTDMI 1.000 52 WMCSRRGKDI 1.000 31 NSQDTSPTDM 0.900 20 PSEAPPNPPL 0.600 10 PTPFQDPLYL 0.600 38 TDMIRKAHAL 0.600 56 RRGKDISWNF 0.560 3 VYSTVEGPTP 0.500 43 KAHALSRPWW 0.200 26 NPPLWNSQDT 0.180 22 EAPPNPPLWN 0.180 35 TSPTDMIRKA 0.165 29 LWNSQDTSPT 0.150 23 APPNPPLWNS 0.150 39 DMIRKAHALS 0.150 45 HALSRPWWMC 0.150 2 HVYSTVEGPT 0.140 9 GPTPFQDPLY 0.120 12 PFQDPLYLPS 0.108 46 ALSRPWWMCS 0.100 53 MCSRRGKDIS 0.100 54 CSRRGKDISW 0.100 44 AHALSRPWWM 0.050 42 RKAHALSRPW 0.024 49 RPWWMCSRRG 0.020 14 QDPLYLPSEA 0.020 5 STVEGPTPFQ 0.018 36 SPTDMIRKAH 0.017 51 WWMCSRRGKD 0.017 18 YLPSEAPPNP 0.015 6 TVEGPTPFQD 0.015 27 PPLWNSQDTS 0.015 15 DPLYLPSEAP 0.015 19 LPSEAPPNPP 0.014 13 FQDPLYLPSE 0.012 21 SEAPPNRPLW 0.012 34 DTSPTDMIRK 0.012 47 LSRPWWMCSR 0.012 55 SRRGKDISWN 0.010 37 PTDMIRKAHA 0.010 11 TPFQDPLYLP 0.010 30 WNSQDTSPTD 0.010 40 MTRKAHALSR 0.010 24 PPNPPLWNSQ 0.003 25 PNPPLWNSQD 0.002 7 VEGPTPFQDP 0.002 48 SRPWWMCSRR 0.002 1 LHVYSTVEGP 0.002 50 PWWMCSRRGK 0.001 28 PLWNSQDTSP 0.001 33 QDTSPTDMIR 0.001 41 IRKAHALSRP 0.001 16 PLYLPSEAPP 0.001 v.3-A24-10 mers: 213P1F11 10 ATLPSPFPYL 8.640 1 VYIIQACRGA 7.500 3 IIQACRGATL 6.000 12 LPSPFPYLSL 4.800 7 CRGATLPSPF 0.240 11 TLPSPFPYLS 0.150 2 YIIQACRGAT 0.150 5 QACRGATLPS 0.100 9 GATLPSPFPY 0.100 8 RGATLPSPFP 0.020 4 IQACRGATLP 0.010 6 ACRGATLPSP 0.010 v.4-A24-10 mers: 213P1F11 12 SVQPEKRTGL 7.200 65 GVGDIVGRDL 6.720 25 NGECGQTFRL 6.000 33 RLKEEQGRAF 4.800 69 IVGRDLSISF 2.400 60 EVFGGGVGDI 1.000 42 FRGSSVHQKL 0.739 61 VFGGGVGDIV 0.700 84 SASEEEKYDM 0.600 6 EYDKSLSVQP 0.600 77 SFRNSETSAS 0.500 2 GKCQEYDKSL 0.400 23 DENGECGQTF 0.360 3 KCQEYDKSLS 0.360 53 NDPRETQEVF 0.300 43 RGSSVHQKLV 0.200 85 ASEEEKYDMS 0.180 57 ETQEVFGGGV 0.180 74 LSISFRNSET 0.165 19 TGLRDENGEC 0.165 52 VNDPRETQEV 0.158 68 DIVGRDLSIS 0.150 67 GDIVGRDLSI 0.150 4 CQEYDKSLSV 0.150 82 ETSASEEEKY 0.110 1 MGKCQEYDKS 0.110 38 QGRAFRGSSV 0.100 44 GSSVHQKLVN 0.100 37 EQGRAFRGSS 0.100 76 ISFRNSETSA 0.100 10 SLSVQPEKRT 0.100 66 VGDIVGRDLS 0.100 75 SISFRNSETS 0.100 31 TFRLKEEQGR 0.060 41 AFRGSSVHQK 0.050 72 RDLSISFRNS 0.036 9 KSLSVQPEKR 0.033 22 RDENGECGQT 0.030 50 KLVNDPRETQ 0.030 79 RNSETSASEE 0.026 18 RTGLRDENGE 0.024 64 GGVGDIVGRD 0.021 40 RAFRGSSVHQ 0.020 36 EEQGRAFRGS 0.018 13 VQPEKRTGLR 0.018 51 LVNDPRETQE 0.018 28 CGQTFRLKEE 0.017 80 NSETSASEEE 0.017 49 QKLVNDPRET 0.017 58 TQEVFGGGVG 0.015 11 LSVQPEKRTG 0.0 15 14 QPEKRTGLRD 0.015 32 FRLKEEQGRA 0.015 45 SSVHQKLVND 0.015 46 SVHQKLVNDP 0.014 71 GRDLSISFRN 0.014 16 EKRTGLRDEN 0.013 29 GQTFRLKEEQ 0.013 70 VGRDLSISFR 0.012 73 DLSISFRNSE 0.012 20 GLRDENGECG 0.012 54 DPRETQEVFG 0.012 24 ENGECGQTFR 0.012 27 ECGQTFRLKE 0.011 30 QTFRLKEEQG 0.010 83 TSASEEEKYD 0.010 63 GGGVGDIVGR 0.010 48 HQKLVNDPRE 0.010 62 FGGGVGDIVG 0.010 56 RETQEVFGGG 0.003 35 KEEQGPAFRG 0.003 47 VHQKLVNDPR 0.002 17 KRTGLRDENG 0.002 34 LKEEQGRAFR 0.002 86 SEEEKYDMSG 0.002 8 DKSLSVQPEK 0.002 59 QEVFGGGVGD 0.002 78 FRNSETSASE 0.002 26 GECGQTFRLK 0.001 7 YDKSLSVQPE 0.001 21 LRDENGECGQ 0.001 5 QEYDKSLSVQ 0.001 81 SETSASEEEK 0.001 39 GRAFRGSSVH 0.001 55 PRETQEVFGG 0.000 15 PEKRTGLRDE 0.000 v.5-A24-10 mers: 213P1F11 5 LALILRVTKA 0.165 2 GARLALILRV 0.120 4 RLALILRVTK 0.024 3 ARLALILRVT 0.021 6 ALILRVTKAR 0.021 10 RVTKAREGSE 0.020 7 LILRVTKARE 0.015 9 LRVTKAREGS 0.015 8 ILRVTKAREG 0.011 1 SGARLALILR 0.010 v.6-A24-10 mers: 213P1F11 9 AMNNKNCQAL 7.200 6 LFEAMNNKNC 0.750 2 KLENLFEAMN 0.300 5 NLFEAMNNKN 0.158 8 EANNNKNCQA 0.150 1 VKLENLFEAM 0.130 4 ENLFEANNNK 0.018 3 LENLFEAMNN 0.015 10 MNNKNCQALR 0.015 7 FEAMNNKNCQ 0.001
[0765] 18 TABLE XV Pos 123456789 Score SeqID v.1-B7-9 mers: 213P1F11 87 MAHGREGFL 12.000 223 KARKTNPEI 12.000 13 DMSGARLAL 6.000 233 STLRKRLYL 6.000 231 IQSTLRKRL 6.000 142 TVGGDEIVM 5.000 112 LNNKNCQAL 4.000 227 TNPEIQSTL 4.000 210 TRRMAEAEL 4.000 183 KGSCFTQTL 4.000 77 DPVSCAFVV 4.000 158 TIPTYTDAL 4.000 15 SGARLALIL 4.000 195 FTKRKGHIL 4.000 57 TAEQFQEEL 3.600 38 ALEHMFRQL 3.600 16 GARLALILC 3.000 135 EQRDPGETV 3.000 3 NPRSLEEEK 2.000 155 SPQTIPTYT 2.000 78 PVSCAFVVL 2.000 123 KPKVYIIQA 2.000 31 GSEEDLDAL 1.200 121 RAKPKVYII 1.200 100 GEMVKLENL 1.200 104 KLENLFEAL 1.200 94 FLKGEDGEM 1.000 73 DSREDPVSC 1.000 79 VSCAFVVLM 1.000 44 RQLRFESTM 1.000 205 LLTEVTRRM 1.000 118 QALRAKPKV 0.600 164 DALHVYSTV 0.600 119 ALRAKPKVY 0.600 208 EVTRRMAEA 0.500 150 MVIKDSPQT 0.500 197 KRKGHILEL 0.400 198 RKGHILELL 0.400 14 MSGARLALI 0.400 151 VIKDSPQTI 0.400 28 AREGSEEDL 0.360 131 ACRGEQRDP 0.300 27 KAREGSEED 0.300 111 ALNNKNCQA 0.300 20 ALILCVTKA 0.300 6 SLEEEKYDM 0.300 50 STMKRDPTA 0.300 55 DPTAEQFQE 0.200 184 GSCFTQTLV 0.200 159 IPTYTDALH 0.200 143 VGGDEIVMV 0.200 201 HILELLTEV 0.200 138 DPGETVGGD 0.200 141 ETVGGDEIV 0.200 71 AIDSREDPV 0.180 24 CVTKAREGS 0.150 148 IVMVIKDSP 0.150 49 ESTMKRDPT 0.150 97 GEDGEMVKL 0.120 144 GGDEIVMVI 0.120 11 KYDMSGARL 0.120 64 ELEKFQQAI 0.120 199 KGHILELLT 0.100 80 SCAFVVLMA 0.100 34 EDLDALEHM 0.100 89 HGREGFLKG 0.100 234 TLRKRLYLQ 0.100 45 QLRFESTMK 0.100 188 IQTLVDVFT 0.100 167 HVYSTVEGY 0.100 18 RLALILCVT 0.100 226 KTNPEIQST 0.100 209 VTRRMAEAE 0.100 157 QTIPTYTDA 0.100 169 YSTVEGYIA 0.100 30 EGSEEDLDA 0.100 193 DVFTKRKGH 0.075 177 AYRHDQKGS 0.060 120 LRAKPKVYI 0.060 211 RRMAEAELV 0.060 17 ARLALILCV 0.060 228 NPEIQSTLR 0.060 218 LVQEGKARK 0.050 125 KVYIIQACR 0.050 102 MVKLENLFE 0.050 84 VVLMAHGRE 0.050 83 FVVLMAHGR 0.050 70 QAIDSREDP 0.045 206 LTEVTRRMA 0.045 140 GETVGGDEI 0.040 168 VYSTVEGYI 0.040 194 VFTKRKGHI 0.040 180 HDQKGSCFI 0.040 86 LMAHGREGF 0.030 19 LALILCVTK 0.030 37 DALEHMFRQ 0.030 12 YDMSGARLA 0.030 165 ALHVYSTVE 0.030 85 VLMAHGREG 0.030 216 AELVQEGKA 0.030 v.2-B7-9 mers 8 GPTPFQDPL 80.000 10 TPFQDPLYL 80.000 38 DMIRKAHAL 4.000 44 HALSRPWWM 3.000 14 DPLYLPSEA 2.000 35 SPTDMIRKA 2.000 22 APPNPPLWN 1.800 20 SEAPPNPPL 0.600 45 ALSRPWWMC 0.450 52 MCSRRGKDI 0.400 31 SQDTSPTDM 0.300 48 RPWWMCSRR 0.200 46 LSRPWWMCS 0.200 53 SCRRGKDIS 0.200 39 MIRKAHALS 0.200 25 NPPLWNSQD 0.200 26 PPLWNSQDT 0.200 18 LPSEAPPNP 0.200 29 WNSQDTSPT 0.100 42 KAHALSRPW 0.060 32 QDTSPTDMI 0.060 23 PPNPPLWNS 0.060 21 EAPPNPPLW 0.060 1 HVYSTVEGP 0.050 37 TDMIRKAHA 0.030 5 TVEGPTPFQ 0.023 54 SRRGKDISW 0.020 56 RGKDISWNF 0.020 4 STVEGPTPF 0.020 17 YLPSEAPPN 0.020 7 EGPTPFQDP 0.015 51 WMCSRRGKD 0.015 33 DTSPTDMIR 0.010 2 VYSTVEGPT 0.010 34 TSPTDMIRK 0.010 30 NSQDTSPTD 0.010 3 YSTVEGPTP 0.010 43 AHALSRPWW 0.009 12 FQDPLYLPS 0.006 50 WWMCSRRGK 0.005 9 PTPFQDPLY 0.002 55 RRGKDISWN 0.002 27 PLWNSQDTS 0.002 15 PLYLPSEAP 0.002 41 RKAHALSRP 0.001 6 VEGPTPFQD 0.001 13 QDPLYLPSE 0.001 40 IRKAHALSR 0.001 47 SRPWWMCSR 0.001 24 PNPPLWNSQ 0.001 16 LYLPSEAPP 0.001 28 LWNSQDTSP 0.001 19 PSEAPPNPP 0.000 36 PTDMIRKAH 0.000 11 PFQDPLYLP 0.000 49 PWWMCSRRG 0.000 v.3-B7-9 mers: 213P1F11 10 TLPSPFPYL 6.000 3 IQACRGATL 4.000 5 ACRGATLPS 0.600 12 PSPFPYLSL 0.600 11 LPSPFPYLS 0.400 2 IIQACRGAT 0.150 1 YIIQACRGA 0.100 9 ATLPSPFPY 0.060 8 GATLPSPFP 0.045 4 QACRGATLF 0.030 7 RGATLPSPF 0.020 6 CRGATLPSP 0.001 v.4-B7-9 mers: 213P1F11 13 VQPEKRTGL 6.000 3 KCQEYDKSL 4.000 54 DPRETQEVF 4.000 43 RGSSVHQKL 4.000 66 VGDIVGRDL 1.200 20 GLRDENGEC 1.000 85 ASEEEKYDM 0.900 26 GECGQTFRL 0.400 68 DIVGRDLSI 0.400 38 QGRAFRGSS 0.300 62 FGGGVGDIV 0.200 70 VGRDLSISF 0.200 44 GSSVHQKLV 0.200 11 LSVQPEKRT 0.150 51 LVNDPRETQ 0.113 77 SFRNSETSA 0.100 69 IVGRDLSIS 0.100 75 SISFRNSET 0.100 33 RLKEEQGRA 0.100 50 KLVNDPRET 0.100 14 QPEKRTGLR 0.060 58 TQEVFGGGV 0.060 60 EVFGGGVGD 0.050 12 SVQPEKRTG 0.050 46 SVHQKLVND 0.050 65 GVGDIVGRD 0.050 61 VFGGGVGDI 0.040 84 SASEEEKYD 0.030 41 AFRGSSVHQ 0.030 40 RAFRGSSVH 0.030 83 TSASEEEKY 0.020 37 EQGRAFRGS 0.020 73 DLSISFRNS 0.020 24 ENGECGQTF 0.020 39 GRAFRGSSV 0.020 53 NDPRETQEV 0.020 5 QEYDKSLSV 0.020 45 SSVHQKLVN 0.020 76 ISFRNSETS 0.020 74 LSISFRNSE 0.015 28 CGQTRFLKE 0.015 27 ECGQTFRLK 0.010 9 KSLSVQPEK 0.010 23 DENGECGQT 0.010 30 QTFRLKEEQ 0.010 10 SLSVQPEKR 0.010 1 MGKCQEYDK 0.010 63 GGGVGDIVG 0.010 82 ETSASEEEK 0.010 57 ETQEVFGGG 0.010 64 GGVGDIVGR 0.010 18 RTGLRDENG 0.010 16 EKRTGLRDE 0.010 48 HQKLVNDPR 0.010 29 GQTFRLKEE 0.010 79 RNSETSASE 0.010 19 TGLRDENGE 0.010 31 TFRLKEEQG 0.010 4 CGEYDKSLS 0.006 67 GDIVGRDLS 0.003 17 KRTGLRDEN 0.003 52 VNDPRETQE 0.003 25 NGECGQTFR 0.003 80 NSETSASEE 0.003 2 GKCQEYDKS 0.002 72 RDLSISFRN 0.002 78 FRNSETSAS 0.002 59 QEVFGGGVG 0.001 47 VHQKLVNDP 0.001 42 FRGSSVHQK 0.001 81 SETSASEEE 0.001 49 QKLVNDPRE 0.001 36 EEQGRAFRG 0.001 8 DKSLSVQPE 0.001 32 FRLKEEQGR 0.001 7 YDKSLSVQP 0.001 56 RETQEVFGG 0.001 34 LKEEQGRAF 0.001 86 SEEEKYDMS 0.001 35 KEEQGRAFR 0.000 6 EYDKSLSVQ 0.000 21 LRDENGECG 0.000 71 GRDLSISFR 0.000 22 RDENGECGQ 0.000 15 PEKRTGLRD 0.000 55 PRETQEVFG 0.000 v.5-B7-9 mers: 213P1F11 1 GARLALILR 0.300 5 ALILRVTKA 0.300 9 RVTKAREGS 0.150 7 ILRVTKARE 0.100 3 RLALILRVT 0.100 2 ARLALILRV 0.060 4 LALILRVTK 0.045 6 LILRVTKAR 0.010 8 LRVTKAREG 0.001 v.6-B7-9 mers: 213P1F11 9 MNNKNCQAL 4.000 8 AMNNKNCQA 0.300 1 KLENLFEAM 0.300 7 EAMNNKNCQ 0.090 3 ENLFEAMNN 0.020 6 FEAMNNKNC 0.010 4 NLFEAMNNK 0.010 2 LENLFEAMN 0.002 5 LFEAMNNKN 0.001
[0766] 19 TABLE XVI Pos 1234567890 Score SeqID v.1-B7-10 mers:213P1F11 27 KAREGSEEDL 120.000 77 DPVSCAFVVL 80.000 209 VTRRMAEAEL 40.000 119 ALRAKPKVYI 18.000 111 ALNNKNCQAL 12.000 37 DALEHMFRQL 12.000 16 GARLALILCV 6.000 232 QSTLRKRLYL 6.000 230 EIQSTLRKRL 6.000 157 QTIPTYTDAL 4.000 3 NPRSLEEEKY 4.000 226 KTNPEIQSTL 4.000 196 TKRKGHILEL 4.000 159 IPTYTDALHV 4.000 30 EGSEEDLDAL 4.000 14 MSGARLALIL 4.000 86 LMAHGREGFL 4.000 193 DVFTKRKGHI 2.000 167 HVYSTVEGYT 2.000 150 MVIKDSPQTI 2.000 123 KPKVYIIQAC 2.000 12 YDMSGARLAL 1.800 73 DSREDPVSCA 1.500 96 KGEDGEMVKL 1.200 99 DGEMVKLENL 1.200 5 RSLEEEKYDM 1.000 204 ELLTEVTRRM 1.000 142 TVGGDEIVMV 1.000 141 ETVGGDEIVM 1.000 70 QAIDSREDPV 0.600 78 PVSCAFVVLM 0.500 102 MVKLENLFEA 0.500 218 LVQEGKARKT 0.500 131 ACRGEQRDPG 0.450 10 EKYDMSGARL 0.400 13 DMSGARLALI 0.400 194 VFTKRKGHIL 0.400 103 VKLENLFEAL 0.400 182 QKGSCFIQTL 0.400 143 VGGDEIVMVI 0.400 56 PTAEQFQEEL 0.400 197 KRKGHILELL 0.400 223 KARKTNPEIQ 0.300 19 LALILCVTKA 0.300 155 SPQTIPTYTD 0.300 177 AYRHDQKGSC 0.300 110 EALNNKNCQA 0.300 183 KGSCFIQTLV 0.200 94 FLKGEDGEMV 0.200 185 SCFIQTLVDV 0.200 138 DPGETVGGDE 0.200 55 DPTAEQFQEE 0.200 210 TRRMAEAELV 0.200 117 CQALRAKPKV 0.200 205 LLTEVTRRMA 0.150 148 IVMVIKDSPQ 0.150 135 EQRDPCETVG 0.100 42 MFRQLRFEST 0.100 79 VSCAFVVLMA 0.100 89 HGREGFLKGE 0.100 113 NNKNCQALRA 0.100 149 VMVIKDSPQT 0.100 181 DQKGSCFIQT 0.100 93 GFLKGEDGEM 0.100 43 FRQLRFESTM 0.100 15 SGARLALILC 0.100 45 QLRFESTMKR 0.100 187 FIQTLVDVFT 0.100 201 HILELLTEVT 0.100 154 DSPQTIPTYT 0.100 49 ESTMKRDPTA 0.100 85 VLMAHGREGF 0.090 215 EAELVQEGKA 0.090 228 NPEIQSTLRK 0.060 176 IAYRHDQKGS 0.060 118 QALPAKPKVY 0.060 84 VVLMAHGREG 0.050 125 KVYIIQACRG 0.050 208 EVTRRMAEAE 0.050 24 CVTKAREGSE 0.050 83 FVVLMAHGRE 0.050 63 EELEKFQQAI 0.040 120 LRAKPKVYII 0.040 222 GKARKTNPEI 0.040 130 QACRGEQRDP 0.030 134 GEQRDPGETV 0.030 23 LCVTKAREGS 0.030 165 ALHVYSTVEG 0.030 17 ARLALILCVT 0.030 20 ALILCVTKAR 0.030 122 AKPKVYIIQA 0.030 81 CAFVVLMAHG 0.030 50 STMKRDPTAE 0.030 164 DALHVYSTVE 0.030 41 HMFRQLRFES 0.030 87 MAHGREGFLK 0.030 121 RAKPKVYIIQ 0.030 33 EEDLDALEHM 0.030 162 YTDALHVYST 0.030 133 RGEQRDPGET 0.030 v.2-B7-10 mers: 213P1F11 8 EGPTPFQDPL 4.000 26 NPPLWNSQDT 2.000 23 APPNPPLWNS 1.800 38 TDMIRKAHAL 1.200 31 NSQDTSPTDM 1.000 2 HVYSTVEGPT 0.500 45 HALSRPWWMC 0.450 9 GPTPFQDPLY 0.400 52 WMCSRRGKDI 0.400 10 PTPFQDPLYL 0.400 36 SPTDMIRKAH 0.300 15 DPLYLPSHAP 0.300 11 TPFQDPLYLP 0.300 44 AHALSRPWWM 0.300 19 LPSEAPPNPP 0.300 49 RPWWMCSRRG 0.200 54 CSRRGKDISW 0.200 32 SQDTSPTDMI 0.180 20 PSEAPPNPPL 0.180 35 TSPTDMIRKA 0.100 40 MIRKAHALSR 0.100 47 LSRPWWMCSR 0.100 43 KAHALSRPWW 0.090 22 EAPPNPPLWN 0.090 46 ALSRPWWMCS 0.060 27 PPLWNSQDTS 0.040 4 YSTVEGPTPF 0.020 55 SRRGKDISWN 0.020 39 DMIRKAHALS 0.020 24 PPNPPLWNSQ 0.020 53 MCSRRGKDIS 0.020 6 TVEGPTPFQD 0.015 5 STVEGPTPFQ 0.015 30 WNSQDTSPTD 0.010 29 LWNSQDTSPT 0.010 18 YLPSEAPPNP 0.100 34 DTSPTDMIRK 0.010 14 QDPLYLPSEA 0.010 51 WWMCSRRGKD 0.005 13 FQDPLYLPSE 0.003 37 PTDMIRKAHA 0.003 56 RRGKDISWNF 0.002 42 RKAHALSRPW 0.002 21 SEAPPNPPLW 0.002 17 LYLPSEAPPN 0.002 7 VEGPTPFQDP 0.002 48 SRPWWMCSRR 0.001 25 PNPPLWNSQD 0.001 33 QDTSPTDMIR 0.001 41 IRKAHALSRP 0.001 3 VYSTVEGPTP 0.001 16 PLYLPSEAPP 0.001 28 PLWNSQDTSP 0.001 1 LHVYSTVEGP 0.001 12 PFQDPLYLPS 0.000 50 PWWMCSRRGK 0.000 v.3-B7-10 mers: 213P1F11 12 LPSPFPYLSL 120.000 10 ATLPSPFPYL 18.000 3 IIQACRGATL 4.000 6 ACRGATLPSP 0.300 2 YIIQACRGAT 0.150 9 GATLPSPFPY 0.060 5 QACRGATLPS 0.060 11 TLPSPFPYLS 0.020 8 RGATLPSPFP 0.015 4 IQACRGATLP 0.010 1 VYIIQACRGA 0.010 7 CRGATLPSPF 0.002 v.4-B7-10 mers: 213P1F11 12 SVQPEKRTGL 30.000 65 GVGDIVGRDL 20.000 84 SASEEEKYDM 3.000 60 EVFGGGVGDI 2.000 54 DPRETQEVFG 2.000 38 QGRAFRGSSV 2.000 25 NGECGQTFRL 1.200 2 GKCQEYDKSL 0.400 42 FRGSSVHQKL 0.400 43 RGSSVHQKLV 0.200 57 ETQEVFGGGV 0.200 10 SLSVQPEKRT 0.150 74 LSISFRNSET 0.100 69 IVGRDLSISF 0.100 76 ISFRNSETSA 0.100 70 VGRDLSISFR 0.100 19 TGLRDENGEC 0.100 20 GLRDENGECG 0.100 14 QPEKRTGLRD 0.060 52 VNDPRETQEV 0.060 4 CQEYDKSLSV 0.060 46 SVHQKLVNDP 0.050 51 LVNDPRETQE 0.050 67 GDIVGRDLSI 0.040 41 AFRGSSVHQK 0.030 37 EQGRAFRGSS 0.030 40 RAFRGSSVHQ 0.030 16 EKRTGLRDEN 0.030 50 KLVNDPRETQ 0.023 33 RLKEEQGRAF 0.020 77 SFRNSETSAS 0.020 44 GSSVHQKLVN 0.020 75 SISFRNSETS 0.020 68 DIVGRDLSIS 0.020 3 KCQEYDKSLS 0.020 82 ETSASEEEKY 0.020 1 MGKCQEYDKS 0.020 61 VFGGGVGDIV 0.020 85 ASEEEKYDMS 0.018 27 ECGQTFRLKE 0.015 73 DLSISFRNSE 0.015 28 CGQTFRLKEE 0.010 24 ENGECGQTFR 0.010 9 KSLSVQPEKR 0.010 30 QTFRLKEEQG 0.010 31 TFRLKEEQGR 0.010 13 VQPEKRTGLR 0.010 83 TSASEEEKYD 0.010 11 LSVQPEKRTG 0.010 62 FGGGVGDIVG 0.010 64 GGVGDIVGRD 0.010 29 GQTFRLKEEQ 0.010 48 HQKLVNDPRE 0.010 45 SSVHQKLVND 0.010 32 FRLKEEQGRA 0.010 63 GGGVGDIVGR 0.010 18 RTGLRDENGE 0.010 79 RNSETSASEE 0.010 49 QKLVNDPRET 0.010 66 VGDIVGRDLS 0.009 80 NSETSASEEE 0.003 58 TQEVFGGGVG 0.003 22 RDENGECGQT 0.003 23 DENGECGQTF 0.002 53 NDPRETQEVF 0.002 72 RDLSISFRNS 0.002 36 EEQGRAFRGS 0.002 81 SETSASEEEK 0.001 39 GRAFRGSSVH 0.001 7 YDKSLSVQPE 0.001 26 GECGQTFRLK 0.001 5 QEYDKSLSVQ 0.001 47 VHQKLVNDPR 0.001 8 DKSLSVQPEK 0.001 56 RETQEVFGGG 0.001 78 FRNSETSASE 0.001 17 KRTGLRDENG 0.001 59 QEVFGGGVGD 0.001 71 GRDLSISFRN 0.001 34 LKEEQGRAFR 0.000 21 LRDENGECGQ 0.000 35 KEEQGRAFRG 0.000 6 EYDKSLSVQP 0.000 86 SEEEKYDMSG 0.000 15 PEKRTGLRDE 0.000 55 PRETQEVFGG 0.000 v.5-B7-10 mers: 213P1F11 2 GARLALILRV 6.000 5 LALILRVTKA 0.300 8 ILRVTKAREG 0.100 10 RVTKAREGSE 0.050 6 ALILRVTKAR 0.030 3 ARLALILRVT 0.030 4 RLALILRVTK 0.015 7 LILRVTKARE 0.010 1 SGARLALILR 0.010 9 LRVTKAREGS 0.003 v.6-B7-10 mers: 213P1F11 9 AMNNKNCQAL 12.000 8 EAMNNKNCQA 0.900 1 VKLENLFEAM 0.100 5 NLFEAMNNKN 0.020 4 ENLFEAMNNK 0.010 10 MNNKNCQALR 0.010 2 KLENLFEAMN 0.006 6 LFEAMNNKNC 0.003 3 LENLFEAMNN 0.002 7 FEAMNNKNCQ 0.001
[0767] 20 TABLE XVII Pos 123456789 Score SeqID v.1-B35-9 mers: 213P1F11 123 KPKVYIIQA 12.000 232 QSTLRKRLY 10.000 79 VSCAFVVLM 10.000 154 DSPQTIPTY 10.000 94 FLKGEDGEM 9.000 121 RAKPKVYTT 7.200 223 KARKTNPEI 7.200 119 ALRAKPKVY 6.000 73 DSREDPVSC 4.500 31 GSEEDLDAL 4.500 205 LLTEVTRRM 4.000 77 DPVSCAFVV 4.000 170 STVEGYIAY 4.000 44 RQLRFESTM 4.000 195 FTKRKGHIL 3.000 142 TVGGDEIVM 3.000 87 MAHGREGFL 3.000 151 VIKDSPQTI 2.400 227 TNPEIQSTL 2.000 183 KGSCFTQTL 2.000 167 HVYSTVEGY 2.000 14 MSGARLALI 2.000 155 SPQTIPTYT 2.000 6 SLEEEKYDM 1.800 135 EQRDPGETV 1.200 231 IQSTLRKRL 1.000 233 STLRKRLYL 1.000 101 EMVKLENLF 1.000 158 TIPTYTDAL 1.000 112 LNNKNCQAL 1.000 187 FIQTLVDVF 1.000 15 SGARLALIL 1.000 184 GSCFIQTLV 1.000 86 LMAHGREGF 1.000 13 DMSGARLAL 1.000 16 GARLALILC 0.900 57 TAEQFQEEL 0.900 169 YSTVEGYIA 0.750 118 QALRAKPKV 0.600 3 NPRSLEEEK 0.600 104 KLENLFEAL 0.600 197 KRKGHILEL 0.600 164 DALHVYSTV 0.600 143 VGGDEIVMV 0.600 49 ESTMKRDPT 0.500 161 TYTDALHVY 0.400 138 DPGETVGGD 0.400 34 EDLDALEHM 0.400 201 HILELLTEV 0.400 27 KAREGSEED 0.360 60 QFQEELEKF 0.300 35 DLDALEHMF 0.300 55 DPTAEQFQE 0.300 210 TRRMAEAEL 0.300 5 RSLEEEKYD 0.300 226 KTNPEIQST 0.300 38 ALEHMFRQL 0.300 30 EGSEEDLDA 0.300 144 GGDEIVMVI 0.240 141 ETVGGDEIV 0.200 199 KGHILELLT 0.200 198 RKGHILELL 0.200 18 RLALILCVT 0.200 159 IPTYTDALH 0.200 150 MVIKDSPQT 0.150 106 ENLFEALNN 0.150 64 ELEKFQQAIL 0.120 24 CVTKAREGS 0.100 40 EHMFRQLRF 0.100 111 ALNNKNCQA 0.100 188 IQTLVDVFT 0.100 147 EIVMVIKDS 0.100 78 PVSCAFVVL 0.100 208 EVTRRMAEA 0.100 100 GEMVKLENL 0.100 20 ALILCVTKA 0.100 157 QTIPTYTDA 0.100 80 SCAFVVLMA 0.100 50 STMKRDPTA 0.100 89 HGREGFLKG 0.060 228 NPEIQSTLR 0.060 70 QAIDSREDP 0.060 95 LKGEDGEMV 0.060 211 RRMAEAELV 0.060 37 DALEHMFRQ 0.060 11 KYDMSGARL 0.060 71 AIDSREDPV 0.060 53 KRDPTAEQF 0.060 179 RHDQKGSCF 0.060 75 REDPVSCAF 0.060 1 MSNPRSLEE 0.050 9 EEKYDMSGA 0.045 177 AYRHDQKGS 0.045 51 TMKRDPTAE 0.045 45 QLRFESTMK 0.045 102 MVKLENLFE 0.045 131 ACRGEQRDP 0.045 97 GEDGEMVKL 0.045 180 HDQKGSCFI 0.040 168 VYSTVEGYI 0.040 v.2-B35-9 mers: 213P1F11 10 TPFQDPLYL 30.000 8 GPTPFQDPL 20.000 56 RGKDISWNF 12.000 44 HALSRPWWM 6.000 35 SPTDMIRKA 4.000 42 KAHALSRPW 3.000 4 STVEGPTPF 2.000 22 APPNPPLWN 2.000 14 DPLYLPSEA 2.000 46 LSRPWWMCS 1.500 53 CSRRGKDIS 1.500 21 EAPPNPPLW 1.500 38 DMIRKAHAL 1.000 31 SQDTSPTDM 0.600 48 RPWWMCSRR 0.400 18 LPSEAPPNP 0.400 52 MCSRRGKDI 0.400 39 MIRKAHALS 0.300 23 PPNPPLWNS 0.200 9 PTPFQDPLY 0.200 26 PPLWNSQDT 0.200 25 NPPLWNSQD 0.200 54 SRRCKDTSW 0.150 17 YLPSEAPPN 0.150 29 WNSQDTSPT 0.150 45 ALSRPWWMC 0.100 20 SEAPPNPPL 0.100 30 NSQDTSPTD 0.100 34 TSPTDMIRK 0.075 3 YSTVEGPTP 0.075 43 AHALSRPWW 0.050 32 QDTSPTDMI 0.040 55 RRGKDISWN 0.030 12 FQDPLYLPS 0.030 33 DTSPTDMIR 0.010 27 PLWNSQDTS 0.010 37 TDMTRKAHA 0.010 2 VYSTVEGPT 0.010 51 WMCSRRGKD 0.010 1 HVYSTVEGP 0.010 7 EGPTPFQDP 0.010 40 IRKALHALSR 0.003 5 TVEGPTPFQ 0.003 41 RKAHALSRP 0.002 19 PSEAPPNPP 0.002 50 WWMCSRRGK 0.001 24 PNPPLWNSQ 0.001 13 QDPLYLPSE 0.001 47 SRPWWMCSR 0.001 16 LYLPSEAPP 0.001 6 VEGPTPFQD 0.001 15 PLYLPSEAP 0.001 28 LWNSQDTSP 0.001 36 PTDMIRKAH 0.000 11 PFQDPLYLP 0.000 49 PWWMCSRRG 0.000 v.3-B35-9 mers 11 LPSPFPYLS 2.000 9 ATLPSPFPY 2.000 7 RGATLPSPF 2.000 10 TLPSPFPYL 1.000 3 IQACRGATL 1.000 12 PSPFPYLSL 0.500 5 ACRGATLPS 0.300 1 YIIQACRGA 0.100 2 IIQACROAT 0.100 4 QACRGATLP 0.030 8 GATLPSPFP 0.030 6 CRGATLPSP 0.001 v.4-B35-9 mers 54 DPRETQEVF 120.000 83 TSASEEEKY 15.000 85 ASEEEKYDM 9.000 70 VGRDLSISF 6.000 3 KCQEYDKSL 4.000 13 VQPEKRTGL 2.000 43 RGSSVHQKL 2.000 24 ENGECGQTF 2.000 33 RLKEEQGRA 1.800 44 GSSVHQKLV 1.000 20 GLRDENGEC 0.900 76 ISFRNSETS 0.500 45 SSVHQKLVN 0.500 11 LSVQPEKRT 0.500 68 DIVGRDLSI 0.400 38 QGRAFRGSS 0.300 66 VGDIVGRDL 0.300 50 KLVNDPRET 0.300 62 FGGGVGDIV 0.200 69 ILVGRDLSIS 0.150 75 SISFRNSET 0.100 37 EQGRAFRGS 0.100 73 DLSISFRNS 0.100 9 KSLSVQPEK 0.100 26 GECGQTFRL 0.100 84 SASEEEKYD 0.090 40 RAFRGSSVH 0.060 14 QPEKRTGLR 0.060 34 LKEEQGRAF 0.060 58 TQEVFGGGV 0.060 74 LSISFRNSE 0.050 4 CQEYDKSLS 0.045 79 RNSETSASE 0.040 61 VFGGGVGDI 0.040 5 QEYDKSLSV 0.040 48 HQKLVNDPR 0.030 77 SFRNSETSA 0.030 53 NDPRETQEV 0.030 1 MGKCQEYDK 0.030 18 RTGLRDENG 0.020 51 LVNDPRETQ 0.020 39 GPAFRGSSV 0.020 72 RDLSISFRN 0.020 57 ETQEVFGGG 0.020 17 KRTGLRDEN 0.020 65 GVGDIVGRD 0.020 23 DENGECGQT 0.015 19 TGLRDENGE 0.015 12 SVQPEKRTG 0.015 80 NSETSASEE 0.015 64 GGVGDIVGR 0.015 78 FRNSETSAS 0.015 2 GKCQEYDKS 0.015 46 SVHQKLVND 0.010 28 CGQTFRLKE 0.010 29 GQTFRLKEE 0.010 30 QTFRLKEEQ 0.010 60 EVFGGGVGD 0.010 82 ETSASEEEK 0.010 10 SLSVQPEKR 0.010 27 ECGQTFRLK 0.010 63 GGGVGDIVG 0.010 67 GDIVGRDLS 0.010 86 SEEEKYDMS 0.006 16 EKRTGLRDE 0.003 7 YDKSLSVQP 0.003 56 RETQEVFGG 0.003 41 AFRGSSVHQ 0.003 31 TFRLKEEQG 0.003 52 VNDPRETQE 0.003 25 NGECGQTFR 0.003 32 FRLKEEQGR 0.002 36 EEQGPAFRG 0.001 81 SETSASEEE 0.001 8 DKSLSVQPE 0.001 49 QKLVNDPRE 0.001 47 VHQKLVNDP 0.001 42 FRGSSVHQK 0.001 59 QEVEGGGVG 0.001 22 RDENGECGQ 0.001 21 LRDENGECG 0.001 35 KEEQGRAFR 0.001 15 PEKRTGLRD 0.000 71 GRDLSISFR 0.000 6 EYDKSLSVQ 0.000 55 PRETQEVFG 0.000 v.5-B35-9 mers: 213P1F11 9 RVTKAREGS 0.200 3 RLALILRVT 0.200 5 ALILRVTKA 0.100 1 GARLALILR 0.090 7 ILRVTKARE 0.030 4 LALILRVTK 0.030 2 ARLALILRV 0.020 6 LILRVTKAR 0.010 8 LRVTKAREG 0.001 v.6-B35-9 mers: 213P1F11 1 KLENLFEAM 1.200 9 MNNKNCQAL 1.000 3 ENLFEAMNN 0.150 8 AMNNKNCQA 0.100 7 EAMNNKNCQ 0.030 4 NLFEAMNNK 0.020 6 FEANNNKNC 0.010 2 LENLFEAMN 0.010 5 LFEAMNNKN 0.003
[0768] 21 TABLE XVIII Pos 1234567890 Score SeqID v.1-B35-10 mers: 213P1F11 3 NPRSLEEEKY 180.000 5 RSLEEEKYDM 60.000 27 KAREGSEEDL 36.000 77 DPVSCAFVVL 20.000 123 KPKVYIIQAC 12.000 169 YSTVEGYIAY 10.000 37 DALEHMFRQL 6.000 118 QALRAKPKVY 6.000 159 IPTYTDALHV 6.000 232 QSTLRKRLYL 5.000 14 MSGARLALIL 5.000 30 EGSEEDLDAL 3.000 209 VTRRMAEAEL 3.000 141 ETVGGDEIVM 3.000 73 DSREDPVSCA 3.000 204 ELLTEVTRRM 2.000 231 IQSTLRKRLY 2.000 226 KTNPEIQSTL 2.000 96 KGEDGEMVKL 1.800 16 GARLALILCV 1.800 59 EQFQEELEKF 1.500 119 ALRAKPKVYI 1.200 70 QAIDSREDPV 1.200 157 QTIPTYTDAL 1.000 85 VLMAHGREGF 1.000 86 LMAHGREGFL 1.000 111 ALNNKNCQAL 1.000 230 EIQSTLRKRL 1.000 94 FLKGEDGEMV 0.900 143 VGGDEIVMVI 0.800 197 KRKGHILELL 0.600 52 MKRDPTAEQF 0.600 154 DSPQTIPTYT 0.500 79 VSCAFVVLMA 0.500 49 ESTMKRDPTA 0.500 176 IAYRHDQKGS 0.450 193 DVFTKRKGHI 0.400 167 HVYSTVEGYI 0.400 183 KGSCFIQTLV 0.400 13 DMSGARLALI 0.400 153 KDSPQTIPTY 0.400 138 DPGETVGGDE 0.400 150 MVIKDSPQTI 0.400 142 TVGGDEIVMV 0.300 110 EALNNKNCQA 0.300 196 TKRKGHILEL 0.300 113 NNKNCQALRA 0.300 19 LALILCVTKA 0.300 102 MVKLENLFEA 0.300 99 DGEMVKLENL 0.300 181 DQKGSCFIQT 0.300 93 GFLKGEDGEM 0.300 160 PTYTDALHVY 0.200 55 DPTAEQFQEE 0.200 10 EKYDMSGARL 0.200 34 EDLDALEHMF 0.200 117 CQALRAKPKV 0.200 155 SPQTIPTYTD 0.200 56 PTAEQFQEEL 0.200 205 LLTEVTRRMA 0.200 78 PVSCAFVVLM 0.200 218 LVQEGKARKT 0.200 201 HILELLTEVT 0.200 107 NLFEALNNKN 0.200 185 SCFTQTLVDV 0.200 166 LHVYSTVEGY 0.200 103 VKLENLFEAL 0.200 178 YRHDQKGSCF 0.200 43 FRQLRFESTM 0.200 121 RAKPKVYIIQ 0.180 223 KARKTNPEIQ 0.180 149 VMVIKDSPQT 0.150 182 QKGSCFIQTL 0.100 187 FIQTLVDVFT 0.100 186 CFIQTLVDVF 0.100 12 YDMSGARLAL 0.100 194 VFTKRKGHIL 0.100 39 LEHMFRQLRF 0.100 100 GEMVKLENLF 0.100 41 HMFRQLRFES 0.100 15 SGARLALILC 0.100 23 LCVTKAREGS 0.100 210 TRRMAEAELV 0.090 215 EAELVQEGKA 0.090 133 RGEQRDPGET 0.090 63 EELEKFQQAIL 0.080 228 NPEIQSTLRK 0.060 212 RMAEAELVQE 0.060 89 HGREGFLKGE 0.060 135 EQRDPGETVG 0.060 151 VIKDSPQTIP 0.060 33 EEDLDALEHM 0.060 74 SREDPVSCAF 0.060 104 KLENLFEALN 0.060 6 SLEEEKYDMS 0.060 1 MSNPRSLEEE 0.050 184 GSCFIQTLVD 0.050 87 MAHGREGFLK 0.045 25 VTKAREGSEE 0.045 130 QACRGEQRDP 0.045 v.2-B35-10 mers: 213P1F11 9 GPTPFQDPLY 40.000 31 NSQDTSPTDM 20.000 54 CSRRGKDISW 7.500 4 YSTVEGPTPF 5.000 43 KAHALSRPWW 3.000 23 APPNPPLWNS 2.000 26 NPPLWNSQDT 2.000 8 EGPTPFQDPL 1.000 35 TSPTDMIRKA 0.500 19 LPSEAPPNPP 0.400 52 WMCSRRGKDI 0.400 36 SPTDMIRKAH 0.400 49 RPWWMCSRRG 0.400 22 EAPPNPPLWN 0.300 45 HALSRPWWMC 0.300 15 DPLYLPSEAP 0.200 56 RRGKDISWNF 0.200 11 TPFQDPLYLP 0.200 27 PPLWNSQDTS 0.200 44 AHALSRPWWM 0.200 10 PTPFQDPLYL 0.150 20 PSEARPNPPL 0.150 47 LSRPWWMCSR 0.150 32 SQDTSPTDMI 0.120 38 TDMIRKAHAL 0.100 2 HVYSTVEGPT 0.100 46 ALSRPWWMCS 0.100 53 MCSRRGKDIS 0.100 39 DMIRKAHALS 0.100 42 RKAHALSRPW 0.100 21 SEAPPNPPLW 0.050 55 SRRGKDISWN 0.045 40 MIRKAHALSR 0.030 5 STVEGPTPFQ 0.020 24 PPNPPLWNSQ 0.020 17 LYLPSEAPPN 0.015 34 DTSPTDMIRK 0.015 29 LWNSQDTSPT 0.015 30 WNSQDTSPTD 0.010 18 YLPSEAPPNP 0.010 14 QDPLYLPSEA 0.010 41 TRKAHALSRP 0.003 13 FQDPLYLPSE 0.003 6 TVEGPTPFQD 0.003 37 PTDMIRKAHA 0.003 12 PFQDPLYLPS 0.002 3 VYSTVEGPTP 0.002 25 PNPPLWNSQD 0.001 7 VEGPTPFQDP 0.001 48 SRPWWMCSRR 0.001 51 WWMCSRRGKD 0.001 33 QDTSPTDMIR 0.001 28 PLWNSQDTSP 0.001 16 PLYLPSEAPP 0.001 1 LHVYSTVEGP 0.001 50 PWWMCSRRGK 0.000 v.3-B35-10 mers: 213P1F11 12 LPSPFPYLSL 20.000 9 GATLPSPFPY 6.000 10 ATLPSPFPYL 1.000 3 IIQACRGATL 1.000 5 QACRGATLPS 0.300 7 CRGATLPSPF 0.100 11 TLPSPFPYLS 0.100 2 YIIQACRGAT 0.100 6 ACRGATLPSP 0.030 8 RGATLPSPFP 0.020 4 IQACRGATLP 0.010 1 VYIIQACRGA 0.010 v.4-B35-10 mers: 213P1F11 84 SASEEEKYDM 18.000 33 RLKEEQGRAF 12.000 82 ETSASEEEKY 3.000 65 GVGDIVGRDL 2.000 54 DPRETQEVFG 1.200 12 SVQPEKRTGL 1.000 69 IVGRDLSISF 1.000 38 QGRAFRGSSV 0.600 3 KCQEYDKSLS 0.600 74 LSISFRNSET 0.500 76 ISFRNSETSA 0.500 44 GSSVHQKLVN 0.500 1 MGKCQEYDKS 0.450 57 ETQEVFGGGV 0.400 43 RGSSVHQKLV 0.400 60 EVFGGGVGDI 0.400 85 ASEEEKYDMS 0.300 25 NGECGQTFRL 0.300 19 TGLRDENGEC 0.150 68 DIVGRDLSIS 0.150 2 GKCQEYDKSL 0.100 37 EQGRAFRGSS 0.100 75 SISFRNSETS 0.100 10 SLSVQPEKRT 0.100 53 NDPRETQEVF 0.100 9 KSLSVQPEKR 0.100 42 FRGSSVHQKL 0.100 23 DENCECGQTF 0.100 52 VNDPRETQEV 0.090 83 TSASEEEKYD 0.075 11 LSVQPEKRTG 0.075 20 GLRDENGECG 0.060 40 RAFRGSSVHQ 0.060 14 QPEKRTGLRD 0.060 70 VGRDLSISFR 0.060 4 CQEYDKSLSV 0.060 45 SSVHQKLVND 0.050 77 SFRNSETSAS 0.045 79 RNSETSASEE 0.040 67 GDIVGRDLSI 0.040 18 RTCLRDENGE 0.030 16 EKRTGLRDEN 0.030 48 HQKLVNDPRE 0.030 66 VGDIVGRDLS 0.030 24 ENGECGQTFR 0.020 61 VFGGGVGDIV 0.020 72 RDLSISFRNS 0.020 51 LVNDPRETQE 0.020 50 KLVNDPRETQ 0.020 13 VQPEKRTGLR 0.020 80 NSETSASEEE 0.015 49 QKLVNDPRET 0.015 32 FRLKEEQGRA 0.015 63 GGGVGDTVGR 0.015 64 GGVGDTVGRD 0.010 30 QTFRLKEEQG 0.010 73 DLSISFRNSE 0.010 28 CGQTFRLKEE 0.010 29 GQTFRLKEEQ 0.010 46 SVHQKLVNDP 0.010 62 FGGGVGDIVG 0.010 36 EEQGRAFRGS 0.010 27 ECGQTFRLKE 0.010 22 RDENGECGQT 0.009 31 TFRLKEEQGR 0.005 7 YDKSLSVQPE 0.003 41 AFRGSSVHQK 0.003 58 TQEVFGGGVG 0.003 71 GRDLSISFRN 0.003 56 RETQEVFGGG 0.002 17 KRTGLRDENG 0.002 5 QEYDKSLSVQ 0.002 47 VHQKLVNDPR 0.001 8 DKSLSVQPEK 0.001 26 GECGQTFRLK 0.001 81 SETSASEEEK 0.001 78 FRNSETSASE 0.001 39 GRAFRGSSVH 0.001 59 QEVFGGGVGD 0.001 21 LRDENGECGQ 0.001 34 LKEEQGRAFR 0.001 86 SEEEKYDMSG 0.001 35 KEEQGRAFRG 0.001 15 PEKRTGLRDE 0.000 6 EYDKSLSVQP 0.000 55 PRETQEVFGG 0.000 v.5-B35-10 mers: 213P1F11 2 GARLALILRV 1.800 5 LALILRVTKA 0.300 8 TLRVTKAREG 0.030 4 RLALILRVTK 0.020 10 RVTKAREGSE 0.020 6 ALILRVTKAR 0.010 7 LILRVTKARE 0.010 3 ARLALILRVT 0.010 9 LRVTKAREGS 0.010 1 SGARLALILR 0.010 v.6-B35-10 mers: 213P1F11 9 AMNNKNCQAL 1.000 1 VKLENLFEAM 0.400 8 EAMNNKNCQA 0.300 5 NLFEAMNNKN 0.200 2 KLENLFEAMN 0.060 3 LENLFEAMNN 0.015 4 ENLFEAMNNK 0.010 10 MNNKNCQALR 0.010 6 LFEAMNNKNC 0.003 7 FEAMNNKNCQ 0.001
[0769] 22 TABLE XIXA MHC Class I Analysis of 213P1F11 (9-mers). Listed are scores which correlate with the ligation strength to a defined HLA type for a sequence of amino acids. The algorithms used are based on the book “MHC Ligands and Peptide Motifs” by H. G. Rammensee, J. Bachmann and S. Stevanovic. The probability of being processed and presented is given in order to predict T-cell epitopes. SEQ. Pos 1 2 3 4 5 6 7 8 9 score ID NO. TABLE XIXA, part 1: MHC Class 1 nonamer analysis of 213P1F11 v.1 (aa 1-242). 213P1F11 v.1: HLA-A*0201 nonamers 201 H I L E L L T E V G 29 20 A L I L C V G T K A 24 104 K L E N L F E A L 22 158 T I P T Y T D A L 22 13 D M S G A R L A L 21 38 A L E H M F R Q L 21 17 A R L A L I L C V 20 18 R L A L I L C V T 20 71 A I D S R E D P V 20 233 S T L R K R L Y L 20 118 Q A L R A K P K V 19 121 R A K P K V Q T I 19 151 V I K D S P Q T I 19 197 K R K G H I L E L 19 205 L L T E V T R R M 19 6 S L E E E K Y D M 18 94 F L K G E D G E M 18 107 N L F E A L N N K 18 111 A L N N K N C Q A 18 143 V G G G D E I V M V G 18 183 K G S C F I Q T L 18 186 C F I Q T L V D V G 18 202 I L E L L T E V T 18 226 K T N P E I Q S T 18 31 G S E E D L D A L 17 97 G E D G E M V K L 17 100 G E M V K L E N L 17 164 D A L H V Y S T V G 17 15 S G A R L A L I L 16 57 T A E Q F Q E E L 16 64 E L E K F Q Q A I 16 87 M A H G R E G F L 16 195 F T K R K G H I L 16 223 K A R K T N P E I 16 86 L M A H G R E G F 15 103 V G K L E N L F E A 15 120 L R A K P K V Y I 15 141 E T V G G D E I V G 15 144 G G D E I V M V I 15 234 T L R K R L Y L Q 15 14 M S G A R L A L I 14 21 L I L C V T K A R 14 22 I L C V T K A R E 14 50 S T M K R D P T A 14 85 V L M A H G R E G 14 95 L K G E D G E M V 14 112 L N N K N C Q A L 14 187 F I Q T L V G D V G F 14 198 R K G H I L E L L 14 21 T R R M A E A E L 14 227 T N P E I Q S T L 14 11 K Y D M S G A R L 13 19 L A L I L C V G T K 13 28 A R E G S E E D L 13 41 H H F R Q L R F E 13 78 P V S C A F V V L 13 227 Y I I Q A C R G E 13 160 P T Y T D A L H V 13 163 T D A L H V Y S T 13 165 A L H V Y S T V E 13 19 T L V D V G F T K R 13 204 E L L T E V T R R 13 212 R M A E A E L V Q 13 214 A E A E L V Q E G 13 79 V S C A F V G V G L M 12 80 S C A F V V L M A 12 119 A L R A K P K V Y 12 128 I I Q A C R G E Q 12 140 G E T V G G D E I 12 157 Q T I P T Y T D A 12 171 T V E G Y I A Y R 12 189 Q T L V D V F T K 12 218 L V Q E G K A R K 12 231 I Q S T L R K R L 12 27 K A R E G S E E D 11 35 D L D A L E H M F 11 74 S R E D P V S C A 11 135 E Q R D P G E T V 11 150 M V I K D S P Q T 11 170 S T V E G Y I A Y 11 175 Y I A Y R H D Q K 11 211 R R M A E A E L V 11 230 E I Q S T L R K R 11 81 C A F V V L M A H 10 142 T V G G D E I V M 10 149 V M V I K D S P Q 10 167 H V Y S T V E G Y 10 180 H D Q K G S C F I 10 184 G S C F I Q T L V 10 200 G H I L E L L T E 10 216 A E L V Q E G K A 10 12 Y D M S G A R L A 9 16 G A R L A L I L C 9 44 R Q L R F E S T M 9 45 Q L R F E S T M K 9 46 L R F E S T M K R 9 76 E D P V S C A F V 9 77 D P V S C A F V V 9 123 K P K V Y I I Q A 9 148 I V M V I K D S P 9 153 K D S P Q T I P T 9 162 Y T D A L H V Y S 9 168 V Y S T V E G Y I 9 176 I A Y R H D Q K G 9 206 L T E V T R R M A 9 213 M A E A E L V Q E 9 219 V Q E G K A R K T 9 2 S N P R S L E E E 8 34 E D L D A L E H M 8 51 T H K R D P T A E 8 52 M K R D P T A E Q 8 60 Q F Q E E L E K F 8 125 K V Y I I Q A C R 8 147 E I V M V I K D S 8 191 L V D V F T K R K 8 194 V F T K R K G H I 8 203 L E L L T E V T R 8 208 E V T R R M A E A 8 37 D A L E H M F R Q 7 43 F R Q L R F E S T 7 56 P T A E Q F Q E E 7 67 K F Q Q A I D S R 7 70 Q A I D S R E D P 7 83 F V V L M A H G R 7 84 V V L M A H G R E 7 89 H G R E G F L K G 7 90 G R E G F L K G E 7 101 E M V K L E N L F 7 134 G E Q R D P G E T 7 137 R D P G E T V G G 7 146 D E I V M V I K D 7 166 L H V Y S T V E G 7 188 I Q T L V D V F T 7 199 K G H I L E L L T 7 217 E L V Q E G K A R 7 222 G K A R K T N P E 7 1 M S N P R S L E E 6 25 V T K A R E G S E 6 63 E E L E K F Q Q A 6 93 G F L K G E D G E 6 105 L E N L F E A L N 6 114 N K N C Q A L R A 6 115 K N C Q A L R A K 6 134 Q A C R G E Q R D 6 138 D P G E T V G G D 6 154 D S P Q T I P T Y 6 155 S P Q T I P T Y T 6 169 Y S T V E G Y I A 6 185 S C F I Q T L V D 6 209 V T R R M A E A E 6 23 L C V T K A R E G 5 32 S E E D L D A L E 5 73 D S R E D P V S C 5 82 A F V V L M A H G 5 108 L F E A L N N K N 5 116 N C Q A L R A K P 5 161 T Y T D A L H V Y S 174 G Y I A Y R H D Q 5 178 Y R H D Q K G S C 5 193 D V F T K R K G H 5 7 L E E E K Y D M S 4 24 C V T K A R E G S 4 26 T K A R E G S E E 4 30 E G S E E D L D A 4 36 L D A L E H M F R 4 69 Q Q A I D S R E D 4 102 M V K L E N L F E 4 110 E A L N N K N C Q 4 129 I Q A C R G E Q R 4 131 A C R G E Q R D P 4 136 Q R D P G E T V G 4 182 Q K G S C F I Q T 4 3 N P R S L E E E K 3 5 R S L E E E K Y D 3 39 L E H M F R Q L R 3 42 M F R Q L R F E S 3 53 K R D P T A E Q F 3 72 I D S R E D P V S 3 75 R E D P V S C A F 3 96 K G E D G E M V K 3 126 V Y I I Q A C R G 3 152 I K D S P Q T I P 3 61 F Q E E L E K F Q 2 65 L E K F Q Q A I D 2 88 A H G R E G F L K 2 91 R E G F L K G E D 2 98 E D G E M V K L E 2 109 F E A L N N K N C 2 122 A K P K V Y I I Q 2 132 C R G E Q R D P G 2 145 G D E I V M V I K 2 156 P Q T I P T Y T D 2 159 I P T Y T D A L H 2 177 A Y R H D Q K G S 2 192 V D V F T K R K G 2 196 T K R K G H I L E 2 229 P E I Q S T L R K 2 48 F E S T M K R D P 1 49 E S T M K R D P T 1 58 A E Q F Q E E L E 1 68 F Q Q A I D S R E 1 99 D G E M V K L E N 1 113 N N K N C Q A L R 1 117 C Q A L R A K P K 1 124 P K V Y I I Q A C 1 133 R G E Q R D P G E 1 172 V E G Y T A Y R E 1 179 R H D Q K G S C F 1 207 T E V T R R M A E 1 215 E A E L V Q E G K 1 220 Q E G K A R K T N 1 224 A R K T N P E I Q 1 4 P R S L E E E K Y −1 8 E E E K Y D M S G −1 62 Q E E L E K F Q Q −1 92 E G F L K G E D G −1 106 E N L F E A L N N −1 228 N P E I Q S T L R −1 232 Q S T L R K R L Y −1 40 E H M F R Q L R F −2 47 R F E S T M K R D −2 55 D P T A E Q F Q E −2 66 E K F Q Q A I D S −2 173 E G Y I A Y R H D −2 139 P G E T V G G D E −4 221 E G K A R K T N P −4 213P1F11 v.1: HLA-A1 nonamers 170 S T V E G Y I A Y 29 232 Q S T L R K R L Y 21 154 D S P Q T I P T Y 19 99 D G E M V K L E N 18 119 A L R A K P K V Y 18 162 Y T D A L H V Y S 18 206 L T E V T R R M A 18 4 P R S L E E E K Y 17 136 Q R D P G E T V G 17 33 E E D L D A L E H 16 75 R E D P V S C A F 16 161 T Y T D A L H V Y 16 167 H V Y S T V E G Y 16 38 A L E H M F R Q L 15 233 S T L R K R L Y L 15 1 M S N P R S L E E 14 31 G S E E D L D A L 14 32 S E E D L D A L E 14 53 K R D P T A E Q F 14 74 S R E D P V S C A 14 104 K L E N L F E A L 14 219 V Q E G K A R K T 14 228 N P E I Q S T L R 14 6 S L E E E K Y D M 13 89 H G R E G F L K G 13 96 K G E D G E M V K 13 97 G E D G E M V K L 13 108 L F E A L N N K N 13 139 P G E T V G G D E 13 28 A R E G S E E D L 12 35 D L D A L E H M F 12 80 S C A F V V L M A 12 144 G G D E I V M V I 12 145 G D E I V M V I K 12 160 P T Y T D A L H V 12 171 T V E G Y T A Y R 12 202 I L E L L T E V T 12 215 E A E L V Q E G K 12 7 L E E E K Y D M S 11 8 E E E K Y D M S G 11 11 K Y D M S G A R L 11 5 P T A E Q F Q E E 11 61 F Q E E L E K F Q 11 64 E L E K F Q Q A I 11 71 A I D S R E D P V 11 90 G R E G F L K G E 11 252 I K D S P Q T I P 11 179 R H D Q K G S C F 11 185 S C F I Q T L V D 11 47 R F E S T M K R D 10 57 T A E Q F Q E E L 10 62 Q E E L E K F Q Q 10 133 R G E Q R D P G E 10 153 K D S P Q T I P T 10 157 Q T I P T Y T D A 10 191 L V D V F T K R K 10 213 M A E A E L V Q E 10 226 K T N P E I Q S T 10 141 E T V G G D E I V 9 13 D M S G A R L A L 8 15 S G A R L A L I L 8 50 S T M K R D P T A 8 79 V S C A F V V L M 8 114 N K N C Q A L R A 8 190 T L V D V F T K R 8 195 F T K R K G H I L 8 196 T K R K G H I L E 8 199 K G H I L E L L T 8 17 A R L A L I L C V 7 25 V T K A R E G S E 7 102 M V K L E N L F E 7 122 A K P K V Y I I Q 7 146 D E I V M V I K D 7 169 Y S T V E G Y I A 7 182 Q K G S C F I Q T 7 189 Q T L V D V F T K 7 197 K R K G H I L E L 7 209 V T R R M A E A E 7 212 R M A E A E L V Q 7 12 Y D M S G A R L A 6 14 M S G A R L A L I 6 16 G A R L A L I L C 6 30 E G S E E D L D A 6 40 E H M F R Q L R F 6 59 E Q F Q E E L E K 6 66 E K F Q Q A I D S 6 106 E N L F E A L N N 6 142 T V G G D E I V M 6 184 G S C F I Q T L V 6 200 G H I L E L L T E 6 229 P E I Q S T L R K 6 20 A L I L C V T K A 5 49 E S T M K R D P T 5 58 A E Q F Q E E L E 5 121 R A K P K V Y I I 5 123 K P K V Y I I Q A 5 216 A E L V Q E G K A 5 225 R K T N P E I Q S 5 5 R S L E E E K Y D 4 43 F R Q L R F E S T 4 73 D S R E D P V S C 4 78 P V S C A F V V L 4 88 A H G R E G F L K 4 165 A L H V Y S T V E 4 178 Y R I I D Q K G S C 4 39 L E H M F R Q L R 3 63 E E L E K F Q Q A 3 85 V L M A H G R E G 3 8 L M A H G R E G F 3 94 F L K G E D G E M 3 98 E D G E M V K L E 3 111 A L N N K N C Q A 3 116 N C Q A L R A K P 3 126 V Y I I Q A C R G 3 132 C R G E Q R D P G 3 140 G E T V G G D E I 3 168 V Y S T V E G Y I 3 187 F I Q T L V D V F 3 192 V D V F T K R K G 3 205 L L T E V T R R M 3 234 T L R K R L Y L Q 3 2 S N P R S L E E E 2 18 R L A L I L C V T 2 21 L I L C V T K A R 2 27 K A R E G S E E D 2 29 R E G S E E D L D 2 41 H M F R Q L R F E 2 45 Q L R F E S T M K 2 46 L R F E S T M K R 2 60 Q F Q E E L E K F 2 65 L E K F Q Q A I D 2 81 C A F V V L M A H 2 93 G F L K G E D G E 2 95 L K G E D G E M V 2 101 E M V K L E N L F 2 105 E N L F E A L N 2 109 F E A L N N K N C 2 113 N N K N C Q A L R 2 118 Q A L R A K P K V 2 127 Y I I Q A C R G E 2 130 Q A C R G E Q R D 2 131 A C R G E Q R D P 2 135 E Q R D P G E T V 2 143 V G G D E I V M V 2 147 E I V M V I K D S 2 155 S P Q T T P T Y T 2 159 I P T Y T D A L H 2 164 D A L H V Y S T V 2 174 G Y I A Y R H D Q 2 175 Y I A Y R H D Q K 2 176 I A Y R H D Q K G 2 180 H D Q K G S C F I 2 188 I Q T L V D V F T 2 198 R K G H I L E L L 2 204 E L L T E V T R R 2 211 R R M A E A E L V 2 214 A E A E L V Q E G 2 217 E L V Q E G K A R 2 218 L V Q E G K A R K 2 230 E I Q S T L R K R 2 22 I L C V T K A R E 1 24 C V T K A R E G S 1 26 T K A R E G S E E 1 42 M F R Q L R F E S 1 48 F E S T M K R D P 1 52 M K R D P T A E Q 1 68 F Q Q A I D S R E 1 70 Q A I D S R E D P 1 72 I D S R E D P V S 1 77 D P V S C A F V V 1 82 A F V V L M A H G 1 83 F V V L M A H G R 1 84 V V L M A H G R E 1 87 M A H G R E G F L 1 92 E G F L K G E D G 1 103 V K L E N L F E A 1 107 N L F E A L N N K 1 120 L R A K P K V Y I 1 128 I I Q A C R G E Q 1 129 I Q A C R G E Q R 1 137 R D P G E T V G G 1 138 D P G E T V G G D 1 149 V M V I K D S P Q 1 151 V I K D S P Q T I 1 151 P Q T T P T Y T D 1 158 T I P T Y T D A L 1 163 T D A L H V Y S T 1 172 V E G Y I A Y R H 1 177 A Y R H D Q K G S 1 181 D Q K G S C F I Q 1 193 D V F T K R K G H 1 194 V F T K R K G H I 1 207 T E V T R R M A E 1 220 Q E G K A R K T N 1 223 K A R K T N P E I 1 224 A R K T N P E I Q 1 231 I Q S T L R K R L 1 213P1F11 v.1: HLA-A26 nonamers 35 D L D A L E H M F 27 170 S T V E G Y I A Y 27 167 H V Y S T V E G Y 26 187 F I Q T L V D V F 25 60 Q F Q E E L E K F 23 78 P V S C A F V V L 23 154 D S P Q T I P T Y 23 104 K L E N L F E A L 22 208 E V T R R M A E A 22 230 E I Q S T L R K R 22 38 A L E H M F R Q L 21 34 E D L D A L E H M 20 56 P T A E Q F Q E E 20 94 F L K G E D G E M 20 142 T V G G D E I V M 20 147 E I V M V I K D S 20 158 T I P T Y T D A L 20 193 D V F T K R K G H 20 195 F T K R K G H I L 20 119 A L R A K P K V Y 19 204 E L L T E V T R R 19 205 L L T E V T R R M 19 233 S T L R K R L Y L 19 6 S L E E E K Y D M 18 101 E M V K L E N L F 18 141 E T V G G D E I V 18 157 Q T I P T Y T D A 18 201 H I L E L L T E V 18 226 K T N P E I Q S T 18 13 D M S G A R L A L 17 40 E H M F R Q L R F 17 64 E L E K F Q Q A I 17 97 G E D G E M V K L 17 107 N L F E A L N N K 17 171 T V E G Y I A Y R 17 197 K R K G H I L E L 16 217 E L V Q E G K A R 16 20 A L I L C V T K A 15 31 G S E E D L D A L 15 63 E E L E K F Q Q A 15 150 M V I K D S P Q T 15 161 T Y T D A L H V Y 15 53 K R D P T A E Q F 14 75 R E D P V S C A F 14 100 G E M V K L E N L 14 127 Y I I Q A C R G E 14 151 V I K D S P Q T I 14 179 R H D Q K G S C F 14 186 C F I Q T L V D V 14 189 Q T L V D V F T K 14 190 T L V D V F T K R 14 218 L V Q E G K A R K 14 9 E E K Y D M S G A 13 18 R L A L I L C V T 13 37 D A L E H M F R Q 13 79 V S C A F V V L M 13 82 A F V V L M A H G 13 138 D P G E T V G G D 13 146 D E I V M V I K D 13 162 Y T D A L H V Y S 13 283 K G S C F I Q T L 13 234 T L R K R L Y L Q 13 21 K Y D M S G A R L 12 24 C V T K A R E G S 12 25 V T K A R E OS E 12 47 R F E S T M K R D 12 50 S T M K R D P T A 12 67 K F Q Q A I D S R 12 71 A I D S R E D P V 12 86 L M A H G R E G F 12 98 E D G E M V K L E 12 175 Y I A Y R H D Q K 12 191 L V D V F T K R K 12 198 R K G H I L E L L 12 227 T N P E I Q S T L 12 4 P R S L E E E K Y 11 21 L I L C V T K A R 11 28 A R E G S E E D L 11 83 F V V L M A H G R 11 84 V V L M A H G R E 11 102 M V K L E N L F E 11 112 L N N K N C Q A L 11 125 K V Y T I Q A C R 11 128 I I Q A C R G E Q 11 148 I V M V I K D S P 11 160 P T Y T D A L H V 11 164 D A L H V Y S T V 11 173 E G Y I A Y R H D 11 206 L T E V T R R M A 11 209 V T R R M A E A E 11 15 S G A R L A L I L L 10 22 I L C V T K A R E 10 57 T A E Q F Q E E L 10 59 E Q F Q E E L E K 10 66 E K F Q Q A I D S 10 73 D S R E D P V S C 10 87 M A H G R E G F L 10 111 A L N N K N C Q A 10 181 D Q K G S C F I Q 10 210 T R R M A E A E L 10 214 A E A E L V Q E G 10 231 I Q S T L R K R L 10 232 Q S T L R K R L Y 10 8 E E E K Y D M S G 9 42 M F R Q L R F E S 9 44 R Q L R F E S T M 9 45 Q L R F E S T M K 9 76 E D P V S C A F V 9 85 V L M A H G R E G 9 92 E G F L K G E D G 9 202 T L E L L T E V T 9 7 L E E E K Y D M S 8 10 E K Y D M S G A R 8 30 E G S E E D L D A 8 33 E E D L D A L E H 8 81 C A F V V L M A H 8 93 G F L K G E D G E 8 108 L F E A L N N K N 8 121 R A K P K V Y I I 8 144 G G D E I V M V I 8 165 A L H V Y S T V E 8 194 V F T K R K G H I 8 221 E G K A R K T N P 8 2 S H P R S L E E E 7 41 H M F R Q L R F E 7 46 L R F E S T M K R 7 49 E S T M K R D P T 7 55 D P T A E Q F Q E 7 77 D P V S C A F V V 7 89 H G R E G F L K G 7 90 G R E G F L K G E 7 99 D G E M V K L E N 7 103 V K L E N L F E A 7 106 E N L F E A L N N 7 135 E Q R D P G E T V 7 143 V G G D E I V M V 7 215 E A E L V Q E G K 7 74 S R E D P V S C A 6 80 S C A F V V L M A 6 110 E A L N N K N C Q 6 115 K N C Q A L R A K 6 122 A K P K V Y I I Q 6 123 K P K V Y I I Q A 6 124 P K V Y I I Q A C 6 163 T D A L H V Y S T 6 182 Q K G S C F I Q T 6 200 G H I L E L L T E 6 213 M A E A E L V Q E 6 14 M S G A R L A L I 5 17 A R L A L I L C V 5 43 F R Q L R F E S T 5 137 R D P G E T V G G 5 145 G D E I V M V I K 5 229 P E I Q S T L R K 5 16 G A R L A L I L C 4 70 Q A I D S R E D P 4 126 V Y I T Q A C R G 4 219 V Q E G K A R K T 4 1 M S N P R S L E E 3 3 N P R S L E E E K 3 26 T K A R E G S E E 3 27 K A R E G S E E D 3 32 S E E D L D A L E 3 52 M K R D P T A E Q 3 61 F Q E E L E K F Q 3 65 L E K F Q Q A I D 3 95 L K G E D G E M V 3 116 N C Q A L R A K P 3 120 L R A K P K V Y I 3 132 C R G E Q R D P G 3 133 R G E Q R D P G E 3 136 Q R D P G E T V G 3 153 K D S P Q T I P T 3 166 L H V Y S T V E G 3 178 Y R H D Q K G S C 3 185 S C F I Q T L V D 3 212 R M A E A E L V Q 3 223 K A R K T N P E I 3 5 R S L E E E K Y D 2 19 L A L I L C V T K 2 23 L C V T K A R E G 2 36 L D A L E H M F R 2 51 T M K R D P T A E 2 54 R D P T A E Q F Q 2 68 F Q Q A I D S R E 2 69 Q Q A I D S R E D 2 91 R E G F L K G E D 2 96 K G E D G E M V K 2 109 F E A L N N K N C 2 113 N N K N C Q A L R 2 114 N K N C Q A L R A 2 118 Q A L R A K P K V 2 129 I Q A C R G E Q R 2 130 Q A C R G E Q R D 2 131 A C R G E Q R D P 2 149 V M V I K D S P Q 2 152 I K D S P Q T I P 2 174 G Y I A Y R H D Q 2 176 I A Y R H D Q K G 2 207 T E V T R R M A E 2 216 A E L V Q E G K A 2 222 G K A R K T N P E 2 12 Y D M S G A R L A 1 29 R E G S E E D L D 1 39 L E H M F R Q L R 1 58 A E Q F Q E E L E 1 62 Q E E L E K F Q Q 1 72 I D S R E D P V S 1 105 L E N L F E A L N 1 117 C Q A L R A K P K 1 134 G E Q R D P G E T 1 140 G E T V G G D E I 1 159 I P T Y T D A L H 1 172 V E G Y I A Y R H 1 177 A Y R H D Q K G S 1 180 H D Q K G S C F I 1 188 T Q T L V D V F T 1 192 V D V F T K R K G 1 196 T K R K G H I L E 1 199 K G H I D E L L T 1 203 L E L L T E V T R 1 220 Q E G K A R K T N 1 224 A R K T N P E I Q 1 225 R K T N P E I Q S 1 228 N P E I Q S T L R 1 213P1F11 v.1: HLA-A3 nonamers 119 A L R A K P K V Y 30 45 Q L R F E S TM K 27 218 L V QE G K AR K 26 125 K V YI I Q AC R 25 175 Y I AY R H DQ K 24 19 L A LI L C VT K 21 96 K G ED G E MV K 21 107 N L FE A L NN K 21 18 R L AL I L CV T 20 167 H V YS T V EG Y 20 191 L V DV F T KR K 20 88 A H GR E G FL K 19 171 T V EG Y I AY R 19 20 A L IL C V TK A 18 78 P V SC A F VV L 18 83 F V VL M A HG R 18 111 A L NN K N CQ A 18 165 A L HV Y S TV E 18 189 Q T LV D V FT K 18 208 E V TR R M AE A 18 217 E L VQ E G KA R 18 35 D L DA L E HM F 17 217 C Q AL R A KP K 17 290 T L VD V F TK R 17 202 I L EL L T EV T 17 204 E L LT E V TR R 17 21 L I LC V T KA R 16 38 A L EH M F RQ L 16 44 R Q LR F E ST M 16 53 K R DP T A EQ F 16 142 T V GG D E IV M 16 145 G D EI V M VI K 16 150 M V IK D S PQ T 16 187 F I QT L V DV F 16 193 D V FT K R KG H 16 229 P E IQ S T LR K 16 22 I L CV T K AR E 15 85 V L MA H G RE G 15 102 M V KL E N LF E 15 104 K L EN L F EA L 15 129 I Q AC R G EQ R 15 151 V I KD S P QT I 15 179 R H DQ K G SC F 15 203 L E LL T E VT R 15 234 T L RK R L YL Q 15 94 F L KG E D GE M 14 115 K N CQ A L RA K 14 128 I I QA C R GE Q 14 148 I V MV I K DS P 14 3 N P RS L E EE K 13 59 E Q FQ E E LE K 13 71 A I DS R E DP V 13 75 R E DP V S CA F 13 84 V V LM A H GR E 13 212 R M AE A E LV Q 13 6 S L EE E K YD H 12 136 Q R DP G E TV G 12 201 H I LE L L TE V 12 205 L L TE V T RR M 12 215 E A EL V Q EG K 12 230 E I QS T L RK R 12 10 E K YD M S GA R 11 15 S G AR L A LI L 11 24 C V TK A R EG S 11 33 E E DL D A LE H 11 64 E L EK F Q QA I 11 73 D S RE D P VS C 11 127 Y I IQ A C RG E 11 135 E Q RD P G ET V 11 161 T Y TD A L HV Y 11 170 S T VE G Y IA Y 11 200 G H IL E L LT E 11 232 Q S TL R K RL Y 11 233 S T LR K R LY L 11 17 A R LA L I LC V 10 26 T K AR E G SE E 10 137 R D PG E T VG G 10 154 D S PQ T I PT Y 10 160 P T YT D A LH V 10 164 D A LH V Y ST V 10 209 V T RR M A EA E 10 11 K Y DM S G AR L 9 27 K A RE G S EE D 9 40 E H MF R Q LR F 9 67 K F QQ A I DS R 9 72 I D SR E D PV S 9 89 H G RE G F LK G 9 113 N N KN C Q AL R 9 121 R A KP K V YI I 9 147 E I VM V T KD S 9 157 Q T IP T Y TD A 9 158 T I PT Y T DA L 9 159 I P TY T D AL H 9 176 I A YR H D QK G 9 185 S C FI Q T LV D 9 197 K R KG H I LE L 9 211 R R MA E A EL V 9 213 M A EA E L VQ E 9 1 M S NP R S LE E 8 25 V T KA R E GS E 8 86 L M AH G R EG F 8 106 E N LF E A LN N 8 131 A C RG E Q RD P 8 188 I Q TL V D VF T 8 195 F T KR K G HI L 8 225 R K TN P E IQ S 8 226 K T NP E I QS T 8 227 T N PE I Q ST L 8 4 P R SL E E EK Y 7 5 R S LE E E KY D 7 13 D M SG A R LA L 7 14 M S GA R L AL I 7 36 L D AL E H MF R 7 50 S T MK R D PT A 7 51 T M KR D P TA E 7 52 M K RD P T AE Q 7 60 Q F QE E L EK F 7 63 E E LE K F QQ A 7 70 Q A ID S R ED P 7 77 D P VS C A FV V 7 80 S C AF V V LM A 7 81 C A FV V L MA H 7 118 Q A LR A K PK V 7 123 K P KV Y I IQ A 7 126 V Y II Q A CR G 7 130 Q A CR G E QR D 7 172 V E GY I A YR H 7 183 K G SC F I QT L 7 186 C F IQ T L VD V 7 199 K G HI L ELL T 7 220 Q E GK A RKT N 7 46 L R FE S TMK R 6 62 Q E EL E KFQ Q 6 79 V S CA F VVL M 6 82 A F VV L M AH G 6 97 G E DG E M VK L 6 114 N K NC Q A LR A 6 116 N C QA L R AK P 6 133 R G EQ R D PG E 6 143 V G GD E I VM V 6 144 G G DE I V MV I 6 153 K D SP Q T TP T 6 162 Y T DA L H VY S 6 174 G Y IA Y R HD Q 6 177 A Y RH D Q KG S 6 210 T R RM A E AE L 6 216 A E LV Q E GK A 6 219 V Q EG K A RK T 6 221 E G KA R K TN P 6 223 K A RK T N PE I 6 228 N P EI Q S TL R 6 12 Y D MS G A RL A 5 28 A R EG S E ED L 5 32 S E ED L D AL E 5 39 L E HM F R QL R 5 43 F R QL R F ES T 5 55 D P TA E Q FQ E 5 68 F Q QA I D SR E 5 74 S R ED P V SC A 5 87 M A HG R E GF L 5 91 R E GF L K GE D 5 92 E G FL K G ED G 5 95 L K GE D G EM V 5 120 L R AK P K VY I 5 134 G E QR D P GE T 5 139 P G ET V G GD E 5 140 G E TV G G DE I 5 149 V M VI K D SP Q 5 156 P Q TI P T YT D 5 173 E G YI A Y RH D 5 182 Q K GS C F IQ T 5 196 T K RK G H IL E 5 207 T E VT R R MA E 5 214 A E AE L V QE G 5 222 G K AR K T NP E 5 224 A R KT N P EI Q 5 2 S N PR S L EE E 4 9 E E KY D M SG A 4 16 G A RL A L IL C 4 23 L C VT K A RE G 4 29 R E GS E E DL D 4 30 E G SE E D LD A 4 42 M F RQ L R FE S 4 47 R F ES T M KR D 4 54 R D PT A E QF Q 4 65 L E KF Q Q AT D 4 69 Q Q AI D S RE D 4 93 G F LK G E DG E 4 99 D G EM V K LE N 4 101 E M VK L E NL F 4 105 L E NL F E AL N 4 122 A K PK V Y II Q 4 141 E T VG G D EI V 4 163 T D AL H V YS T 4 166 L H VY S T VE G 4 178 Y R HD Q K GS C 4 198 R K GH I L EL L 4 7 L E EE K Y DM S 3 8 E E EK Y D MS G 3 34 E D LD A L EH H 3 37 D A LE H MF R Q 3 41 H M FR Q LR F E 3 58 A E QF Q EE L E 3 66 E K FQ Q A ID S 3 76 E D PV S C AF V 3 103 V K LE N L FE A 3 110 E A LN N K NC Q 3 146 D E IV M V IK D 3 152 I K DS P Q TI P 3 155 S P QT I P TY T 3 169 Y S TV E G YI A 3 181 D Q KG S C FI Q 3 194 V F TK R K GH I 3 231 I Q ST L R KR L 3 31 G S EE D L DA L 2 49 E S TM K R DP T 2 56 P T AE Q F QE E 2 90 G R EG F L KG E 2 108 L F EA L N NK N 2 109 F E AL N N KN C 2 132 C R GE Q R DP G 2 138 D P GE T V GG D 2 168 V Y ST V E GY I 2 180 H D QK G S CF I 2 206 L T EV T R RM A 2 61 F Q EE L E KF Q 1 98 E D GE M V KL E 1 100 G E MV K L EN L 1 112 L N NK N C QA L 1 124 P K VY I I QA C 1 184 G S CF I Q TL V 1 213P1F11 v.1: HLA-B*0702 nonamers 13 D H S G A R L A L 18 77 D P V S C A F V V 17 123 K P K V Y I T Q A 17 155 S P Q T I P T Y T 17 78 P V S C A F V V L 16 97 G E D G E M V K L 15 197 K R K G H I L E L 15 11 K Y D M S G A R L 14 28 A R E G S E E D L 14 231 I Q S T L R K R L 14 15 S G A R L A L I L 13 38 A L E H M F R Q L 13 87 M A H G R E G F L 13 104 K L E N L F E A L 13 120 L R A K P K V Y I 13 135 E Q R D P G E T V 13 183 K G S C F I Q T L 13 210 T R R M A E A E L 13 233 S T L R K R L Y L 13 3 N P R S L E E E K 12 112 L N N K N C Q A L 12 138 D P G E T V G G D 12 153 K D S P Q T I P T 12 158 T I P T Y T D A L 12 159 I P T Y T D A L H 12 198 R K G H I L E L L 12 17 A R L A L I L C V 11 30 E G S E E D L D A 11 31 G S E E D L D A L 11 55 D P T A E Q F Q E 11 100 G E M V K L E N L 11 195 F T K R K G H I L 11 223 K A R K T N P E I 11 228 N P E I Q S T L R 11 20 A L I L C V T K A 10 40 E H M F R Q L R F 10 57 T A E Q F Q E E L 10 71 A I D S R E D P V 10 74 S R E D P V S C A 10 75 R E D P V S C A F 10 79 V S C A F V V L M 10 80 S C A F V V L M A 10 242 T V G G D E I V M 10 188 I Q T L V D V F T 10 227 T N P E I Q S T L 10 14 M S G A R L A L I 9 18 R L A L I L C V T 9 49 E S T M K R D P T 9 50 S T M K R D P T A 9 53 K R D P T A E Q F 9 64 E L E K F Q Q A I 9 76 E D P V S C A F V 9 121 R A K P K V Y I I 9 141 E T V G G D E I V 9 143 V G G D E I V M V 9 144 G G D E I V M V I 9 179 R H D Q K G S C F 9 199 K G H I L E L L T 9 202 I L E L L T E V T 9 211 R R M A E A E L V 9 35 D L D A L E H M F 8 63 E E L E K F Q Q A 8 86 L M A H G R E G F 8 94 F L K G E D G E M 8 95 L K G E D G E M V 8 101 E M V K L E N L F 8 111 A L N N K N C Q A 8 114 N I C N C Q A L R A 8 150 M V I K D S P Q T 8 157 Q T I P T Y T D A 8 160 P T Y T D A L H V 8 163 T D A L H V Y S T 8 168 V Y S T V E G Y I 8 180 H D Q K G S C F I 8 182 Q K G S C F T Q T 8 186 C F I Q T L V D V 8 187 F I Q T L V D V F 8 208 E V T R R M A E A 8 216 A E L V Q E G K A 8 219 V Q E G K A R K T 8 226 K T N P E I Q S T 8 9 E E K Y D M S G A 7 12 Y D M S G A R L A 7 34 E D L D A L E H M 7 43 F R Q L R F E S T 7 44 R Q L R F E S T M 7 52 M K R D P T A E Q 7 118 Q A L R A K P K V 7 140 G E T V G G D E I 7 169 Y S T V E G Y I A 7 184 G S C F I Q T L V 7 194 V F T K R K G H I 7 201 H I L E L L T E V 7 205 L L T E V T R R M 7 206 L T E V T R R M A 7 6 S L E E E K Y D M 6 60 Q F Q E E l E K F 6 103 V K L E N L F E A 6 119 A L R A K P K V Y 6 131 A C R G E Q R D P 6 134 G E Q R D P G E T 6 137 R D P G E T V G G 6 151 V I K D S P Q T I 6 152 I L K D S P Q T I P 6 164 D A L H V Y S T V 6 72 I D S R E D P V S 5 89 H G R E G F L K G 5 212 R M A E A E L V Q 5 33 E E D L D A L E H 4 73 D S R E D P V S C 4 88 A H G R E G F L K 4 136 Q R D P G E T V G 4 156 P Q T I P T Y T D 4 162 Y T D A L H V Y S 4 165 A L H V Y S T V E 4 177 A Y R H D Q K G S 4 185 S C F I Q T L V D 4 214 A E A E L V Q E G 4 225 R K T N P E I Q S 4 1 M S N P R S L E E 3 27 K A R E G S E E D 3 42 M F R Q L R F E S 3 45 Q L R F E S T M K 3 59 E Q F Q E E L E K 3 82 A F V V L M A H G 3 85 V L M A H G R E G 3 98 E D G E M V K L E 3 102 M V K L E N L F E 3 106 E N L F E A L N N 3 116 N C Q A L R A K P 3 122 A K P K V Y I I Q 3 128 I I Q A C R G E Q 3 129 I Q A C R G E Q R 3 132 C R G E Q R D P G 3 166 L H V Y S T V E G 3 171 T V E G Y I A Y R 3 196 T K R K G H I L E 3 204 E L L T E V T R R 3 209 V T R R M A E A E 3 213 M A E A E L V Q E 3 217 E L V Q E G K A R 3 220 Q E G K A R K T N 3 221 E G K A R K T N P 3 222 G K A R K T N P E 3 229 P E I Q S T L R K 3 234 T L R K R L Y L Q 3 4 P R S L E E E K Y 2 8 E E E K Y D M S G 2 10 E K Y D M S G A R 2 16 G A R L A L I L C 2 19 L A L I L C V T K 2 21 L I L C V T K A R 2 22 I L C V T K A R E 2 24 C V T K A R E G S 2 26 T K A R E G S E E 2 29 R E G S E E D L D 2 36 L D A L E H M F R 2 48 F E S T M K R D P 2 51 T M K R D P T A E 2 54 R D P T A E Q F Q 2 56 P T A E Q F Q E E 2 58 A E Q F Q E E L E 2 90 G R E G F L K G E 2 91 R E G F L K G E D 2 92 E G F L K G E D G 2 96 K G E D G E M V K 2 99 D G E M V K L E N 2 115 K N C Q A L R A K 2 117 C Q A L R A K P K 2 125 K V Y I I Q A C R 2 133 R G E Q R D P G E 2 145 G D E I V M V I K 2 148 T V M V I K D S P 2 174 G Y I A Y R H D Q 2 181 D Q K G S C F I Q 2 190 T L V D V F T K R 2 191 L V D V F T K R K 2 200 G H I L E L L T E 2 203 L E L L T E V T R 2 207 T E V T R R M A E 2 224 A R K T N P E I Q 2 5 R S L E E E K Y D 1 25 V T K A R E G S E 1 32 S E E D L D A L E 1 39 L E H M F R Q L R 1 41 H M F R Q L R F E 1 46 L R F E S T M K R 1 47 R F E S T M K R D 1 61 F Q E E L E K F Q 1 65 L E K F Q Q A I D 1 66 E K F Q Q A I D S 1 67 K F Q Q A T D S R 1 68 F Q Q A I D S R E 1 69 Q Q A I D S R E D 1 70 Q A I D S R E D P 1 81 C A F V V L M A H 1 93 G F L K G E D G E 1 105 L E N L F E A L N 1 108 L F E A L N N K N 1 109 F E A L N N K N C 1 110 E A L N N K N C Q 1 113 N N K N C Q A L R 1 124 P K V Y I I Q A C 1 139 P G E T V G G D E 1 146 D E I V M V I K D 1 247 E I V M V I K D S 1 249 V M V I K D S P Q 1 254 D S P Q T I P T Y 1 261 T Y T D A L H V Y 1 267 H V Y S T V E G Y 1 270 S T V E G Y I A Y 1 272 V E C Y I A Y R H 1 273 E G Y I A Y R H D 1 275 Y I A Y R H D Q K 1 276 I A Y R H D Q K G 1 289 Q T L V D V F T K 1 293 D V F T K R K G H 1 215 E A E L V Q E G K 1 218 L V Q E G K A R K 1 230 E I Q S T L R K R 1 213P1F11 v.1: HLA-B*08 nonamers 121 R A K P K V Y I I 31 195 F T K R K G H I L 31 119 A L R A K P K V Y 23 87 M A H G R E G F L 22 100 G E M V K L E N L 22 197 K R K G H I L E L 22 233 S T L R K R L Y L 22 151 V I K D S P Q T I 21 221 E G K A R K T N P 20 234 T L R K R L Y L Q 20 25 V T K A R E G S E 19 94 F L K G E D C E M 18 123 K P K V Y I I Q A 18 223 K A R K T N P E I 18 104 K L E N L F E A L 17 111 A L N N K N C Q A 17 210 T R R M A E A E L 17 38 A L E H M F R Q L 16 40 E H M F R Q L R F 16 179 R H D Q K G S C F 16 57 T A E Q F Q E E L 15 64 E L E K F Q Q A I 15 158 T I P T Y T D A L 15 194 V F T K R K G H I 15 14 M S G A R L A L I 14 31 G S E E D L D A L 14 63 E E L E K F Q Q A 14 3 N P R S L E E E K 13 9 E E K Y D M S G A 13 27 K A R E G S E E D 13 92 E G F L K G E D G 13 175 Y I A Y R H D Q K 13 7 L E E E K Y D M S 12 15 S G A R L A L I L 12 16 G A R L A L I L C 12 35 D L D A L E H M F 12 45 Q L R F E S T M K 12 49 E S T M K R D P T 12 71 A I D S R E D P V 12 97 G E D G E M V K L 12 102 M V K L E N L F E 12 187 F I Q T L V D V F 12 227 T N P E I Q S T L 12 231 T Q S T L R K R L 12 13 D M S G A R L A L 11 23 L C V T K A R E G 11 51 T M K R D P T A E 11 78 P V S C A F V V L 11 112 L N N K N C Q A L 11 149 V M V I K D S P Q 11 183 K G S C F I Q T L 11 193 D V F T K R K G H 11 208 E V T R R M A E A 11 219 V Q E G K A R K T 11 222 G K A R K T N P E 11 6 S L E E E K Y D M 10 11 K Y D M S G A R L 10 28 A R E G S E E D L 10 43 F R Q L R F E S T 10 50 S T M K R D P T A 10 65 L E K F Q Q A I D 10 113 N N K N C Q A L R 10 117 C Q A L R A K P K 10 144 G C D E I V M V I 10 181 D Q K G S C F I Q 10 198 R K G H I L E L L 10 217 E L V Q E G K A R 10 224 A R K T N P E I Q 10 1 M S N P R S L E E 9 73 D S R E D P V S C 9 89 H G R E G F L K G 9 129 I Q A C R G E Q R 9 138 D P G E T V G G D 9 140 G E T V G G D E I 9 204 E L L T E V T R R 9 207 T E V T R R M A E 9 232 Q S T L R K R L Y 9 20 A L I L C V T K A 8 22 I L C V T K A R E 8 60 Q F Q E E L E K F 8 85 V L M A H G R E Q 8 101 E M V K L E N L F 8 107 N L F E A L N N K 8 133 R G E Q R D P G E 8 135 E Q R D P G E T V 8 147 E I V M V I K D S 8 155 S P Q T I P T Y T 8 159 I P T Y T D A L H 8 201 H I L E L L T E V 8 202 I L E L L T E V T 8 205 L L T E V T R R M 8 209 V T R R M A E A E 8 18 R L A L I L C V T 7 42 M F R Q L R F E S 7 52 M K R D P T A E Q 7 75 R E D P V S C A P 7 120 L R A K P K V Y I 7 165 A L H V Y S T V E 7 168 V Y S T V E G Y I 7 177 A Y R H D Q K G S 7 180 H D Q K G S C F I 7 215 E A E L V Q E G K 7 21 L I L C V T K A R 6 37 D A L E H M F R Q 6 53 K R D P T A E Q F 6 55 D P T A E Q F Q E 6 77 D P V S C A F V V 6 81 C A F V V L M A H 6 86 L M A H G R E G F 6 98 E D G E M V K L E 6 110 E A L N N K N C Q 6 128 I I Q A C R G E Q 6 131 A C R G E Q R D P 6 190 T L V D V F T K R 6 196 T K R K G H I L E 6 228 N P E I Q S T L R 6 230 E I Q S T L R K R 6 19 L A L I L C V T K 5 170 S T V E G Y I A Y 5 176 I A Y R H D Q K G S 213 M A E A E L V Q E 5 30 E G S E E D L D A 4 33 E E D L D A L E H 4 61 F Q E E L E K F Q 4 70 Q A I D S R E D P 4 103 V K L E N L F E A 4 109 F E A L N N K N C 4 118 Q A L R A K P K V 4 127 Y I I Q A C R G E 4 130 Q A C R G E Q R D 4 164 D A L H V Y S T V 4 214 A E A E L V Q E G 4 2 S N P R S L E E E 3 5 R S L E E E K Y D 3 10 E K Y D M S G A R 3 56 P T A E Q F Q E E 3 59 E Q F Q E E L E K 3 74 S R E D P V S C A 3 76 E D P V S C A F V 3 79 V S C A F V V L M 3 80 S C A F V V L M A 3 124 P K V Y I I Q A C 3 143 V G G D E I V M V 3 166 L H V Y S T V E G 3 188 I Q T L V D V F T 3 8 E E E K Y D M S G 2 32 S E E D L D A L E 2 34 E D L D A L E H H 2 46 L R F E S T M K R 2 66 E K F Q Q A I D S 2 83 F V V L M A H G R 2 90 G R E G F L K G E 2 95 L K G E D G E M V 2 106 E N L F E A L N N 2 132 C R G E Q R D P G 2 134 G E Q R D P G E T 2 141 E T V G G D E I V 2 145 G D E I V M V I K 2 146 D E I V M V I K D 2 148 I V M V I K D S P 2 163 T D A L H V Y S T 2 167 H V Y S T V E G Y 2 172 V E G Y I A Y R H 2 173 E G Y T A Y R H D 2 185 S C F I Q T L V D 2 189 Q T L V D V F T K 2 191 L V D V F T K R K 2 200 G H I L E L L T E 2 203 L E L L T E V T R 2 212 R M A E A E L V Q 2 218 L V Q E G K A R K 2 4 P R S L E E E K Y 1 17 A R L A L I L C V 1 26 T K A R E G S E E 1 36 L D A L E H M F R 1 41 H M F R Q L R F E 1 47 R F E S T M K R D 1 48 F E S T H K R D P 1 62 Q E E L E K F Q Q 1 67 K F Q Q A I D S R 1 68 F Q Q A I D S R E 1 69 Q Q A I D S R E D 1 72 I D S R E D P V S 1 82 A F V V L M A H G 1 84 V V L M A H G R E 1 91 R E G F L K G E D 1 93 G F L K G E D G E 1 99 D G E M V K L E N 1 105 L E N L F E A L N 1 108 L F E A L N N K N 1 115 K N C Q A L R A K 1 126 V Y I I Q A C R G 1 137 R D P G E T V G G 1 152 I K D S P Q T I P 1 154 D S P Q T I P T Y 1 162 Y T D A L H V Y S I 169 Y S T V E G Y I A 1 171 T V E G Y I A Y R 1 174 G Y I A Y R H D Q 1 184 G S C F I Q T L V 1 186 C F I Q T L V D V 1 192 V D V F T K R K G 1 206 T V E V T R R M A 1 216 A E L V Q E G K A 1 220 Q E G K A R K T N I 226 K T N P E I Q S T 1 213P1F11 v.1: HLA-B*1510 nonamers 40 E H M F R Q L R F 19 179 R H D Q K G S C F 17 231 I Q S T L R K R L 16 78 P V S C A F V V L 15 97 G E D G E M V K L 15 13 D H S G A R L A L 14 31 G S E E D L D A L 14 57 T A E Q F Q E E L 14 38 A L E H M F R Q L 13 112 L N N K N C Q A L 13 166 L H V Y S T V E G 13 183 K G S C F I Q T L 13 197 K R K G H I L E L 13 227 T N P E I Q S T L 13 11 K Y D M S G A R L 12 28 A R E G S E E D L 12 100 G E M V K L E N L 12 104 K L E N L F E A L 12 158 T I P T Y T D A L 12 200 G H I L L E L L T E 12 210 T R R M A E A E L 12 15 S G A R L A L I L 11 87 M A H G R E G F L 11 205 L L T E V T R R M 11 233 S T L R K R L Y L 11 88 A H G R E G F L K 10 142 T V G G D E I V M 10 195 F T K R K G H I L 10 198 R K G H I L E L L 10 6 S L E E E K Y D M 9 75 R E D P V S C A F 9 79 V S C A F V V L M 9 86 L M A H G R E G F 9 187 F I Q T L V D V F 9 34 E D L D A L E H M 8 53 K R D P T A E Q F 8 94 F L K G E D G E H 8 101 E H V K L E N L F 8 44 R Q L R F E S T M 7 60 Q F Q E E L E K F 7 22 I L C V T K A R E 6 35 D L D A L E H M F 6 72 I D S R E D P V S 6 120 L R A K P K V Y I 6 96 K G E D G E M V K 5 136 Q R D P G E T V G 5 144 G G D E I V M V I 5 145 G D E I V M V I K 5 202 I L E L L T E V T 5 206 L T E V T R R M A 5 218 L V Q E G K A R K 5 12 Y D M S G A R L A 4 37 D A L E H M F R Q 4 47 R F E S T M K R D 4 48 F E S T M K R D P 4 64 E L E K F Q Q A I 4 69 Q Q A I D S R E D 4 73 D S R E D P V S C 4 74 S R E D P V S C A 4 115 K N C Q A L R A K 4 127 Y I I Q A C R G E 4 128 I I Q A C R G E Q 4 135 E Q R D P G E T V 4 140 G E T V G G D E I 4 143 V G G D E I V M V 4 148 I V M V I K D S P 4 154 D S P Q T I P T Y 4 161 T Y T D A L H V Y 4 171 T V E G Y I A Y R 4 188 I Q T L V D V F T 4 204 E L L T E V T R R 4 212 R M A E A E L V Q 4 214 A E A E L V Q E G 4 219 V Q E G K A R K T 4 1 M S N P R S L E E 3 8 E E E K Y D M S G 3 10 E K Y D M S G A R 3 18 R L A L I L C V T 3 19 L A L I L C V T K 3 23 L C V T K A R E G 3 26 T K A R E G S E E 3 27 K A R E G S E E D 3 30 E G S E E D L D A 3 42 M F R Q L R F E S 3 50 S T M K R D P T A 3 51 T M K R D P T A E 3 52 M K R D P T A E Q 3 56 P T A E Q F Q E E 3 59 E Q F Q E E L E K 3 85 V L M A H G R E G 3 90 G R E G F L K G E 3 93 G F L K G E D G E 3 98 E D G E M V K L E 3 99 D G E M V K L E N 3 103 V K L E N L F E A 3 110 E A L N N K N C Q 3 119 A L R A K P K V Y 3 121 R A K P K V Y I I 3 129 I Q A C R G E Q R 3 130 Q A C R G E Q R D 3 131 A C R G E Q R D P 3 133 R G E Q R D P G E 3 134 G E Q R D P G E T 3 137 R D P G E T V G G 3 141 E T V G G D E I V 3 151 V I K D S P Q T I 3 152 I K D S P Q T I P 3 153 K D S P Q T I P T 3 162 Y T D A L H V Y S 3 163 T D A L H V Y S T 3 165 A L H V Y S T V E 3 170 S T V E G Y I A Y 3 173 E G Y I A Y R H D 3 178 Y R H D Q K G S C 3 186 C F I Q T L V D V 3 189 Q T L V D V F T K 3 191 L V D V F T K R K 3 192 V D V F T K R K G 3 196 T K R K G H I L E 3 203 L E L L T E V T R 3 207 T E V T R R M A E 3 208 E V T R R M A E A 3 213 M A E A E L V Q E 3 217 E L V Q E G K A R 3 220 Q E G K A R K T N 3 223 K A R K T N P E I 3 226 K T N P E I Q S T 3 229 P E I Q S T L R K 3 230 E I Q S T L R K R 3 232 Q S T L R K R L Y 3 7 L E E E K Y D M S 2 9 E E K Y D M S G A 2 24 C V T K A R E G S 2 32 S E E D L D A L E 2 33 E E D L D A L E H 2 41 H M F R Q L R F E 2 49 E S T M K R D P T 2 61 F Q E E L E K F Q 2 62 Q E E L E K F Q Q 2 63 E E L E K F Q Q A 2 66 E K F Q Q A I D S 2 67 K F Q Q A I D S R 2 68 F Q Q A I D S R E 2 70 Q A I D S R E D P 2 76 E D P V S C A F V 2 77 D P V S C A F V V 2 80 S C A F V V L M A 2 81 C A F V V L M A H 2 82 A F V V L M A H G 2 84 V V L M A H C R E 2 89 H G R E G F L K G 2 91 R E G F L K G E D 2 109 F E A L N N K N C 2 114 N K N C Q A L R A 2 118 Q A L R A K P K V 2 123 K P K V Y I I Q A 2 124 P K V Y I T Q A C 2 126 V Y I I Q A C R G 2 132 C R G E Q R D P G 2 138 D P G E T V G G D 2 146 D E I V M V I L K D 2 147 E I V M V I K D S 2 150 M V I K D S P Q T 2 156 P Q T I P T Y T D 2 157 Q T I P T Y T D A 2 159 I P T Y T D A L H 2 169 Y S T V E G Y I A 2 172 V E G Y I A Y R H 2 174 G Y I A Y R H D Q 2 175 Y I A Y R H D Q K 2 176 I A Y R H D Q K G 2 180 H D Q K G S C F I 2 185 S C F I Q T L V D 2 190 T L V D V F T K R 2 194 V F T K R K G H I 2 201 H I L E L L T E V 2 215 E A E L V Q E G K 2 221 E G K A R K T N P 2 222 G K A R K T N P E 2 224 A R K T N P E I Q 2 234 T L R K R L Y L Q 2 2 S N P R S L E E E 1 3 N P R S L E E E K 1 4 P R S L E E E K Y 1 5 R S L E E E K Y D 1 16 G A R L A L I L C 1 17 A R L A L I L C V 1 20 A L I L C V T K A 1 21 L I L C V T K A R 1 36 L D A L E H M F R 1 39 L E H M F R Q L R 1 46 L R F E S T M K R 1 92 E G F L K G E D G 1 95 L K G E D G E M V 1 106 E N L F E A L N N 1 107 N L F E A L N N K 1 108 L F E A L N N K N 1 116 N C Q A L R A K P 1 117 C Q A L R A K P K 1 122 A K P K V Y I I Q 1 139 P G E T V G G D E 1 155 S P Q T I P T Y T 1 164 D A L H V Y S T V 1 167 H V Y S T V E G Y 1 168 V Y S T V E G Y I 1 181 D Q K G S C F I Q 1 184 G S C F I Q T L V 1 193 D V F T K R K G H 1 209 V T R R M A E A E 1 216 A E L V Q E G K A 1 225 R K T N P E I Q S 1 228 N P E I Q S T L R 1 213P1F11 v.1: HLA-B*2705 nonamers 197 K R K G H I L E L 29 46 L R F E S T M K R 28 53 K R D P T A E Q F 25 28 A R E G S E E D L 24 4 P R S L E E E K Y 22 210 T R R M A E A E L 22 120 L R A K P K V Y I 21 97 G E D G E M V K L 19 17 A R L A L I L C V 18 59 E Q F Q E E L E K 18 67 K F Q Q A I D S R 18 107 N L F E A L N N K 18 125 K V Y I I Q A C R 18 179 R H D Q K G S C F 18 204 E L L T E V T R R 18 229 P E I Q S T L R K 18 75 R E D P V S C A F 17 100 G E M V K L E N L 17 218 L V Q E G K A R K 17 11 K Y D M S G A R L 16 44 R Q L R F E S T M 16 90 G R E G F L K G E 16 96 K G E D G E M V K 16 136 Q R D P G E T V G 16 171 T V E G Y I A Y R 16 183 K G S C F I Q T I A 16 190 T L V D V F T K R 16 198 R K C H I L E L L 16 211 R R M A E A E L V 16 227 T N P E I Q S T L 16 19 L A L T L C V T K 15 31 G S E E D L D A L 15 40 E H M F R Q L R F 15 57 T A E Q F Q E E L 15 60 Q F Q E E L E K F 15 101 E M V K L E N L F 15 115 K N C Q A L R A K 15 145 G D E I V M V I K 15 203 L E L L T E V T R 15 45 Q L R F E S T M K 14 81 C A F V V L M A H 14 121 R A K P K V Y I I 14 144 G G D E I V M V I 14 189 Q T L V D V F T K 14 215 E A E L V Q E G K 14 217 E L V Q E G K A R 14 231 I Q S T L R K R L 14 233 S T L R K R L Y L 14 10 E K Y D M S G A R 13 13 D M S G A R L A L 13 15 S G A R L A L I L 13 21 L I L C V T K A R 13 36 L D A L E H M F R 13 74 S R E D P V S C A 13 83 F V V L M A H G R 13 94 F L K G E D G E M 13 104 K L E N L F E A L 13 113 N N K N C Q A L R 13 170 S T V E G Y I A Y 13 172 V E G Y I A Y R H 13 187 F I Q T L V D V F 13 223 K A R K T N P E I 13 151 V I K D S P Q T I 8 228 N P E I Q S T L R 13 230 E I Q S T L R K R 12 3 N P R S L E E E K 12 6 S L E E E K Y D M 12 33 E E D L D A L E H 12 38 A L E H M F R Q L 12 43 F R Q L R F E S T 12 78 L M A H G R E G F 12 86 M A H G R E G F L 12 87 M A H G R E G F L 12 88 A H G R E G F L K 12 112 L N N K N C Q A L 12 117 C Q A L R A K P K 12 129 I Q A C R G E Q R 12 140 G E T V G G D E I 12 142 T V G G D E I V M 12 178 Y R H D Q K G S C 12 191 L V D V F T K R K 12 193 D V F T K R K G H 12 205 L L T E V T R R M 12 34 E D L D A L E H M 11 35 D L D A L E H M F 11 119 A L R A K P K V Y 11 132 C R G E Q R D P G 11 159 I P T Y T D A L H 11 167 H V Y S T V E G Y 11 175 Y I A Y R H D Q K 11 180 H D Q K G S C F I 11 195 F T K R K G H I L 11 224 A R K T N P E I Q 11 39 L E H M F R Q L R 10 64 E L E K F Q Q A I 10 79 V S C A F V V L M 10 158 T I P T Y T D A L 10 161 T Y T D A L V Y 10 200 G H I L E L L T E 10 232 Q S T L R K R L Y 10 20 A L I L C V T K A 9 93 G F L K G E D G E 9 194 V F T K R K G H I 9 5 R S L E E E K Y D 8 14 M S G A R L A L I 8 18 R L A L I L C V T 8 133 R G E Q R D P G E 8 137 R D P G E T V G G 8 151 V I K D S P Q T I 8 184 G S C F I Q T L V 8 201 H I L E L L T E V 8 226 K T N P E I Q S T 8 27 K A R E G S E E D 8 66 E K F Q Q A I D S 7 91 R E G F L K G E D 7 106 E N L F E A L N N 7 123 K P K V Y I I Q A 7 150 M V I K D S P Q T 7 168 V Y S T V E G Y I 7 212 R M A E A E L V Q 7 225 R K T N P E I Q S 7 16 G A R L A L I L C 6 47 R F E S T M K R D 6 89 H G R E G F L K G 6 118 Q A L R A K P K V 6 131 A C R G E Q R D P 6 141 E T V G G D E I V 6 146 D E I V M V I K D 6 152 I K D S P Q T I P 6 176 I A Y R H D Q K G 6 186 C F I Q T L V D V 6 219 V Q E G K A R K T 6 8 E E E K Y D M S G 5 22 I L C V T K A R E 5 29 R E G S E E D L D 5 37 D A L E H M F R Q 5 50 S T M K R D P T A 5 63 E E L E K F Q Q A 5 68 F Q Q A I D S R E 5 72 I D S R E D P V S 5 92 E G F L K G E D G 5 103 V K L E N L F E A 5 108 L F E A L N N K N 5 122 A K P K V Y I I Q 5 124 P K V Y T I Q A C 5 126 V Y I I Q A C R G 5 143 V G G D E I V M V 5 147 E I V M V I K D S 5 157 Q T I P T Y T D A 5 164 D A L H V Y S T V 5 165 A L H V Y S T V E 5 174 G Y I A Y R H D Q 5 185 S C F I Q T L V D 5 188 T Q T L V D V F T 5 196 T K R K G H I L E 5 214 A E A E L V Q E G 5 216 A E L V Q E G K A 5 221 E G K A R K T N P 5 222 G K A R K T N P E 5 1 M S N P R S L E E 4 23 L C V T K A R E G 4 30 E G S E E D L D A 4 41 H M F R Q L R F E 4 42 M F R Q L R F E S 4 54 R D P T A E Q F Q 4 55 D P T A E Q F Q E 4 62 Q E E L E K F Q Q 4 82 A F V V L M A H G 4 84 V V L M A H G R E 4 99 D G E M V K L E N 4 102 M V K L E N L F E 4 109 F E A L N N K N C 4 110 E A L N N K N C Q 4 111 A L N N K N C Q A 4 114 N K N C Q A L R A 4 116 N C Q A L R A K P 4 127 Y I I Q A C R G E 4 130 Q A C R G E Q R D 4 134 G E Q R D P G E T 4 148 I V M V I K D S P 4 149 V M V I K D S P Q 4 153 K D S P Q T I P T 4 160 P T Y T D A L H V 4 163 T D A L H V Y S T 4 166 L H V Y S T V E G 4 192 V D V F T K R K G 4 199 K G H I L E L L T 4 202 I L E L L T E V T 4 213 M A E A E L V Q E 4 234 T L R K R L Y L Q 4 2 S N P R S L E E E 3 12 Y D M S G A R L A 3 26 T K A R E G S E E 3 32 S E E D L D A L E 3 52 M K R D P T A E Q 3 65 L E K F Q Q A L D 3 70 Q A I D S R E D P 3 73 D S R E D P V S C 3 77 D P V S C A F V V 3 80 S C A F V V L M A 3 155 S P Q T I P T Y T 3 156 P Q T I P T Y T D 3 177 A Y R H D Q K G S 3 181 D Q K G S C F I Q 3 220 Q E G K A R K T N 3 7 L E E E K Y D M S 2 24 C V T K A R E G S 2 51 T M K R D P T A E 2 56 P T A E Q F Q E E 2 61 F Q E E L E K F Q 2 69 Q Q A I D S R E D 2 76 E D P V S C A F V 2 98 E D G E M V K L E 2 105 L E N L F E A L N 2 128 I I Q A C R G E Q 2 135 E Q R D P G E T V 2 138 D P G E T V G G D 2 162 Y T D A L H V Y S 2 169 Y S T V E G Y I A 2 173 E G Y I A Y R H D 2 182 Q K G S C F I Q T 2 207 T E V T R R M A E 2 208 E V T R R M A E A 2 9 E E K Y D M S G A 1 25 V T K A R E G S E 1 48 F E S T M K R D P 1 49 E S T M K R D P T 1 58 A E Q F Q E E L E 1 71 A I D S R E D P V 1 85 V L M A H G R E G 1 95 L K G E D G E M V 1 139 P G E T V G G D E 1 209 V T R R M A E A E 1 213P1F11 v.1: HLA-B*2709 nonamers 53 K R D P T A E Q F 23 197 K R K G H I L E L 23 211 R R M A E A E L V 23 17 A R L A L I L C V 22 28 A R E G S E E D L 21 210 T R R M A E A E L 20 120 L R A K P K V Y I 19 121 R A K P K V Y I I 15 198 R K G H I L E L L 15 31 G S E E D L D A L 14 44 R Q L R F E S T M 14 97 G E D G E M V K L 14 100 G E M V K L E N L 14 11 K Y D M S G A R L 13 75 R E D P V S C A P 13 90 G R E G F L K G E 13 144 G G D E I V M V I 13 160 P T Y T D A L H V 13 233 S T L R K R L Y L 13 15 S G A R L A L I L 12 38 A L E H M F R Q L 12 46 L R F E S T M K R 12 104 K L E N L F E A L 12 140 G E T V G G D E I 12 179 R H D Q K G S C F 12 183 K G S C F I Q T L 12 184 G S C F T Q T L V 12 231 I Q S T L R K R L 12 13 D M S G A R L A L 11 74 S R E D P V S C A 11 77 D P V S C A F V V 11 78 P V S C A F V V L 11 118 Q A L R A K P K V 11 136 Q R D P G E T V G 11 223 K A R K T N P E I 11 224 A R K T N P E I Q 11 227 T N P E I Q S T L 11 4 P R S L E E E K Y 10 34 E D L D A L E H M 10 40 E H M F R Q L R F 10 43 F R Q L R F E S T 10 57 T A E Q F Q E E L 10 71 A I D S R E D P V 10 79 V S C A F V V L M 10 87 M A H G R E G F L 10 112 L N N K N C Q A L 10 132 C R G E Q R D P G 10 158 T I P T Y T D A L 10 164 D A L H V Y S T V 10 178 Y R H D Q K G S C 10 186 C F I Q T L V D V 10 195 F T K R K G H I L 10 201 H I L E L L T E V 10 205 L L T E V T R R M 10 6 S L E E E K Y D M 9 95 L K G E D G E M V 9 101 E M V K L E N L F 9 141 E T V G G D E I V 9 142 T V G G D E T V M 9 143 V G G D E I V M V 9 187 F I Q T L V D V F 9 194 V F T K R K G H I 9 14 M S G A R L A L I 8 35 D L D A L E H M F 8 60 Q F Q E E L E K F 8 64 E L E K F Q Q A I 8 76 E D P V S C A F V 8 86 L M A H G R E G F 8 94 F L K G E D G E M 8 135 E Q R D P G E T V 8 151 V I K D S P Q T I 8 168 V Y S T V E G Y I 8 180 H D Q K G S C F I 8 5 R S L E E E K Y D 6 47 R F E S T M K R D 6 225 R K T N P E I Q S 6 18 R L A L I L C V T 5 29 R E G S E E D L D 5 93 G F L K G E D G E 5 106 E N L F E A L N N 5 125 K V Y I I Q A C R 5 133 R G E Q R D P G E 5 137 R D P C E T V G G 5 200 G H I L E L L T E 5 212 R M A E A E L V Q 5 54 R D P T A E Q F Q 4 91 R E G F L K G E D 4 134 G E Q R D P G E T 4 145 G D E I V M V I K 4 150 M V I K D S P Q T 4 167 H V Y S T V E G Y 4 172 V E G Y I A Y R H 4 174 G Y I A Y R H D Q 4 189 Q T L V D V F T K 4 204 E L L T E V T R R 4 16 G A R L A L I L C 3 19 L A L I L C V T K 3 20 A L I L C V T K A 3 33 E E D L D A L E H 3 37 D A L E H M F R Q 3 59 E Q F Q E E L E K 3 63 E E L E K F Q Q A 3 66 E K F Q Q A I D S 3 84 V V L M A H G R E 3 107 N L F E A L N N K 3 114 N K N C Q A L R A 3 123 K P K V Y I I Q A 3 126 V Y I I Q A C R O 3 153 K D S P Q T I P T 3 176 I A Y R H D Q K G 3 185 S C F I Q T L V D 3 188 I Q T L V D V F T 3 199 K G H I L E L L T 3 203 L E L L T E V T R 3 216 A E L V Q E G K A 3 222 G K A R K T N P E 3 226 K T N P E I Q S T 3 229 P E I Q S T L R K 3 1 M S N P R S L E E 2 10 E K Y D M S G A R 2 12 Y D M S G A R L A 2 21 L I L C V T K A R 2 22 I L C V T K A R E 2 23 L C V T K A R E G 2 27 K A R E G S E E D 2 41 H M F R Q L R F E 2 55 D P T A E Q F Q E 2 67 K F Q Q A I D S R 2 68 F Q Q A I D S R E 2 72 I D S R E D P V S 2 73 D S R E D P V S C 2 80 S C A F V V L M A 2 81 C A F V V L M A H 2 82 A F V V L M A H G 2 83 F V V L M A H G R 2 92 E G F L K G E D G 2 96 K G E D G E M V K 2 103 V K L E N L F E A 2 110 E A L N N K N C Q 2 111 A L N N K N C Q A 2 115 K N C Q A L R A K 2 124 P K V Y I I Q A C 2 129 I Q A C R G E Q R 2 146 D E I V M V I K D 2 148 I V M V I K D S P 2 152 I K D S P Q T I P 2 156 P Q T I P T Y T D 2 157 Q T I P T Y T D A 2 159 I P T Y T D A L H 2 163 T D A L H V Y S T 2 166 L H V Y S T V E G 2 169 Y S T V E G Y I A 2 173 E G Y I A Y R H D 2 177 A Y R H D Q K G S 2 182 Q K G S C F I Q T 2 193 D V F T K R K G H 2 213 M A E A E L V Q E 2 214 A E A E L V Q E G 2 3 N P R S L E E E K 1 9 E E K Y D M S G A 1 24 C V T K A R E G S 1 30 E G S E E D L D A 1 49 E S T M K R D P T 1 50 S T M K R D P T A 1 51 T M K R D P T A E 1 58 A E Q F Q E E L E 1 62 Q E E L E K F Q Q 1 69 Q Q A I D S R E D 1 70 Q A I D S R E D P 1 88 A H G R E G F L K 1 89 H G R E G F L K G 1 98 E D G E M V K L E 1 99 D G E M V K L E N 1 102 M V K L E N L F E 1 109 F E A L N N K N C I 117 C Q A L R A K P K 1 119 A L R A K P K V Y 1 122 A K P K V Y I I Q 1 127 Y I I Q A C R G E 1 128 I I Q A C R G E Q 1 130 Q A C R G E Q R D 1 131 A C R G E Q R D P 1 138 D P G E T V G G D 1 147 E I V M V I K D S 1 149 V M V I K D S P Q 1 154 D S P Q T I P T Y 1 155 S P Q T I P T Y T 1 161 T Y T D A L H V Y 1 162 Y T D A L H V Y S 1 165 A L H V Y S T V E 1 170 S T V E G Y I A Y 1 175 Y I A Y R H D Q K 1 190 T L V D V F T K R 1 191 L V D V F T K R K 1 192 V D V F T K R K G 1 202 I L E L L T E V T 1 207 T E V T R R M A E 1 208 E V T R R M A E A 1 209 V T R R M A E A E 1 217 E L V Q E G K A R 1 218 L V Q E G K A R K 1 219 V Q E G K A R K T 1 221 E G K A R K T N P 1 230 E I Q S T L R K R 1 232 Q S T L R K R L Y 1 234 T L R K R L Y L Q 1 213P1F11 v.1: HLA-B*4402 nonamers 75 R E D P V S C A F 26 97 G E D G E M V K L 24 100 G E M V K L E N L 22 140 G E T V G G D E I 18 53 K R D P T A E Q F 17 119 A L R A K P K V Y 17 13 D M S G A R L A L 16 33 E E D L D A L E H 16 38 A L E H M F R Q L 16 146 D E I V M V I K D 16 183 K G S C F I Q T L 16 197 K R K G H I L E L 16 40 E H M F R Q L R F 15 63 E E L E K F Q Q A 15 104 K L E N L F E A L 15 154 D S P Q T I P T Y 15 158 T I P T Y T D A L 15 207 T E V T R R M A E 15 216 A E L V Q E G K A 15 229 P E I Q S T L R K 15 231 I Q S T L R K R L 15 233 S T L R K R L Y L 15 15 S G A R L A L I L 14 28 A R E G S E E D L 14 58 A E Q F Q E E L E 14 78 P V S C A F V V L 14 101 E M V K L E N L F 14 109 F E A L N N K N C 14 161 T Y T D A L H V Y 14 170 S T V E G Y I A Y 14 203 L E L L T E V T R 14 214 A E A E L V Q E G 14 220 Q E G K A R K T N 14 232 Q S T L R K R L Y 14 4 P R S L E E E K Y 13 31 G S E E D L D A L 13 32 S E E D L D A L E 13 48 F E S T M K R D P 13 112 L N N K N C Q A L 13 187 F I Q T L V D V F 13 198 R I C G H I L E L L 13 227 T N P E I Q S T L 13 8 E E E K Y D M S G 12 9 E E K Y D M S G A 12 11 K Y D M S G A R L 12 35 D L D A L E H M F 12 39 L E H M F R Q L R 12 60 Q F Q E E L E K F 12 62 Q E E L E K F Q Q 12 64 E L E K F Q Q A I 12 87 M A H G R E G F L 12 105 L E N L F E A L N 12 121 R A K P K V Y I I 12 134 G E Q R D P G E T 12 144 G G D E I V M V I 12 172 V E G Y I A Y R E 12 195 F T K R K G H I L 12 14 M S G A R L A L I 11 29 R E G S E E D L D 11 86 L M A H G R E G F 11 151 V I K D S P Q T I 11 167 H V Y S T V E G Y 11 179 R H D Q K G S C F 11 7 L E E E K Y D M S 10 57 T A E Q F Q E E L 10 65 L E K F Q Q A I D 10 91 R E G F L K G E D 10 168 V Y S T V E G Y I 10 210 T R R M A E A E L 10 20 A L I L C V T K A 9 120 L R A K P K V Y I 9 194 V F T K R K G H I 9 223 K A R K T N P E I 9 17 A R L A L I L C V 8 180 H D Q K G S C F I 8 147 E I V M V I K D S 7 153 K D S P Q T I P T 7 200 G H I L E L L T E 7 226 K T N P E I Q S T 7 21 L I L C V T K A R 6 66 E K F Q Q A I D S 6 98 E D G E M V K L E 6 110 E A L N N K N C Q 6 123 K P K V Y I I Q A 6 124 P K V Y I I Q A C 6 157 Q T I P T Y T D A 6 185 S C F I Q T L V D 6 193 D V F T K R K G H 6 217 E L V Q E G K A R 6 224 A R K T N P E I Q 6 1 M S N P R S L E E 5 10 E K Y D M S G A R 5 34 E D L D A L E H M 5 41 H M F R Q L R F E 5 59 E Q F Q E E L E K 5 70 Q A I D S R E D P 5 71 A I D S R E D P V 5 81 C A F V V L M A H 5 88 A H G R E G F L K 5 92 E G F L K G E D G 5 106 E N L F E A L N N 5 107 N L F E A L N N K 5 111 A L N N K N C Q A 5 115 K N C Q A L R A K 5 117 C Q A L R A K P K 5 122 A K P K V Y I I Q 5 131 A C R G E Q R D P 5 136 Q R D P G E T V G 5 143 V G G D E I V M V 5 174 G Y I A Y R H D Q 5 177 A Y R H D Q K G S 5 186 C F I Q T L V D V 5 204 E L L T E V T R R 5 230 E I Q S T L R K R 5 2 S N P R S L E E E 4 12 Y D M S G A R L A 4 16 G A R L A L I L C 4 18 R L A L I L C V T 4 30 E G S E E D L D A 4 46 L R F E S T M K R 4 50 S T M K R D P T A 4 51 T M K R D P T A E 4 67 K F Q Q A T D S R 4 90 G R E G F L K G E 4 126 V Y I I Q A C R G 4 127 Y I I Q A C R G E 4 135 E Q R D P G E T V 4 137 R D P G E T V G G 4 150 M V I K D S P Q T 4 155 S P Q T I P T Y T 4 165 A L H V Y S T V E 4 171 T V E G Y I A Y R 4 191 L V D V F T K R K 4 208 E V T R R M A E A 4 209 V T R R M A E A E 4 219 V Q E G K A R K T 4 5 R S L E E E K Y D 3 19 L A L I L C V T K 3 23 L C V T K A R E G 3 43 F R Q L R F E S T 3 44 R Q L R F E S T M 3 49 E S T M K R D P T 3 72 I D S R E D P V S 3 74 S R E D P V S C A 3 76 E D P V S C A F V 3 79 V S C A F V V L M 3 80 S C A F V V L M A 3 82 A F V V L M A H G 3 83 F V V L M A H G R 3 89 H G R E G F L K G 3 96 K G E D G E M V K 3 103 V K L E N L F E A 3 113 N N K N C Q A L R 3 114 N K N C Q A L R A 3 116 N C Q A L R A K P 3 118 Q A L R A K P K V 3 141 E T V G G D E I V 3 142 T V G G D E I V N 3 160 P T Y T D A L H V 3 173 E G Y I A Y R H D 3 182 Q K G S C F I Q T 3 189 Q T L V D V F T K 3 190 T L V D V F T K R 3 199 K G H I L E L L T 3 202 I L E L L T E V T 3 211 R R M A E A E L V 3 213 M A E A E L V Q E 3 215 E A E L V Q E G K 3 221 E G K A R K T N P 3 222 G K A R K T N P E 3 225 R K T N P E I Q S 3 234 T L R K R L Y L Q 3 47 R F E S T M K R D 2 52 M K R D P T A E Q 2 54 R D P T A E Q F Q 2 61 F Q E E L E K F Q 2 73 D S R E D P V S C 2 77 D P V S C A F V V 2 85 V L M A H G R E G 2 102 M V K L E N L F E 2 108 L F E A L N N K N 2 125 K V Y I I Q A C R 2 129 I Q A C R G E Q R 2 148 I V M V I K D S P 2 156 P Q T I P T Y T D 2 162 Y T D A L H V Y S 2 163 T D A L H V Y S T 2 164 D A L H V Y S T V 2 166 L H V Y S T V E G 2 175 Y I A Y R H D Q K 2 176 I A Y R H D Q K G 2 184 G S C F I Q T L V 2 188 I Q T L V D V F T 2 192 V D V F T K R K G 2 196 T K R K G H I L E 2 201 H I L E L L T E V 2 205 L L T E V T R R M 2 206 L T E V T R R M A 2 212 R M A E A E L V Q 2 228 N P E I Q S T L R 2 3 N P R S L E E E K 1 6 S L E E E K Y D M 1 24 C V T K A R E G S 1 25 V T K A R E G S E 1 26 T K A R E G S E E 1 27 K A R E G S E E D 1 36 L D A L E H M F R 1 37 D A L E H M F R Q 1 42 M F R Q L R F E S 1 55 D P T A E Q F Q E 1 56 P T A E Q F Q E E 1 68 F Q Q A I D S R E 1 69 Q Q A I D S R E D 1 84 V V L M A H G R E 1 93 G F L K G E D G E 1 94 F L K G E D G E M 1 99 D G E M V K L E N 1 128 I I Q A C R G E Q 1 130 Q A C R G E Q R D 1 132 C R G E Q R D P G 1 133 R G E Q R D P G E 1 138 D P G E T V G G D 1 139 P G E T V G G D E 1 145 G D E I V M V I K 1 152 I K D S P Q T I P 1 159 I P T Y T D A L H 1 178 Y R H D Q K G S C 1 181 D Q K G S C F I Q 1 213P1F11 v.1: HLA-B*5101 nonamers 164 D A L H V Y S T V 28 77 D P V S C A F V V 26 121 R A K P K V Y I I 23 144 G G D E I V M V I 23 223 K A R K T N P E I 23 118 Q A L R A K P K V 22 37 D A L E H M F R Q 19 138 D P G E T V G G D 19 87 M A H G R E G F L 18 143 V G G D E T V M V 18 176 I A Y R H D Q K G 18 19 L A L I L C V T K 17 57 T A E Q F Q E E L 17 151 V I K D S P Q T I 16 160 P T Y T D A L H V 16 55 D P T A E Q F Q E 15 183 K G S C F I Q T L 15 15 S G A R L A L I L 14 81 C A F V V L M A H 14 120 L R A K P K V Y I 14 168 V Y S T V E G Y I 14 201 H I L E L L T E V 14 213 M A E A E L V Q E 14 14 M S G A R L A L I 13 17 A R L A L I L C V 13 99 D G E M V K L E N 13 110 E A L N N K N C Q 13 123 K P K V Y I I Q A 13 155 S P Q T I P T Y T 13 159 I P T Y T D A L H 13 194 V F T K R K G H I 13 13 D M S G A R L A L 12 16 G A R L A L I L C 12 27 K A R E G S E E D 12 70 Q A I D S R E D P 12 89 H G R E G F L K G 12 95 L K G E D G E M V 12 173 E G Y I A Y R H D 12 215 E A E L V Q E G K 12 227 T N P E I Q S T L 12 228 N P E I Q S T L R 12 3 N P R S L E E E K 11 64 E L E K F Q Q A I 11 78 P V S C A F V V L 11 130 Q A C R G E Q R D 11 135 E Q R D P G E T V 11 180 H D Q K G S C F I 11 186 C F I Q T L V D V 11 76 E D P V S C A F V 10 96 K G E D G E M V K 10 97 G E D G E M V K L 10 140 G E T V G G D E I 10 146 D E I V M V T K D 10 158 T I P T Y T D A L 10 211 R R M A E A E L V 10 233 S T L R K R L Y L 10 30 E G S E E D L D A 9 31 G S E E D L D A L 9 38 A L E H M F R Q L 9 112 L N N K N C Q A L 9 139 P G E T V G G D E 9 141 E T V G G D E I V 9 154 D S P Q T I P T Y 9 184 G S C F I Q T L V 9 197 K R K G H I L E L 9 11 K Y D M S G A R L 8 28 A R E G S E E D L 8 46 L R F E S T M K R 8 71 A I D S R E D P V 8 73 D S R E D P V S C 8 92 E G F L K G E D G 8 100 G E M V K L E N L 8 190 T L V D V F T K R 8 193 D V F T K R K G H 8 195 F T K R K G H I L 8 203 L E L L T E V T R 8 205 L L T E V T R R M 8 221 E G K A R K T N P 8 21 L I L C V T K A R 7 104 K L E N L F E A L 7 108 L F E A L N N K N 7 119 A L R A K P K V Y 7 167 H V Y S T V E G Y 7 187 F I Q T L V D V F 7 198 R K G H I L E L L 7 199 K G H I L E L L T 7 204 E L L T E V T R R 7 210 T R R M A E A E L 7 219 V Q E G K A R K T 7 10 E K Y D M S G A R 6 23 L C V T K A R E G 6 63 E E L E K F Q Q A 6 98 E D G E M V K L E 6 103 V K L E N L F E A 6 107 N L F E A L N N K 6 133 R G E Q R D P G E 6 142 T V G G D E T V M 6 161 T Y T D A L H V Y 6 181 D Q K G S C F I Q 6 189 Q T L V D V F T K 6 212 R H A E A E L V Q 6 5 R S L E E E K Y D 5 7 L E E E K Y D M S 5 20 A L I L C V T K A 5 34 E D L D A L E H H 5 35 D L D A L E H M F 5 60 Q F Q E E L E K F 5 72 I D S R E D P V S 5 79 V S C A F V V L M 5 80 S C A F V V L M A 5 122 A K P K V Y I I Q 5 165 A L H V Y S T V E 5 185 S C F I Q T L V D 5 188 I Q T L V D V F T 5 192 V D V F T K R K G 5 202 I L E L L T E V T 5 218 L V Q E G K A R K 5 230 E I Q S T L R K R 5 12 Y D M S G A R L A 4 18 R L A L I L C V T 4 22 I L C V T K A R E 4 44 R Q L R F E S T M 4 48 F E S T M K R D P 4 61 F Q E E L E K F Q 4 84 V V L M A H G R E 4 106 E N L F E A L N N 4 114 N K N C Q A L R A 4 116 N C Q A L R A K P 4 125 K V Y I I Q A C R 4 136 Q R D P G E T V G 4 147 E I V M V I K D S 4 162 Y T D A L H V Y S 4 166 L H V Y S T V E G 4 171 T V E G Y T A Y R 4 191 L V D V F T K R K 4 200 G H I L E L L T E 4 206 L T E V T R R M A 4 214 A E A E L V Q E G 4 216 A E L V Q E G K A 4 220 Q E G K A R K T N 4 2 S N P R S L E E E 3 41 H M F R Q L R F E 3 47 R F E S T M K R D 3 50 S T M K R D P T A 3 51 T M K R D P T A E 3 56 P T A E Q F Q E E 3 66 E K F Q Q A T D S 3 68 F Q Q A I D S R E 3 74 S R E D P V S C A 3 83 F V V L M A H G R 3 85 V L M A H G R E G 3 90 G R E G F L K G E 3 93 G F L K G E D G E 3 102 M V K L E N L F E 3 109 F E A L N N K N C 3 126 V Y I I Q A C R G 3 127 Y I I Q A C R G E 3 128 I I Q A C R G E Q 3 137 R D P G E T V G G 3 145 G D E I V M V I K 3 152 I K D S P Q T T P 3 163 T D A L H V Y S T 3 170 S T V E G Y I A Y 3 172 V E G Y I A Y R H 3 178 Y R H D Q K G S C 3 196 T K R K G H I L E 3 209 V T R R M A E A E 3 229 P E I Q S T L R K 3 234 T L R K R L Y L Q 3 1 M S N P R S L E E 2 4 P R S L E E E K Y 2 6 S L E E E K Y D M 2 8 E E E K Y D M S G 2 25 V T K A R E G S E 2 33 E E D L D A L E H 2 36 L D A L E H M F R 2 39 L E H M F R Q L R 2 40 E H M F R Q L R F 2 43 F R Q L R F E S T 2 52 M K R D P T A E Q 2 53 K R D P T A E Q F 2 54 R D P T A E Q F Q 2 59 E Q F Q E E L E K 2 65 L E K F Q Q A I D 2 75 R E D P V S C A F 2 86 L M A H G R E G F 2 94 F L K G E D G E M 2 101 E M V K L E N L F 2 105 L E N L F E A L N 2 111 A L N N K N C Q A 2 115 K N C Q A L R A K 2 117 C Q A L R A K P K 2 129 I Q A C R G E Q R 2 131 A C R G E Q R D P 2 132 C R G E Q R D P G 2 148 I V M V I K D S P 2 149 V M V I K D S P Q 2 150 M V I K D S P Q T 2 156 P Q T I P T Y T D 2 157 Q T I P T Y T D A 2 169 Y S T V E G Y I A 2 174 G Y I A Y R H D Q 2 175 Y I A Y R H D Q K 2 182 Q K G S C F I Q T 2 207 T E V T R R M A E 2 222 G K A R K T N P E 2 224 A R K T N P E I Q 2 24 C V T K A R E G S 1 26 T K A R E G S E E 1 32 S E E D L D A L E 1 42 M F R Q L R F E S 1 45 Q L R F E S T M K 1 49 E S T M K R D P T 1 67 K F Q Q A I D S R 1 69 Q Q A I D S R E D 1 82 A F V V L M A H G 1 113 N N K N C Q A L R 1 124 P K V Y I I Q A C 1 134 G E Q R D P G E T 1 153 K D S P Q T I P T 1 177 < TABLE XIXA, part 2: MHC Class 1 nonamer analysis of 2213P1F11 v.2 (aa 1-230) 213P1F11 v.2: HLA-A*0201 nonamers 38 D M I R K AH A L 22 20 S E A P P NP P L 18 8 G P T P F QD P L 13 17 Y L P S E AP P N 13 45 A L S R P WW M C 13 10 T P F Q D PL Y L 12 35 S P T D M IR K A 12 1 H V Y S T VE G P 10 4 S T V E G PT P F 10 15 P L Y L P SE A P 10 27 P L W N S QD T S 10 32 Q D T S P TD M I 10 39 M I R K A HA L S 10 44 H A L S R PW W M 10 51 W M C S R RG K D 10 5 T V E G P TP F Q 9 13 Q D P L Y LP S E 8 16 L Y L P S EA P P 8 14 D P L Y L PS E A 7 37 T D M I R KA H A 7 52 M C S R R GK D I 7 54 S R R G K DT S W 7 29 W N S Q D TS P T 6 31 S Q D T S PT D M 6 41 R K A H A LS R P 6 42 K A H A L SR P W 6 2 V Y S T V EG P T 5 12 F Q D P L YL P S 5 18 L P S E A PP N P 5 23 P P N P P LW N S 5 28 L W N S Q DT S P 5 33 D T S P T DM I R 5 43 A H A L S RP W W 5 3 Y S T V E GP T P 4 11 P F Q D P LY L P 4 21 E A P P N PP L W 4 22 A P P N P PL W N 4 30 N S Q D T SP T D 4 40 I R K A H AL S R 4 46 L S R P W WM C S 4 47 S R P W W MC S R 4 55 R R G K D IS W N 4 6 V E G P T PF Q D 3 24 P N P P L WN S Q 3 26 P P L W N SQ D T 3 50 W W M C S RR G K 2 53 C S R R G KD T S 2 9 P T P F Q DP L Y 1 36 P T D M I RK A H 1 48 R P W W M CS R R 1 7 E G P T P FQ D P −2 19 P S E A P PN P P −2 49 P W W M C SR R G −2 213P1F11 v.2: HLA-A1 nonamers 9 P TP F Q D PL Y 25 12 F QD P L Y LP S 21 36 P TD M I R KA H 17 19 P SE A P P NP P 15 5 T VE G P T PF Q 12 31 S QD T S P TD M 12 4 S TV E G P TP F 10 33 D TS P T D MI R 10 20 S EA P P N PP L 8 22 A PP N P P LW N 8 34 T SP T D M IR K 8 46 L SR P W W MC S 8 3 Y ST V E G PT P 7 54 S RR G K D IS W 7 10 T PF Q D P LY L 6 21 E AP P N P PL W 6 40 I RK A H A LS R 6 53 C SR R G K DI S 6 6 V EG P T P FQ D 5 23 P PN P P L WN S 5 7 E GP T P F QD P 4 8 G PT P F Q DP L 4 16 L YL P S E AP P 4 24 P NP P L W NS Q 4 30 N SQ D T S PT D 4 35 S PT D M I RK A 4 45 A LS R P W WM C 4 2 V YS T V E GP T 3 11 P FQ D P L YL P 3 17 Y LP S E A PP N 3 51 W MC S R R GK D 3 15 P LY L P S EA P 2 28 L WN S Q D TS P 2 32 Q DT S P T DM I 2 39 M IR K A H AL S 2 43 A HA L S R PW W 2 47 S RP W W M CS R 2 1 H VY S T V EG P 1 13 Q DP L Y L PS E 1 25 N PP L W N SQ D 1 27 P LW N S Q DT S 1 50 W WH C S R RG K 1 52 M CS R R G KD I 1 213P1F11 v.2: HLA-A26 nonamers 9 P T P F Q D P L Y 23 4 S T V E G P T P F 22 33 D T S P T D M I R 19 38 D M I R K A H A L 18 1 H V Y S T V E G P 16 5 T V E G P T P F Q 13 56 R G K D I S W N F 13 7 E G P T P F Q D P 12 20 S E A P P N P P L 12 36 P T D M I R K A H 12 39 M I R K A H A L S 12 10 T P F Q D P L Y L 11 11 P F Q D P L Y L P 11 17 Y L P S E A P P N 11 8 G P T P F Q D P L 10 21 E A P P N P P L W 10 31 S Q D T S P T D M 10 14 D P L Y L P S E A 9 15 P L Y L P S E A P 9 27 P L W N S Q D T S 9 44 H A L S R P W W M 9 45 A L S R P W W M C 9 12 F Q D P L Y L P S 8 13 Q D P L Y L P S E 7 23 P P N P P L W N S 7 41 R K A H A L S R P 7 34 T S P T D M I R K 6 35 S P T D M I R K A 6 47 S R P W W M C S R 6 55 R R G K D I S W N 6 24 P N P P L W N S Q 5 16 L Y L P S E A P P 4 18 L P S E A P P N P 4 54 S R R G K D I S W 4 6 V E G P T P F Q D 3 25 N P P L W N S Q D 3 28 L W N S Q D T S P 3 43 A H A L S R P W W 3 46 L S R P W W M C S 3 29 W N S Q D T S P T 2 30 N S Q D T S P T D 2 32 Q D T S P T D M I 2 37 T D M I R K A H A 2 40 I R K A H A L S R 2 48 R P W W M C S R R 2 50 W W M C S R R G K 2 51 W M C S R R G K D 2 3 Y S T V E G P T P 1 22 A P P N P P L W N 1 26 P P L W N S Q D T 1 42 K A H A L S R P W 1 49 P W W M C S R R G 1 213P1F11 v.2: HLA-A3 nonamers 40 I R KA H A LS R 17 45 A L SR P W WM C 17 15 P L YL P S EA P 16 27 P L WN S Q DT S 15 1 H V YS T V EG P 14 39 M I RK A H AL S 14 17 Y L PS E A PP N 13 5 T V EG P T PF Q 12 38 D M IR K A HA L 11 48 R P WW M C SR R 11 50 W W MC S R RG K 11 4 S T VE G P TP F 10 34 T S PT D M IR K 10 56 R G KD I S WN F 10 22 A P PN P P LW N 9 54 S R RG K D IS W 9 16 L Y LP S E AP P 8 33 D T SP T D MI R 8 41 R K AH A L SR P 8 52 M C SR R G KD I 8 53 C S RR G K DI S 8 55 R R GK D I SW N 8 3 Y S TV E G PT P 7 6 V E GP T P FQ D 7 25 N P PL W N SQ D 7 43 A H AL S R PW W 7 46 L S RP W W MC S 7 47 S R PW W M CS R 7 9 P T PF Q D PL Y 6 12 F Q DP L Y LP S 6 14 D P LY L P SE A 6 36 P T DM I R KA H 6 37 T D MI R K AH A 6 44 H A LS R P WW M 6 13 Q D PL Y L PS E 5 20 S E AP P N PP L 5 24 P N PP L W NS Q 5 30 N S QD T S PT D 5 42 K A HA L S RP W 5 10 T P FQ D P LY L 4 21 E A PP N P PL W 4 51 W M CS R R GK D 4 8 G P TP F Q DP L 3 19 P S EA P P NP P 3 23 P P NP P L WN S 3 28 L W NS Q D TS P 3 29 W N SQ D T SP T 3 31 S Q DT S P TD M 3 32 Q D TS P T DM I 3 2 V Y ST V E GP T 2 18 L P SE A P PN P 2 26 P P LW N S QD T 2 35 S P TD M I RK A 2 11 P F QD P L YL P 1 213P1F11 v.2: HLA-B*0702 nonamers 8 G P T P F Q D P L 22 10 T P F Q D P L Y L 22 20 S E A P P N P P L 17 22 A P P N P P L W N 17 14 D P L Y L P S E A 16 26 P P L W N S Q D T 16 35 S P T D M I R K A 16 23 P P N P P L W N S 14 18 L P S E A P P N P 13 38 D M I R K A H A L 11 48 R P W W M C S R R 11 25 N P P L W N S Q D 10 29 W N S Q D T S P T 10 32 Q D T S P T D M I 10 2 V Y S T V E G P T 9 52 M C S R R G K D I 9 4 S T V E C P T P F 8 5 T V E G P T P F Q 8 31 S Q D T S P T D M 8 37 T D M I R K A H A 8 45 A L S R P W W M C 7 56 R G K D I S W N F 7 44 H A L S R P W W M 6 7 E G P T P F Q D P 5 12 F Q D P L Y L P S 5 19 P S E A P P N P P 5 43 A H A L S R P W W 5 15 P L Y L P S E A P 4 39 M I R K A H A L S 4 40 I R K A H A L S R 4 54 S R R G K D I S W 4 11 P F Q D P L Y L P 3 16 L Y L P S E A P P 3 21 E A P P N P P L W 3 33 D T S P T D M I R 3 36 P T D M I R K A H 3 42 K A H A L S R P W 3 46 L S R P W W M C S 3 55 R R G K D I S W N 3 3 Y S T V E G P T P 2 6 V E G P T P F Q D 2 17 Y L P S E A P P N 2 41 R K A H A L S R P 2 50 W W M C S R R G K 2 53 C S R R G K D I S 2 1 H V Y S T V E G P 1 9 P T P F Q D P L Y 1 13 Q D P L Y L P S E 1 24 P N P P L W N S Q 1 28 L W N S Q D T S P 1 30 N S Q D T S P T D 1 51 W M C S R R G K D 1 213P1F11 v.2: HLA-B*08 nonamers 38 D M I R K A H A L 21 54 S R R G K D T S W 18 8 G P T P F Q D P L 17 10 T P F Q D P L Y L 16 56 R G K D I S W N F 16 52 M C S R R G K D I 15 20 S E A P P N P P L 13 44 H A L S R P W W M 13 40 I R K A H A L S R 11 4 S T V E G P T P F 10 39 M I R K A H A L S 10 35 S P T D M I R K A 9 14 D P L Y L P S E A 8 15 P L Y L P S E A P 8 18 L P S E A P P N P 8 25 N P P L W N S Q D 8 37 T D M I R K A H A 8 51 W M C S R R G K D 8 17 Y L P S E A P P N 6 21 E A P P N P P L W 6 22 A P P N P P L W N 6 23 P P N P P L W N S 6 26 P P L W N S Q D T 6 27 P L W N S Q D T S 6 32 Q D T S P T D M I 6 45 A L S R P W W M C 6 46 L S R P W W M C S 6 48 R P W W M C S R R 6 53 C S R R G K D I S 6 42 K A H A L S R P W 4 1 H V Y S T V E G P 2 7 E G P T P F Q D P 2 31 S Q D T S P T D M 2 36 P T D M I R K A H 2 47 S R P W W M C S R 2 2 V Y S T V E G P T 1 3 Y S T V E G P T P 1 6 V E G P T P F Q D 1 12 F Q D P L Y L P S 1 13 Q D P L Y L P S E 1 43 A H A L S R P W W 1 55 R R G K D I S W N 1 213P1F11 v.2: HLA-B*1510 nonamers 20 S E A P P N P P L 15 8 G P T P F Q D P L 13 10 T P F Q D P L Y L 13 43 A H A L S R P W W 13 38 D M I R K A H A L 11 4 S T V E G P T P F 9 31 S Q D T S P T D M 7 44 H A L S R P W W M 7 56 R G K D I S W N F 7 21 E A P P H P P L W 6 5 T V E G P T P F Q 5 3 Y S T V E G P T P 4 18 L P S E A P P N P 4 34 T S P T D M I R K 4 35 S P T D M I R K A 4 49 P W W M C S R R G 4 6 V E G P T P F Q D 3 19 P S E A P P N P P 3 23 P P N P P L W N S 3 30 N S Q D T S P T D 3 33 D T S P T D M I R 3 41 R K A H A L S R P 3 50 W W M C S R R G K 3 7 E G P T P F Q D P 2 11 P F Q D P L Y L P 2 12 F Q D P L Y L P S 2 14 D P L Y L P S E A 2 15 P L Y L P S E A P 2 16 L Y L P S E A P P 2 17 Y L P S E A P P N 2 22 A P P N P P L W N 2 24 P N P P L W N S Q 2 29 W N S Q D T S P T 2 36 P T D M I R K A H 2 37 T D M I R K A H A 2 40 I R K A H A L S R 2 42 K A H A L S R P W 2 45 A L S R P W W M C 2 46 L S R P W W M C S 2 48 R P W W M C S R R 2 53 C S R R G K D I S 2 55 R R G K D I S W N 2 1 H V Y S T V E G P 1 2 V Y S T V E G P T 1 9 P T P F Q D P L Y 1 13 Q D P L Y L P S E 1 26 P P L W N S Q D T 1 27 P L W N S Q D T S 1 32 Q D T S P T D M I 1 39 M I R K A H A L S 1 47 S R P W W M C S R 1 51 W M C S R R G K D 1 52 M C S R R G K D I 1 54 S R R G K D I S W 1 213P1F11 v.2: HLA-B*2705 nonamers 40 I R K A H A L S R 24 47 S R P W W M C S R 21 55 R R G K D I S W N 20 56 R G K D I S W N F 19 48 R P W W M C S R R 17 4 S T V E G P T P F 16 8 G P T P F Q D P L 16 10 T P F Q D P L Y L 16 54 S R R G K D I S W 16 20 S E A P P N P P L 14 38 D M I R K A H A L 14 33 D T S P T D M I R 13 34 T S P T D M I R K 13 31 S Q D T S P T D M 11 44 H A L S R P W W M 11 50 W W M C S R R G K 11 9 P T P F Q D P L Y 10 36 P T D M I R K A H 9 41 R K A H A L S R P 8 32 Q D T S P T D M I 7 23 P P N P P L W N S 6 52 M C S R R G K D I 6 14 D P L Y L P S E A 5 15 P L Y L P S E A P 5 16 L Y L P S E A P P 5 35 S P T D M I R K A 5 5 T V E G P T P F Q 4 13 Q D P L Y L P S E 4 18 L P S E A P P N P 4 24 P N P P L W N S Q 4 25 N P P L W N S Q D 4 27 P L W N S Q D T S 4 28 L W N S Q D T S P 4 42 K A H A L S R P W 4 43 A H A L S R P W W 4 1 H V Y S T V E G P 3 3 Y S T V E G P T P 3 11 P F Q D P L Y L P 3 12 F Q D P L Y L P S 3 17 Y L P S E A P P N 3 22 A P P N P P L W N 3 26 P P L W N S Q D T 3 29 W N S Q D T S P T 3 30 N S Q D T S P T D 3 39 M I R K A H A L S 3 45 A L S R P W W M C 3 49 P W W M C S R R G 3 53 C S R R G K D I S 3 6 V E G P T P F Q D 2 19 P S E A P P N P P 2 21 E A P P N P P L W 2 37 T D M I R K A H A 2 46 L S R P W W M C S 2 51 W M C S R R G K D 2 2 V Y S T V E G P T 1 7 E G P T P F Q D P 1 213P1F11 v.2: HLA-B*2709 nonamers 8 G P T P F Q D P L 15 55 R R G K D I S W N 15 10 T P F Q D P L Y L 14 56 R G K D I S W N F 14 40 I R K A H A L S R 13 20 S E A P P N P P L 12 38 D M I R K A H A L 12 44 H A L S R P W W M 11 32 Q D T S P T D M I 10 47 S R P W W M C S R 10 54 S R R G K D I S W 10 4 S T V E G P T P F 9 31 S Q D T S P T D M 8 52 M C S R R G K D I 8 41 R K A H A L S R P 5 48 R P W W M C S R R 5 1 H V Y S T V E G P 4 16 L Y L P S E A P P 3 17 Y L P S E A P P N 3 3 Y S T V E G P T P 2 6 V E G P T P F Q D 2 12 F Q D P L Y L P S 2 14 D P L Y L P S E A 2 15 P L Y L P S E A P 2 18 L P S E A P P N P 2 21 E A P P N P P L W 2 22 A P P N P P L W N 2 23 P P N P P L W N S 2 2 P P L W N S Q D T 2 34 T S P T D M I R K 2 42 K A H A L S R P W 2 43 A H A L S R P W W 2 5 T V E G P T P F Q 1 9 P T P F Q D P L Y 1 11 P F Q D P L Y L P 1 13 Q D P L Y L P S E 1 24 P I I P P L W N S Q 1 25 N P P L W N S Q D 1 28 L W N S Q D T S P 1 29 W N S Q D T S P T 1 3 N S Q D T S P T D 1 33 D T S P T D M I R 1 35 S P T D M I R K A 1 3 P T D M I R K A H 1 45 A L S R P W W M C 1 4 L S R P W W M C S 1 49 P W W M C S R R G 1 51 W M C S R R G K D 1 213P1F11 v.2: HLA-B*4402 nonamers 20 S E A P P N P P L 24 21 E A P P N P P L W 19 38 D M I R K A H A L 18 6 V E G P T P F Q D 15 43 A H A L S R P W W 15 10 T P F Q D P L Y L 14 8 G P T P F Q D P L 13 52 M C S R R G K D I 13 54 S R R G K D I S W 13 4 S T V E G P T P F 12 9 P T P F Q D P L Y 12 42 K A H A L S R P W 12 56 R G K D I S W N F 12 32 Q D T S P T D M I 9 22 A P P N P P L W N 7 35 S P T D M I R K A 7 36 P T D M I R K A H 7 7 E G P T P F Q D P 5 12 F Q D P L Y L P S 5 24 P N P P L W N S Q 5 45 A L S R P W W M C 5 16 L Y L P S E A P P 4 23 P P N P P L W N S 4 25 N P P L W N S Q D 4 31 S Q D T S P T D H 4 33 D T S P T D M I R 4 11 P F Q D P L Y L P 3 13 Q D P L Y L P S E 3 15 P L Y L P S E A P 3 30 N S Q D T S P T D 3 34 T S P T D M I R K 3 44 H A L S R P W W M 3 46 L S R P W W M C S 3 50 W W M C S R R G K 3 51 W M C S R R G K D 3 55 R R G K D T S W N 3 2 V Y S T V E G P T 2 5 T V E G P T P F Q 2 14 D P L Y L P S E A 2 17 Y L P S E A P P N 2 18 L P S E A P P N P 2 19 P S E A P P N P P 2 26 P P L W N S Q D T 2 27 P L W N S Q D T S 2 29 W N S Q D T S P T 2 37 T D M I R K A H A 2 40 I R K A H A L S R 2 47 S R P W W M C S R 2 1 H V Y S T V E G P 1 3 Y S T V E G P T P 1 39 M I R K A H A L S 1 41 R K A H A L S R P 1 49 P W W M C S R R G 1 53 C S R R G K D I S 1 213P1F11 v.2: HLA-B *5101 nonamers 10 T P F Q D P L Y L 20 14 D P L Y L P S E A 17 8 G P T P F Q D P L 16 35 S P T D M I R K A 16 18 L P S E A P P N P 15 22 A P P N P P L W N 13 25 N P P L W N S Q D 13 44 H A L S R P W W M 13 48 R P W W M C S R R 13 21 E A P P N P P L W 12 26 P P L W N S Q D T 12 38 D M I R K A H A L 12 42 K A H A L S R P W 12 52 M C S R R G K D I 12 23 P P N P P L W N S 11 32 Q D T S P T D M I 10 7 E G P T P F Q D P 9 1 H V Y S T V E G P 7 20 S E A P P N P P L 7 56 R G K D I S W N F 7 16 L Y L P S E A P P 5 30 N S Q D T S P T D 5 33 D T S P T D M I R 5 34 T S P T D M T R K 5 40 I R K A H A L S R 5 2 V Y S T V E G P T 4 3 Y S T V E G P T P 4 4 S T V E G P T P F 4 15 P L Y L P S E A P 4 17 Y L P S E A P P N 4 27 P L W M S Q D T S 4 31 S Q D T S P T D M 4 6 V E G P T P F Q D 3 13 Q D P L Y L P S E 3 19 P S E A P P N P P 3 28 L W H S Q D T S P 3 49 P W W M C S R R G 3 54 S R R G K D I S W 3 5 T V E G P T P F Q </t TABLE XIXA, part 3: MHC Class I nonamer analysis of 213P1F11 v.3(aa1-146) 213P1F11 v.3: HLA-A*0201 nonamers 10 T L P S P F P Y L 22 3 I Q A C R G A T L 18 1 Y I I Q A C R G A 17 9 A T L P S P F P Y 10 12 P S P F P Y L S L 10 5 A C R G A T L P S 7 6 C R G A T L P S P 7 4 Q A C R G A T L P 6 8 G A T L P S P F P 6 11 L P S P F P Y L S 6 7 R G A T L P S P F 3 213P1F11 v.3: HLA-A1 nonamers 9 A T L P S P F P Y 26 12 P S P F P Y L S L 11 5 A C R G A T L P S 8 11 L P S P F P Y L S 6 10 T L P S P F P Y L 5 4 Q A C R G A T L P 4 1 Y T I Q A C R G A 2 2 I I Q A C R G A T 1 3 I Q A C R G A T L 1 8 G A T L P S P F P 1 213P1F11 v.3: HLA-A26 nonamers 9 A T L P S P F P Y 23 10 T L P F P Y L 23 1 Y I I Q A C R G A 14 12 P S P F P Y L S L 14 7 R G A T L P S P F 13 2 I I Q A C R G A T 11 3 I Q A C R G A T L 10 6 C R G A T L P S P 6 5 A C R G A T L P S 2 11 L P S P F P Y L S 2 8 G A T L P S P F P 1 213P1F11v.3: HLA-A3 nonamers 2 I I QA C R G A T 15 3 I Q AC R G A T L 15 9 A T LP S P F P Y 15 7 R G AT L P S P F 13 1 Y I IQ A C R G A 11 4 Q A CR G A T L P 11 5 A C RG A T L P S 11 10 T L PS P F P Y L 10 12 P S PF P Y L S L 5 6 C R GA T L P S P 4 11 L P SP F P Y L 2 4 8 G A TL P S P F P 1 213P1F11 v.3: HLA-B*0702 nonamers 10 T L P S P F P Y L 15 12 P S P F P Y L S L 15 3 I Q A C R G A T L 14 11 L P S P F P Y L S 13 2 I I Q A C R G A T 10 5 A C R G A T L P S 10 7 R G A T L P S P F 9 1 Y I I Q A C R G A 6 8 G A T L P S P F P 5 9 A T L P S P F P Y 4 6 C R G A T L P S P 3 4 Q A C R G A T L P 1 213P1F11 v.3: HLA-B*08 nonamers 3 I Q A C R G A T L 19 10 T L P S P F P Y L 16 12 P S P F P Y L S L 10 2 I I Q A C R G A T 6 5 A C R G A T L P S 6 7 R G A T L P S P F 6 8 G A T L P S P F P 6 11 L P S P F P Y L S 6 1 Y I I Q A C R G A 4 4 Q A C R G A T L P 4 213P1F11 v.3: HLA-B*1510 nonamers 3 I Q A C R G A T L 14 10 T L P S P F P Y L 13 12 P S P F P Y L S L 11 7 R G A T L P S P F 8 1 Y I I Q A C R G A 4 2 I I Q A C R G A T 4 11 L P S P F P Y L S 4 8 G A T L P S P F P 3 9 A T L P S P F P Y 3 4 Q A C R G A T L P 1 5 A C R G A T L P S 1 6 C R G A T L P S P 1 213P1F11 v.3: HLA-B*2705 nonamers 7 R G A T L P S P F 16 3 I Q A C R G A T L 13 6 C R G A T L P S P 13 9 A T L P S P F P Y 13 12 P S P F P Y L S L 13 10 T L P S P F P Y L 12 8 G A T L P S P F P 7 4 Q A C R G A T L P 5 1 Y I I Q A C R G A 4 5 A C R G A T L P S 4 11 L P S P F P Y L S 2 2 I I Q A C R G A T 1 213P1F11 v.3: HLA-B*2709 nonamers 7 R G A T L P S P F 12 10 T L P S P F P Y L 12 3 I Q A C R G A T L 11 6 C R G A T L P S P 11 12 P S P F P Y L S L 11 8 G A T L P S P F P 5 9 A T L P S P F P Y 4 5 A C R G A T L P S 2 11 L P S P F P Y L S 2 1 Y I I Q A C R G A 1 2 I I Q A C R G A T 1 4 Q A C R G A T L P 1 213P1F11 v.3: HLA-B*4402 nonamers 9 A T L P S P F P Y 18 3 I Q A C R G A T L 12 7 R G A T L P S P F 12 10 T L P S P F P Y L 12 12 P S P F P Y L S L 12 5 A C R G A T L P S 6 11 L P S P F P Y L S 6 1 Y I I Q A C R G A 4 2 I I Q A C R G A T 3 4 Q A C R G A T L P 3 6 C R G A T L P S P 1 8 G A T L P S P F P 1 213P1F11 v.3: HLA-B*5101 nonamers 11 L P S P F P Y L S 13 4 Q A C R G A T L P 12 3 I Q A C R G A T L 10 8 G A T L P S P F P 10 10 T L P S P F P Y L 9 7 R G A T L P S P F 8 12 P S P F P Y L S L 8 9 A T L P S P F P Y 4 1 Y I I Q A C R G A 3 TABLE XIXA, part 4: MHC Class 1 nonamer analysis of 213P1F11 v.4 (aa 1-321) 213P1F11 v.4: HLA-A*0201 nonamers 68 D I V G R DL S I 20 50 K L V N D PR E T 17 61 V F G G G VG D I 17 5 Q E Y D K SL S V 16 3 K C Q E Y DK S L 15 13 V Q P E K RT G L 15 26 G E C G Q TF R L 15 33 R L K E E QG R A 15 75 S I S F R NS E T 15 10 S L S V Q PE K R 14 20 G L R D E NG E C 14 39 G R A F R GS S V 14 53 N D P R E TQ E V 14 46 S V H Q K LV N D 13 62 F G G G V GD I V 13 43 R G S S V HQ K L 12 51 L V N D P RE T Q 11 58 T Q E V F GG G V 11 66 V G D I V GR D L 11 12 S V Q P E KR T G 10 44 G S S V H QK L V 10 64 G G V G D IV G R 10 65 G V G D I VG R D 10 69 I V G R D LS I S 10 60 E V F G G GV G D 9 73 D L S I S FR N S 9 30 Q T F R L KE E Q 8 74 L S I S F RN S E 8 77 S F R N S ET S A 8 85 A S E E E KY D M 8 40 R A F R G SS V H 7 47 V H Q K L VN D P 7 84 S A S E E EK Y D 7 9 K S L S V QP E K 6 11 L S V Q P EK R T 6 23 D E N G E CG Q T 6 41 A F R G S SV H Q 6 42 F R G S S VH Q K 6 79 R N S E T SA S E 6 17 K R T G L RD E N 5 67 G D I V G RD L S 5 70 V G R D L SI S F 5 71 G R D L S IS F R 5 76 I S F R N SE T S 5 78 F R N S E TS A S 5 7 Y D K S L SV Q P 4 18 R T G L R DE N G 4 19 T G L R D EN G E 4 21 L R D E N GE C C 4 28 C G Q T F RL K E 4 29 G Q T F R LK E E 4 32 F R L K E EQ G R 4 34 L K E E Q GR A F 4 35 K E E Q G RA F R 4 56 R E T Q E VF G G 4 57 E T Q E V FG G G 4 83 T S A S E EE K Y 4 86 S E E E K YD M S 4 2 G K C Q E YD K S 3 8 D K S L S VQ P E 3 16 E K R T G LR D E 3 38 Q G R A F RG S S 3 45 S S V H Q KL V N 3 49 Q K L V N DP R E 3 52 V N D P R ET Q E 3 63 G G G V G DI V G 3 81 S E T S A SE E E 3 82 E T S A S EE E K 3 22 R D E N G EC G Q 2 72 R D L S I SF R N 2 6 E Y D K S LS V Q 1 14 Q P E K R TG L R 1 24 E N G E C GQ T F 1 25 N G E C G QT F R 1 31 T F R L K EE Q G 1 54 D P R E T QE V F 1 59 Q E V F G GG V G 1 80 N S E T S AS E E 1 36 E E Q G R AF R G −1 48 H Q K L V ND P R −1 4 C Q E Y D KS L S −2 37 E Q G R A FR G S −2 55 P R E T Q EV F G −2 15 P E K R T GL R D −3 27 E C G Q T FR L K −3 213P1F11 v.4: HLA-A1 nonamers 83 T SA S E E EK Y 21 45 S SV H Q K LV N 15 52 V ND P R E TQ E 15 85 A SE E E K YD H 15 66 V GD I V G RD L 14 80 N SE T S A SE E 14 34 L KE E Q G RA F 13 35 K EE Q G R AF R 13 86 S EE E K Y DM S 13 4 C QE Y D K SL S 12 14 Q PE K R T GL R 12 22 R DE N G E CG Q 12 25 N GE C G Q TF R 12 55 P RE T Q E VF G 12 6 E YD K S L SV Q 11 21 L RD E N G EC G 11 57 E TQ E V F GG G 11 58 T QE V F G GG V 11 71 G RD L S I SF R 10 28 C GQ T F R LK E 9 10 S LS V Q P EK R 7 15 P EK R T G LR D 7 63 G GG V G D IV G 7 68 D IV G R D LS I 7 5 Q EY D K S LS V 6 9 K SL S V Q PE K 6 12 S VQ P E K RT G 6 18 R TG L R D EN G 6 30 Q TF R L K EE Q 6 44 G SS V H Q KL V 6 82 E TS A S E EE K 6 11 L SV Q P E KR T 5 27 E CG Q T F RL K 5 61 V FG G G V GD I 5 62 F GG G V G DI V 5 67 G DI V G R DL S 5 70 V GR D L S IS F 5 74 L SI S F R NS E 5 43 R GS S V H QK L 4 59 Q EV F G G GV G 4 60 E VF G G G VG D 4 73 D LS I S F RN S 4 76 I SF R N S ET S 4 26 G EC G Q T FR L 3 37 E QG R A F RG S 3 40 R AF R G S SV H 3 48 H QK L V N DP R 3 75 S IS F R N SE T 3 1 M GK C Q E YD K 2 2 G KC Q E Y DK S 2 7 Y DK S L S VQ P 2 13 V QP E K R TG L 2 16 E KR T G L RD E 2 17 K RT G L R DE N 2 20 G LR D E N GE C 2 41 A FR G S S VH Q 2 42 F RG S S V HQ K 2 46 S VH Q K L VN D 2 47 V HQ K L V ND P 2 51 L VN D P R ET Q 2 77 S FR N S E TS A 2 81 S ET S A S EE E 2 84 S AS E E E KY D 2 3 K CQ E Y D KS L 1 8 D KS L S V QP E 1 23 D EN G E C GQ T 1 24 E NG E C G QT F 1 32 F RL K E E QG R 1 33 R LK E E Q GR A 1 36 E EQ G R A FR G 1 38 Q GR A F R GS S 1 39 G RA F R G SS V 1 50 K LV N D P RE T 1 54 D PR E T Q EV F 1 56 R ET Q E V FG G 1 64 G GV G D I VG R 1 65 G VG D T V GR D 1 78 F RN S E T SA S 1 213P1F11 v.4: HLA-A26 nonamers 57 E T Q E V F G G G 23 60 E V F G G G V G D 22 73 D L S I S F R N S 20 24 E N G E C G Q T F 19 68 D I V G R D L S I 19 46 S V H Q K L V N D 18 54 D P R E T Q E V F 18 82 E T S A S E E E K 18 65 G V G D I V G R D 16 69 I V G R D L S I S 15 6 E Y D K S L S V Q 14 30 Q T F R L K E E Q 14 34 L K E E Q G R A F 14 13 V Q P E K R T G L 13 70 V G R D L S I S F 13 83 T S A S E E E K Y 13 8 D K S L S V Q P E 12 12 S V Q P E K R T G 12 26 G E C G Q T F R L 12 33 R L K E E Q G R A 12 61 V F G G G V G D I 12 75 S I S F R N S E T 12 3 K C Q E Y D K S L 11 18 R T C L R D E N C 11 27 E C G Q T F R L K 11 37 E Q G R A F R G S 11 41 A F R C S S V H Q 11 43 R G S S V H Q K L 11 51 L V N D P R E T Q 11 10 S L S V Q P E K R 10 16 E K R T G L H D E 10 20 G L R D E N G E C 10 85 A S E E E K Y D M 10 23 D E N G E C G Q T 9 36 E E Q G R A F R G 9 50 K L V N D P R E T 9 66 V G D I V G R D L 9 31 T F R L K E E Q G 8 64 G G V C D I V G R 8 77 S F R N S E T S A 8 86 S E E E K Y D M S 8 42 F R G S S V H Q K 7 71 G R D L S I S F R 7 2 G K C Q E Y D K S 6 47 V H Q K L V N D P 6 56 R E T Q E V F G G 6 7 Y D K S L S V Q P 5 29 G Q T F R L K E E 5 17 K R T G L R D E N 4 52 V N D P R E T Q E 4 74 L S I S F R N S E 4 79 R N S E T S A S E 4 5 Q E Y D K S L S V 3 9 K S L S V Q P E K 3 21 L R D E N G E C G 3 32 F R L K E E Q G R 3 39 G R A F R G S S V 3 40 R A F R G S S V H 3 53 N D P R E T Q E V 3 78 F R N S E T S A S 3 80 N S E T S A S E E 3 1 M G K C Q E Y D K 2 11 L S V Q P E K R T 2 15 P E K R T G L R D 2 35 K E E Q G R A F R 2 49 Q K L V N D P R E 2 55 P R E T Q E V F G 2 62 F G G G V G D I V 2 67 G D I V G R D L S 2 72 R D L S I S F R N 2 76 I S F R N S E T S 2 81 S E T S A S E E E 2 84 S A S E E E K Y D 2 4 C Q E Y D K S L S 1 14 Q P E K R T G L R 1 19 T G L R D E N G E 1 22 R D E N G E C G Q 1 28 C G Q T F R L K E 1 38 Q G R A F R G S S 1 45 S S V H Q K L V N 1 48 H Q K L V N D P R 1 58 T Q E V F G G G V 1 213P1F11 v.4: HLA-A3 nonamers 60 E V FG G G VG D 21 40 R A FR G S SV H 20 12 S V QP E K RT G 18 69 I V GR D L ST S 18 33 R L KE E Q GR A 17 68 D I VG R D LS I 17 9 K S LS V Q PE K 16 10 S L SV Q P EK R 16 46 S V HQ K L VN D 16 51 L V ND P R ET Q 16 20 G L RD E N GE C 15 50 K L VN D P RE T 15 75 S I SF R N SE T 14 5 Q E YD K S LS V 13 213P1F11 v.4: HLA-A3 nonamers 60 E V FG G G VG D 21 40 R A FR G S SV H 20 12 S V QP E K RT G 18 69 I V GR D L SI S 18 33 R L KE E Q GR A 17 68 D I VG R D LS I 17 9 K S LS V Q PE K 16 10 S L SV Q P EK R 16 46 S V HQ K L VN D 16 51 L V ND P R ET Q 16 20 G L RD E N GE C 15 50 K L VN D P RE T 15 75 S I SF R N SE T 14 5 Q E YD K S LS V 13 35 K E EQ G R AF R 13 42 F R GS S V HQ K 13 24 E N GE C G QT F 12 41 A F RG S S VH Q 12 54 D P RE T Q EV F 12 59 Q E VF G G GV G 12 65 G V GD I V GR D 12 82 E T SA S E EE K 12 1 M G KC Q E YD K 11 38 Q G RA F R GS S 11 39 G R AF R G SS V 11 73 D L SI S F RN S 11 15 P E KR T G LR D 10 27 E C GQ T F RL K 10 64 G G VG D I VG R 10 70 V G RD L S IS F 10 79 R N SE T S AS E 10 14 Q P EK R T GL R 9 34 L K EE Q G RA F 9 52 V N DP R E TQ E 9 76 I S FR N S ET S 9 7 Y D KS L S VQ P 8 31 T F RL K E EQ G 8 45 S S VH Q K LV N 8 71 G R DL S I SF R 8 77 S F RN S E TS A 8 6 E Y DK S L SV Q 7 18 R T GL R D EN G 7 22 R D EN G E CG Q 7 25 N G EC G Q TF R 7 36 E E QG R A FR G 7 48 H Q KL V N DP R 7 83 T S AS E E EK Y 7 3 K C QE Y D KS L 6 17 K R TG L R DE N 6 23 D E NG E C GQ T 6 32 F R LK E E QG R 6 62 F G GG V G DI V 6 63 G G GV G D IV G 6 67 G D IV G R DL S 6 72 R D LS I S FR N 6 74 L S IS F R NS E 6 80 N S ET S A SE E 6 16 E K RT G L RD E 5 19 T G LR D E NG E 5 30 Q T FR L K EE Q 5 43 R G SS V H QK L 5 61 V F GG G V GD I 5 66 V G DI V G RD L 5 13 V Q PE K R TG L 4 21 L R DE N G EC G 4 28 C G QT F R LK E 4 49 Q K LV N D PR E 4 55 P R ET Q E VF G 4 56 R E TQ E V FG G 4 58 T Q EV F G GG V 4 78 F R NS E T SA S 4 85 A S EE E K YD M 4 86 S E EE K Y DM S 4 4 C Q EY D K SL S 3 37 E Q GR A F RG S 3 53 N D PR E T QE V 3 57 E T QE V F GG G 3 26 G E CG Q T FR L 2 29 G Q TF R L KE E 2 84 S A SE E E KY D 2 8 D K SL S V QP E 1 81 S E TS A S EE E 1 213P1F11 v.4: HLA-B*0702 nonamers 54 D P R E T Q E V F 19 26 G E C G Q T F R L 13 43 R G S S V H Q K L 13 13 V Q P E K R T G L 12 14 Q P E K R T G L R 12 66 V G D I V G R D L 12 3 K C Q E Y D K S L 11 61 V F G G G V G D I 10 62 F G G G V G D I V 10 68 D I V G R D L S I 10 11 L S V Q P E K R T 9 24 E N G E C G Q T F 9 77 S F R N S E T S A 9 5 Q E Y D K S L S V 8 23 D E N G E C G Q T 8 34 L K E E Q G R A F 8 39 G R A F R G S S V 8 41 A F R G S S V H Q 8 44 G S S V H Q K L V 8 70 V G R D L S I S F 8 75 S I S F R N S E T 8 85 A S E E E K Y D M 8 33 R L K E E Q G R A 7 50 K L V N D P R E T 7 53 N D P R E T Q E V 7 58 T Q E V F G G G V 7 60 E V F G G G V G D 6 6 E Y D K S L S V Q 4 8 D K S L S V Q P E 4 17 K R T G L R D E N 4 35 K E E Q G R A F R 4 38 Q G R A F R G S S 4 51 L V N D P R E T Q 4 64 G G V G D T V G R 4 79 R N S E T S A S E 4 82 E T S A S E E E K 4 15 P E K R T G L R D 3 16 E K R T G L R D E 3 27 E C G Q T F R L K 3 28 C G Q T F R L K E 3 36 E E Q G R A F R G 3 37 E Q G R A F R G S 3 45 S S V H Q K L V N 3 52 V N D P R E T Q E 3 55 P R E T Q E V F G 3 73 D L S I S F R N S 3 7 Y D K S L S V Q P 2 9 K S L S V Q P E K 2 10 S L S V Q P E K R 2 18 R T G L R D E N G 2 20 G L R D E N G E C 2 21 L R D E N G E C G 2 25 N G E C G Q T F R 2 31 T F R L K E E Q G 2 40 R A F R G S S V H 2 42 F R G S S V H Q K 2 46 S V H Q K L V N D 2 47 V H Q K L V N D P 2 56 R E T Q E V F G G 2 57 E T Q E V F G G G 2 59 Q E V F G G G V G 2 63 G G G V G D I V G 2 65 G V G D I V G R D 2 69 I V G R D L S T S 2 71 G R D L S I S F R 2 78 F R N S E T S A S 2 84 S A S E E E K Y D 2 12 S V Q P E K R T G 1 22 R D E N G E C G Q 1 32 F R L K E E Q G R 1 48 H Q K L V N D P R 1 49 Q K L V N D P R E 1 67 G D I V G R D L S 1 72 R D L S I S F R N 1 74 L S I S F R N S E 1 76 I S F R N S E T S 1 83 T S A S E E E K Y 1 213P1F11 v.4: HLA-B*08 nonamers 13 V Q P E K R T G L 23 54 D P R E T Q E V F 20 33 R L K E E Q G R A 18 68 D I V G R D L S I 18 31 T F R L K E E Q G 17 75 S I S F R N S E T 16 20 G L R D E N G E C 15 14 Q P E K R T G L R 14 86 S E E E K Y D M S 14 3 K C Q E Y D K S L 13 46 S V H Q K L V N D 13 48 H Q K L V N D P R 13 70 V G R D L S I S F 13 7 Y D K S L S V Q P 11 26 G E C G Q T F R L 11 29 G Q T F R L K E E 11 66 V G D I V G R D L 11 1 M G K C Q E Y D K 10 5 Q E Y D K S L S V 10 15 P E K R T G L R D 10 24 E N G E C G Q T F 10 36 E E Q G R A F R G 10 43 R G S S V H Q K L 10 10 S L S V Q P E K R 9 18 R T G L R D E N G 9 34 L K E E Q G R A F 9 39 G R A F R G S S V 9 52 V H D P R E T Q E 9 84 S A S E E E K Y D 9 16 E K R T G L R D E 8 61 V F G G G V G D I 8 77 S F R N S E T S A 8 41 A F R G S S V H Q 7 50 K L V N D P R E T 7 38 Q G R A F R G S S 6 73 D L S I S F R N S 6 40 R A F R G S S V H 4 57 E T Q E V F G G G 4 44 G S S V H Q K L V 3 60 E V F G G G V G D 3 64 G G V G D I V G R 3 71 G R D L S I S F R 3 81 S E T S A S E E E 3 82 E T S A S E E E K 3 6 E Y D K S L S V Q 2 8 D K S L S V Q P E 2 12 S V Q P E K R T G 2 21 L R D E N G E C G 2 27 E C G Q T F R L K 2 37 E Q G R A F R G S 2 42 F R G S S V H Q K 2 45 S S V H Q K L V N 2 47 V H Q K L V N D P 2 63 G G G V G D I V G 2 65 G V G D I V G R D 2 67 G D I V G R D L S 2 78 F R N S E T S A S 2 79 R N S E T S A S E 2 85 A S E E E K Y D M 2 2 G K C Q E Y D K S 1 4 C Q E Y D K S L S 1 9 K S L S V Q P E K 1 11 L S V Q P E K R T 1 17 K R T G L R D E N 1 30 Q T F R L K E E Q 1 32 F R L K E E Q G R 1 49 Q K L V N D P R E 1 53 N D P R E T Q E V 1 55 P R E T Q E V F G 1 56 R E T Q E V F G G 1 58 T Q E V F G G G V 1 62 F G G G V G D I V 1 69 I V G R D L S I S 1 76 I S F R N S E T S 1 80 N S E T S A S E E 1 213P1F11 v.4: HLA-B*1510 nonamers 26 G E C G Q T F R L 14 13 V Q P E K R T G L 12 47 V H Q K L V N D P 12 66 V G D I V G R D L 12 3 K C Q E Y D K S L 11 34 L K E E Q G R A F 11 43 R G S S V H Q K L 11 24 E N G E C G Q T F 9 54 D P R E T Q E V F 9 85 A S E E E K Y D M 9 70 V G R D L S I S F 7 12 S V Q P E K R T G 6 49 Q K L V N D P R E 5 60 E V F G G G V G D 5 63 G G G V G D T V G 5 64 G G V G D I V G R 5 65 G V G D I V G R D 5 6 E Y D K S L S V Q 4 11 L S V Q P E K R T 4 16 E K R T G L R D E 4 27 E C G Q T F R L K 4 35 K E E Q G R A F R 4 36 E E Q G R A F R G 4 50 K L V N D P R E T 4 51 L V N D P R E T Q 4 67 G D I V G R D L S 4 76 I S F R N S E T S 4 7 Y D K S L S V Q P 3 10 S L S V Q P E K R 3 17 K R T G L R D E N 3 20 G L R D E N G E C 3 33 R L K E E Q G R A 3 37 E Q G R A F R G S 3 40 R A F R G S S V H 3 41 A F R G S S V H Q 3 44 G S S V H Q K L V 3 45 S S V H Q K L V N 3 46 S V H Q K L V N D 3 55 P R E T Q E V F G 3 58 T Q E V F G G G V 3 59 Q E V F G G G V G 3 61 V F G G G V G D I 3 73 D L S I S F R N S 3 82 E T S A S E E E K 3 2 G K C Q E Y D K S 2 4 C Q E Y D K S L S 2 8 D K S L S V Q P E 2 9 K S L S V Q P E K 2 14 Q P E K R T G L R 2 15 P E K R T G L R D 2 21 L R D E N G E C G 2 22 R D E N G E C G Q 2 25 N G E C G Q T F R 2 29 G Q T F R L K E E 2 30 Q T F R L K E E Q 2 39 G R A F R G S S V 2 52 V N D P R E T Q E 2 56 R E T Q E V F G G 2 57 E T Q E V F G G G 2 69 I V G R D L S I S 2 71 G R D L S I S F R 2 72 R D L S I S F R N 2 75 S I S F R N S E T 2 79 R N S E T S A S E 2 80 N S E T S A S E E 2 83 T S A S E E E K Y 2 84 S A S E E E K Y D 2 86 S E E E K Y D M S 2 5 Q E Y D K S L S V 1 19 T G L R D E N G E 1 23 D E N G E C G Q T 1 28 C G Q T F R L K E 1 31 T F R L K E E Q G 1 32 F R L K E E Q G R 1 38 Q G R A F R G S S 1 42 F R G S S V H Q K 1 53 N D P R E T Q E V 1 62 F G G G V G D I V 1 68 D I V G R D L S I 1 74 L S I S F R N S E 1 78 F R N S E T S A S 1 81 S E T S A S E E E 1 213P1F11 v.4: HLA-B*2705 nonamers 71 G R D L S I S F R 29 32 F R L K E E Q G R 25 42 F R G S S V H Q K 23 40 R A F R G S S V H 20 64 G G V G D I V G R 20 9 K S L S V Q P E K 19 26 G E C G Q T F R L 17 35 K E E Q G R A F R 17 43 R G S S V H Q K L 17 17 K R T G L R D E N 16 25 N G E C G Q T F R 16 3 K C Q E Y D K S L 15 39 G R A F R G S S V 15 13 V Q P E K R T G L 14 54 D P R E T Q E V F 14 70 V G R D L S 1S F 14 48 H Q K L V N D P R 13 85 A S E E E K Y D M 13 10 S L S V Q P E K R 12 14 Q P E K R T G L R 12 24 E N G E C G Q T F 12 27 E C G Q T F R L C 12 68 D I V G R D L S I 12 78 F R N S E T S A S 12 82 E T S A S E E E K 12 1 M G K C Q E Y D K 11 21 L R D E N G E C G 11 34 L K E E Q G R A F 11 55 P R E T Q E V F G 11 61 V F G G G V G D I 11 66 V G D I V G R D L 11 83 T S A S E E E K Y 11 33 R L K E E Q G R A 10 72 R D L S I S F R N 10 2 G K C Q E Y D K S 8 18 R T G L R D E N G 8 56 R E T Q E V F G G 8 60 E V F G G G V G D 8 63 G G G V G D I V G 8 79 R N S E T S A S E 8 46 S V H Q K L V N D 7 65 G V G D I V G R D 7 67 G D I V G R D L S 7 5 Q E Y D K S L S V 6 6 E Y D K S L S V Q 6 11 L S V Q P E K R T 6 20 G L R D E N G E C 6 22 R D E N G E C G Q 6 30 Q T F R L K E E Q 6 41 A F R G S S V H Q 6 49 Q K L V N D P R E 6 76 I S F R K S E T S 6 29 G Q T F R L K E E 5 36 E E Q G R A F R G 5 44 G S S V H Q K L V 5 47 V H Q K L V N D P 5 7 Y D K S L S V Q P 4 12 S V Q P E K R T G 4 23 D E N G E C G Q T 4 45 S S V H Q K L V N 4 50 K L V N D P R E T 4 52 V N D P R E T Q E 4 59 Q E V F G G G V G 4 74 L S I S F R N S E 4 77 S F R N S E T S A 4 80 N S E T S A S E E 4 8 D K S L S V Q P E 3 15 P E K R T G L R D 3 16 E K R T G L R D E 3 19 T G L R D E N G E 3 28 C G Q T F R L K E 3 31 T F R L K E E Q G 3 62 F G G G V G D I V 3 69 I V G R D L S I S 3 73 D L S I S F R N S 3 75 S I S F R N S E T 3 4 C Q E Y D K S L S 2 51 L V N D P R E T Q 2 53 N D P R E T Q E V 2 57 E T Q E V F G G G 2 81 S E T S A S E E E 2 84 S A S E E E K Y D 2 86 S E E E K Y D M S 2 38 Q G R A F R G S S 1 213P1F11 v.4: HLA-B*2709 nonamers 39 G R A F R G S S V 21 43 R G S S V H Q K L 15 26 G E C G Q T F R L 14 17 K R T G L R D E N 13 42 F R G S S V H Q K 13 71 G R D L S I S F R 13 3 K C Q E Y D K S L 12 32 F R L K E E Q G R 12 5 Q E Y D K S L S V 11 44 G S S V H Q K L V 11 55 P R E T Q E V F G 11 78 F R N S E T S A S 11 13 V Q P E K R T G L 10 21 L R D E N G E C G 10 66 V G D I V G R D L 10 68 D I V G R D L S I 10 85 A S E E E K Y D M 10 61 V F G G G V G D I 9 72 R D L S I S F R N 9 24 E N G E C G Q T F 8 34 L K E E Q G R A F 8 53 N D P R E T Q E V 8 54 D P R E T Q E V F 8 58 T Q E V F G G G V 8 62 F G G G V G D I V 8 70 V G R D L S 1S F 8 9 K S L S V Q P E K 6 40 R A F R G S S V H 6 56 R E T Q E V F G G 6 64 G G V G D I V G R 6 65 G V G D I V G R D 6 18 R T G L R D E N G 5 33 R L K E E Q G R A 5 22 R D E N G E C G Q 4 29 G Q T F R L K E E 4 49 Q K L V N D P R E 4 50 K L V N D P R E T 4 67 G D I V G R D L S 4 79 R N S E T S A S E 4 2 G K C Q E Y D K S 3 7 Y D K S L S V Q P 3 19 T G L R D E N G E 3 20 G L R D E N G E C 3 60 E V F G G G V G D 3 63 G G G V G D I V G 3 76 I S F R N S E T S 3 11 L S V Q P E K R T 2 15 P E K R T G L R D 2 30 Q T F R L K E E Q 2 35 K E E Q G R A F R 2 36 E E Q G R A F R G 2 41 A F R G S S V H Q 2 45 S S V H Q K L V N 2 46 S V H Q K L V N D 2 52 V N D P R E T Q E 2 69 I V G R D L S I S 2 74 L S I S F R N S E 2 81 S E T S A S E E E 2 1 M G K C Q E Y D K 1 4 C Q E Y D K S L S 1 8 D K S L S V Q P E 1 12 S V Q P E K R T G 1 23 D E N G E C G Q T 1 28 C G Q T F R L K E 1 31 T F R L K E E Q G 1 47 V H Q K L V N D P 1 59 Q E V F G G G V G 1 73 D L S I S F R N S 1 80 N S E T S A S E E 1 83 T S A S E E E K Y 1 213P1F11 v.4: HLA-B*4402 nonamers 26 G E C G Q T F R L 22 36 E E Q G R A F R G 15 3 K C Q E Y D K S L 13 24 E N G E C G Q T F 13 34 L K E E Q G R A F 13 5 Q E Y D K S L S V 12 13 V Q P E K R T G L 12 15 P E K R T G L R D 12 23 D E N G E C G Q T 12 35 K E E Q G R A F R 12 43 R G S S V H Q K L 12 66 V G D I V G R D L 12 70 V G R D L S I S F 12 54 D P R E T Q E V F 11 56 R E T Q E V F G G 11 59 Q E V F G G G V G 11 68 D I V G R D L S I 11 81 S E T S A S E E E 11 83 T S A S E E E K Y 11 86 S E E E K Y D M S 11 61 V F G G G V G D I 10 12 S V Q P E K R T G 6 52 V N D P R E T Q E 6 60 E V F G G G V G D 6 6 E Y D K S L S V Q 5 16 E K R T G L R D E 5 27 E C G Q T F R L K 5 37 E Q G R A F R G S 5 41 A F R G S S V H Q 5 64 G G V G D I V G R 5 67 G D I V G R D L S 5 71 G R D L S I S F R 5 74 L S I S F R N S E 5 76 I S F R N S E T S 5 10 S L S V Q P E K R 4 28 C G Q T F R L K E 4 29 G Q T F R L K E E 4 40 R A F R G S S V H 4 44 G S S V H Q K L V 4 50 K L V N D P R E T 4 53 N D P R E T Q E V 4 84 S A S E E E K Y D 4 8 D K S L S V Q P E 3 14 Q P E K R T G L R 3 17 K R T G L R D E N 3 19 T G L R D E N G E 3 30 Q T F R L K E E Q 3 42 F R G S S V H Q K 3 45 S S V H Q K L V N 3 46 S V H Q K L V N D 3 51 L V N D P R E T Q 3 57 E T Q E V F G G G 3 65 G V G D I V G R D 3 73 D L S I S F R N S 3 75 S I S F R N S E T 3 78 F R N S E T S A S 3 80 N S E T S A S E E 3 82 E T S A S E E E K 3 85 A S E E E K Y D M 3 7 Y D K S L S V Q P 2 9 K S L S V Q P E K 2 11 L S V Q P E K R T 2 18 R T G L R D E N G 2 21 L R D E N G E C G 2 31 T F R L K E E Q G 2 47 V H Q K L V N D P 2 49 Q K L V N D P R E 2 55 P R E T Q E V F G 2 63 G G G V G D I V G 2 69 I V G R D L S I S 2 72 R D L S I S F R N 2 77 S F R N S E T S A 2 2 G K C Q E Y D K S 1 4 C Q E Y D K S L S 1 20 G L R D E N G E C 1 22 R D E N G E C G Q 1 25 N G E C G Q T F R 1 32 F R L K E E Q G R 1 38 Q G R A F R G S S 1 39 G R A F R G S S V 1 48 H Q K L V N D P R 1 62 F G G G V G D I V 1 79 R N S E T S A S E 1 213P1F11 v.4: HLA-B*5101 nonamers 54 D P R E T Q E V F 20 62 F G G G V G D I V 18 43 R G S S V H Q K L 16 66 V G D I V G R D L 16 68 D I V G R D L S I 16 5 Q E Y D K S L S V 15 40 R A F R G S S V H 14 61 V F G G G V G D I 14 84 S A S E E E K Y D 13 13 V Q P E K R T G L 12 14 Q P E K R T G L R 11 3 K C Q E Y D K S L 10 19 T G L R D E N G E 10 28 C G Q T F R L K E 10 53 N D P R E T Q E V 10 63 G G G V G D I V G 10 44 G S S V H Q K L V 9 58 T Q E V F G G G V 9 64 G G V G D I V G R 9 70 V G R D L S I S F 9 25 N G E C G Q T F R 8 26 G E C G Q T F R L 8 39 G R A F R G S S V 8 1 M G K C Q E Y D K 7 8 D K S L S V Q P E 7 38 Q G R A F R G S S 7 73 D L S I S F R N S 7 6 E Y D K S L S V Q 6 23 D E N G E C G Q T 5 34 L K E E Q G R A F 5 47 V H Q K L V N D P 5 51 L V N D P R E T Q 5 57 E T Q E V F G G G 5 76 I S F R N S E T S 5 9 K S L S V Q P E K 4 11 L S V Q P E K R T 4 21 L R D E N G E C G 4 24 E N G E C G Q T F 4 32 F R L K E E Q G R 4 41 A F R G S S V H Q 4 42 F R G S S V H Q K 4 45 S S V H Q K L V N 4 65 G V G D I V G R D 4 80 N S E T S A S E E 4 7 Y D K S L S V Q P 3 10 S L S V Q P E K R 3 12 S V Q P E K R T G 3 15 P E K R T G L R D 3 16 E K R T G L R D E 3 27 E C G Q T F R L K 3 33 R L K E E Q G R A 3 36 E E Q G R A F R G 3 46 S V H Q K L V N D 3 48 H Q K L V N D P R 3 49 Q K L V N D P R E 3 59 Q E V F G G G V G 3 60 E V F G G G V G D 3 69 I V G R D L S I S 3 72 R D L S I S F R N 3 74 L S I S F R N S E 3 79 R N S E T S A S E 3 83 T S A S E E E K Y 3 86 S E E E K Y D M S 3 2 G K C Q E Y D K S 2 20 G L R D E N G E C 2 29 G Q T F R L K E E 2 31 T F R L K E E Q G 2 37 E Q G R A F R G S 2 50 K L V N D P R E T 2 52 V N D P R E T Q E 2 55 P R E T Q E V F G 2 56 R E T Q E V F G G 2 71 G R D L S I S F R 2 77 S F R N S E T S A 2 82 E T S A S E E E K 2 85 A S E E E K Y D M 2 17 K R T G L R D E N 1 30 Q T F R L K E E Q 1 75 S I S F R N S E T 1 78 F R N S E T S A S 1 213P1F11 v.4: RT1.A1 nonamers 76 I S F R N S E T S 18 83 T S A S E E E K Y 18 60 E V F G G G V G D 17 85 A S E E E K Y D M 17 40 R A F R G S S V H 16 9 K S L S V Q P E K 14 30 Q T F R L K E E Q 13 24 E N G E C G Q T F 12 54 D P R E T Q E V F 12 12 S V Q P E K R T G 11 34 L K E E Q G R A F 11 5 Q E Y D K S L S V 10 70 V G R D L S I S F 10 51 L V N D P R E T Q 9 84 S A S E E E K Y D 9 3 K C Q E Y D K S L 8 13 V Q P E K R T G L 8 32 F R L K E E Q G R 8 45 S S V H Q K L V N 8 46 S V H Q K L V N D 8 74 L S I S F R N S E 8 11 L S V Q P E K R T 7 19 T G L R D E N G E 7 26 G E C G Q T F R L 7 66 V G D I V G R D L 7 69 I V G R D L S I S 7 80 N S E T S A S E E 7 43 R G S S V H Q K L 6 44 G S S V H Q K L V 6 49 Q K L V H D P R E 6 65 G V G D I V G R D 6 72 R D L S I S F R N 6 17 K R T G L R D E N 5 2 G K C Q E Y D K S 3 35 K E E Q G R A F R 3 50 K L V N D P R E T 3 81 S E T S A S E E E 3 86 S E E E K Y D M S 3 4 C Q E Y D K S L S 2 10 S L S V Q P E K R 2 18 R T G L R D E N G 2 20 G L R D E N G E C 2 21 L R D E N G E C G 2 23 D E N G E C G Q T 2 28 C G Q T F R L K E 2 31 T F R L K E E Q G 2 33 R L K E E Q G R A 2 38 Q G R A F R G S S 2 41 A F R G S S V H Q 2 47 V H Q K L V N D P 2 48 H Q K L V N D P R 2 53 N D P R E T Q E V 2 56 R E T Q E V F G G 2 61 V F G G G V G D I 2 62 F G G G V G D I V 2 64 G G V G D I V G R 2 67 G D I V G R D L S 2 75 S I S F R N S E T 2 82 E T S A S E E E K 2 1 M G K C Q E Y D K 1 6 E Y D K S L S V Q 1 8 D K S L S V Q P E 1 14 Q P E K R T G L R 1 16 E K R T G L R D E TABLE XIXA, part 5: MHC Class 1 nonamer analysis of 213P1F11 v.5 (aa 1-242) 213P1F11 v.5: HLA-A *0201 nonamers 5 A L I L R VT K A 24 2 A R L A L IL R V 20 3 R L A L I LR V T 20 6 L I L R V TK A R 14 7 I L R V T KA R E 14 4 L A L I L RV T K 13 1 G A R L A LI L R 9 8 L R V T K AR E G 5 9 R V T K A RE G S 4 213P1F11 v.5: HLA-A1 nonamers 2 A RL A L I LR V 7 1 G AR L A L IL R 6 5 A LI L R V TK A 5 3 R LA L I L RV T 2 6 L IL R V T KA R 2 4 L AL I L R VT K 1 7 I LR V T K AR E 1 9 R VT K A R EG S 1 213P1F11 v.5: HLA-A26 nonamers 5 A L I L R V T K A 15 3 R L A L I L R V T 13 9 R V T K A R E G S 12 6 L I L R V T K A R 11 7 I L R V T K A R E 10 2 A R L A L I L R V 6 1 G A R L A L I L R 4 4 L A L I L R V T K 2 8 L R V T K A R E G 2 213P1F11 v.5: HLA-A3 nonamers 4 L A LI L R VT K 22 3 R L AL I L RV T 20 5 A L IL R V TK A 20 6 L I LR V T KA R 19 7 I L RV T K AR E 18 9 R V TK A R EG S 15 2 A R LA L I LR V 10 1 C A RL A L IL R 8 8 L R VT K A RE G 4 213P1F11 v.5: HLA-B*0702 nonamers 2 A R L A L I L R V 11 5 A L I L R V T K A 10 3 R L A L I L R V T 9 7 I L R V T K A R E 4 4 L A L I L R V T K 3 9 R V T K A R E G S 3 1 G A R L A L I L R 2 6 L I L R V T K A R 2 213P1F11 v.5: HLA-B*08 nonamers 5 A L I L R V T K A 16 7 I L R V T K A R E 14 1 G A R L A L I L R 12 8 L R V T K A R E G 11 3 R L A L I L R V T 7 6 L I L R V T K A R 6 4 L A L I L R V T K 5 2 A R L A L I L R V 1 213P1F11 v.5: HLA-B*1510 nonamers 7 I L R V T K A R E 6 3 R L A L I L R V T 5 4 L A L I L R V T K 4 2 A R L A L I L R V 3 8 L R V T K A R E G 3 9 R V T K A R E G S 2 1 G A R L A L I L R 1 5 A L I L R V T K A 1 6 L I L R V T K A R 1 213P1F11 v.5: HLA-B*2705 nonamers 2 A R L A L I L R V 20 1 G A R L A L I L R 16 4 L A L I L R V T K 15 8 L R V T K A R E G 14 6 L I L R V T K A R 13 5 A L I L R V T K A 9 3 R L A L I L R V T 8 9 R V T K A R E G S 6 7 I L R V T K A R E 5 213P1F11 v.5: HLA-B*2709 nonamers 2 A R L A L I L R V 23 8 L R V T K A R E G 12 3 R L A L I L R V T 5 9 R V T K A R E G S 5 1 G A R L A L I L R 3 4 L A L I L R V T K 3 5 A L I L R V T K A 3 6 L T L R V T K A R 2 7 I L R V T K A R E 2 213P1F11 v.5: HLA-B*4402 nonamers 5 A L l L R V T K A 10 2 A R L A L I L R V 8 6 L I L R V T K A R 7 2 R L A L I L R TABLE XIXA, part 6: MHC Class 1 nonamer analysis of 213P1F11 v.6(aa 1-242) 213P1F11 v.6: HLA-A*0201 nonamers 1 K L E N L FE A M 16 8 A M N N K NC Q A 16 4 N L F E A MN N K 14 9 M N N K N CQ A L 12 2 L E N L F EA M N 6 5 L F E A M NN K N 4 7 E A M N N KN C Q 3 6 F E A M N NK N C 2 3 E N L F E AM N N −2 213P1F11 v.6: HLA-A1 nonamers 1 K L E N L F EA M 14 5 L F E A M N NK N 13 6 F E A M N N KN C 2 8 A M N N K N CQ A 2 2 L E N L F E AM N 1 4 N L F E A M NN K 1 213P1F11 v.6: HLA-A26 nonamers 1 K L E N L F E A M 22 4 N L F E A M N N K 17 9 M N N K N C Q A L 11 3 E N L F E A M N N 8 5 L F E A M N N K N 7 7 E A M N N K N C Q 6 2 L E N L F E A M N 2 6 F E A M N N K N C 2 8 A M N N K N C Q A 2 213P1F11 v.6: HLA-A3 nonamers 4 N L FE A M NN K 21 1 K L EN L F EA M 15 8 A M NN K N CQ A 8 3 E N LF E A MN N 6 2 L E NL F E AM N 5 5 L F EA M N NK N 2 6 F E AM N N KN C 1 7 E A MN N K NC Q 1 9 M N NK N C QA L 1 213P1F11 v.6: HLA-B*0702 nonamers 9 M N N K N C Q A L 12 1 K L E N L F E A M 9 8 A M N N K N C Q A 8 7 E A M N N K N C Q 2 2 L E N L F E A M N 1 3 E N L F E A M N N 1 5 L F E A M N N K N 1 6 F E A M N N K N C 1 213P1F11 v.6: HLA-B*08 nonamers 8 A M N N K N C Q A 11 9 M N N K N C Q A L 11 4 N L F E A M N N K 8 1 K L E N L F E A M 7 7 E A M N N K N C Q 6 6 F E A M N N K N C 3 3 E N L F E A M N N 2 2 L E N L F E A M N 1 5 L F E A M N N K N 1 213P1F11 v.6: HLA-B*1510 nonamers 9 M N N K N C Q A L 13 1 K L E N L F E A M 8 7 E A M N N K N C Q 3 4 N L F E A M N N K 2 6 F E A M N N K N C 2 3 E N L F E A M N N 1 5 L F E A M N N K N 1 213P1F1 v.6: HLA-B*2705 nonamers 4 N L F E A M N N K 17 9 M N N K N C Q A L 12 1 K L E N L F E A M 11 3 E N L F E A M N N 7 5 L F E A M N N K N 4 8 A M N N K N C Q A 4 6 F E A M N N K N C 3 2 L E N L F E A M N 2 7 E A M N N K N C Q 2 213P1F11 v.6: HLA-B*2709 nonamers 1 K L E N L F E A M 10 9 M N N K N C Q A L 10 3 E N L F E A M N N 4 4 N L F E A M N N K 3 8 A M N N K N C Q A 2 6 F E A M N N K N C 1 213P1F11 v.6: HLA-B*4402 nonamers 6 F E A M N N K N C 13 9 M N N K N C Q A L 13 2 L E N L F E A M N 11 7 E A M N N K N C Q 6 1 K L E N L F E A M 5 8 A M N N K N C Q A 5 3 E N L F E A M N N 4 4 N L F E A M N N K 4 5 L F E A M N N K N 2
[0770] 23 TABLE XIXB MHC Class I Analysis of 213P1F11 (decamers). SEQ. Pos 1 2 3 4 5 6 7 8 9 0 score ID NO. TABLE XIXB, part 1: MHC Class I decamer analysis of 213P1F11 v.1 (aa 1-242). 213P1F11 v.1: HLA-A*0201 decamers 111 A L N N K N C Q A L 24 13 D M S G A R L A L I 23 86 L M A H G R E G F L 22 94 F L K G E D G E M V 22 119 A L R A K P K V Y I 22 142 T V G G D E I V M V 22 16 G A R L A L I L C V 20 226 K T N P E I Q S T L 20 157 Q T I P T Y T D A L 19 185 S C F I Q T L V D V 19 200 G H I L E L L T E V 19 209 V T R R M A E A E L 19 19 L A L I L C V T K A 18 212 R M A E A E L V Q E 18 27 K A R E G S E E D L 17 37 D A L E H M F R Q L 17 56 P T A E Q F Q E E L 17 70 Q A I D S R E D P V 17 120 L R A K P K V Y I I 17 162 Y T D A L H V Y S T 17 196 T K R K G H I L E L 17 205 L L T E V T R R M A 17 18 R L A L I L C V T K 16 103 V K L E N L F E A L 16 107 N L F E A L N N K N 16 150 M V I K D S P Q T I 16 165 A L H V Y S T V E G 16 182 Q K G S C F I Q T L 16 197 K R K G H I L E L L 16 201 H I L E L L T E V T 16 12 Y D M S G A R L A L 15 96 K G E D G E M V K L 15 117 C Q A L R A K P K V 15 187 F I Q T L V D V F T 15 202 I L E L L T E V T R 15 20 A L E L L V T K A R 14 21 L I L C V T K A R E 14 22 I L C V T K A R E G 14 30 E G S E E D L D A L 14 75 R E D P V S C A F V 14 127 Y I I Q A C R G E Q 14 143 V G G D E I V M V I 14 149 V M V I K D S P Q T 14 163 T D A L H V Y S T V 14 170 S T V E G Y I A Y R 14 218 L V Q E G K A R K T 14 222 G K A R K T N P E I 14 230 E I Q S T L R K R L 14 6 S L E E E K Y D M S 13 85 V L M A H G R E G F 13 102 M V K L E N L F E A 13 134 G E Q R D P G E T V 13 167 H V Y S T V E G Y I 13 175 Y I A Y R H D Q K G 13 204 E L L T E V T R R M 13 233 S T L R K R L Y L Q 13 17 A R L A L I L C V T 12 99 D G E M V K L E N L 12 104 K L E N L F E A L Q 13 193 D V F T K R K G H I 12 10 E K Y D M S G A R L 12 14 M S G A R L A L I L 11 38 A L E H M F R Q L R 11 41 H H F R Q L R F B S 11 45 Q L R F E S T M K R 11 51 T H K R D P T A E Q 11 71 A I D S R E D P V S 11 77 D P V S C A F V V L 11 78 P V S C A F V V L M 11 159 I P T Y T D A L H V 11 183 K G S C F I Q T L V 11 194 V F T K R K G H I L 11 63 E E L E K F Q Q A I 10 79 V S C A F V V L M A 10 122 A K P K V Y I I Q A 10 128 I I Q A C R G E Q R 10 140 G E T V G G D E I V 10 153 K D S P Q T I P T Y 10 158 T I P T Y T D A L H 10 190 T L V D V F T K R K 10 210 T R R M A E A E L V 10 232 Q S T L R K R L Y L 10 1 M S N P R S L E E E 9 5 R S L E E E K Y D M 9 35 D L D A L E H M F R 9 73 D S R E D P V S C A 9 84 V V L M A H G R E G 9 93 G F L K G E D G E M 9 151 V I K D S P Q T I P 9 213 M A E A E L V Q E G 9 217 E L V Q E G K A R K 9 225 R K T N P E I Q S T 9 15 S G A R L A L I L C 8 5 S T M K R D P T A E 8 64 E L E K F Q Q A I D 8 80 S C A F V V L M A H 8 179 R H D Q K G S C F I 8 189 Q T L V D V F T K R 8 191 L V D V F T K R K G 8 199 K G H I L E L L T E 8 203 L E L L T E V T R R 8 207 T E V T R R M A E A 8 43 F R Q L R F E S T M 7 76 E D P V S C A F V V 7 81 C A F V V L M A H G 7 110 E A L N N K N C Q A 7 137 R D P G E T V G G D 7 141 E T V G G D E I V M 7 147 E I V M V I K D S P 7 148 I V M V I K D S P Q 7 160 P T Y T D A L H V Y 7 176 I A Y R H D Q K G S 7 188 I Q T L V D V F T K 7 214 A E A E L V Q E G K 7 25 V T K A R E G S E E 6 42 M F R Q L R F E S T 6 72 I D S R E D P V S C 6 87 M A H G R E G F L K 6 89 H G R E G F L K G E 6 97 G E D G E M V K L E 6 101 E M V K L E N L F E 6 114 N K N C Q A L R A K 6 125 K V Y I I Q A C R G 6 139 P G E T V G G D E I 6 145 G D E I V M V I K D 6 152 T K D S P Q T I P T 6 166 L H V Y S T V E G Y 6 168 V Y S T V E G Y I A 6 171 T V E G Y I A Y R H 6 186 C F I Q T L V D V F 6 206 L T E V T R R M A E 6 215 E A E L V Q E G K A 6 7 L E E E K Y D M S G 5 11 K Y D M S G A R L A 5 24 C V T K A R E G S E 5 29 R E G S E E D L D A 5 32 S E E D L D A L E H 5 33 E E D L D A L E H M 5 48 F E S T M K R D P T 5 83 F V V L M A H G R E 5 88 A H G R E G F L K G 5 112 L N N K N C Q A L R 5 115 K N C Q A L R A K P 5 118 Q A L R A K P K V Y 5 121 R A K P K V Y I I Q 5 123 K P K V Y I I Q A C 5 133 R G E Q R D P G E T 5 13 Q R D P G E T V G G 5 14 G G D E I V M V I K 5 155 S P Q T I P T Y T D 5 164 D A L H V Y S T V E 5 198 R K G H I L E L L T 5 223 K A R K T N P E I Q 5 229 P E I Q S T L R K R 5 2 S N P R S L E E E K 4 2 T K A R E G S E E D 4 31 G S E E D L D A L E 4 3 L D A L E H M F R Q 4 4 L R F E S T M K R D 4 58 A E Q F Q E E L E K 4 59 E Q F Q E E L E K F 4 6 E K F Q Q A I D S R 4 82 A F V V L M A H G R 4 9 G R E G F L K G E D 4 95 L K G E D G E M V K 4 105 L E N L F E A L N N 4 109 F E A L N N K N C Q 4 113 N N K N C Q A L R A 4 12 V Y I I Q A C R G E 4 129 I Q A C R G E Q R D 4 13 Q A C R G E Q R D P 4 131 A C R G E Q R D P G 4 132 C R G E Q R D P G E 4 14 D E I V M V I K D S 4 177 A Y R H D Q K G S C 4 181 D Q K G S C F I Q T 4 195 F T K R K G H I L E 4 21 A E L V Q E G K A R 4 23 L C V T K A R E G S 3 28 A R E G S E E D L D 3 3 L E H M F R Q L R F 3 4 E S T M K R D P T A 3 53 K R D P T A E Q F Q 3 57 T A E Q F Q E E L E 3 62 Q E E L E K F Q Q A 3 68 F Q Q A I D S R E D 3 7 S R E D P V S C A F 3 10 G E M V K L E N L F 3 10 E N L F E A L N N K 3 15 D S P Q T I P T Y T 3 17 G Y I A Y R H D Q K 3 178 Y R H D Q K G S C F 3 219 V Q E G K A R K T N 3 3 N P R S L E E E K Y 2 4 R Q L R F E S T M K 2 61 F Q E E L E K F Q Q 2 69 Q Q A I D S R E D P 2 98 E D G E M V K L E N 2 161 T Y T D A L H V Y S 2 169 Y S T V E G Y I A Y 2 211 R R M A E A E L V Q 2 227 T N P E I Q S T L R 2 231 I Q S T L R K R L Y 2 8 E E E K Y D M S G A 1 34 E D L D A L E H M F 2 40 E H M F R Q L R F E 1 52 M K R D P T A E Q F 1 60 Q F Q E E L E K F Q 1 67 K F Q Q A I D S R E 1 138 D P G E T V G G D E 1 184 G S C F I Q T L V D 1 208 E V T R R M A E A E 1 224 A R K T N P E I Q S 1 47 R F E S T M K R D P −1 91 R E G F L K G E D G −1 92 E G F L K G E D G E −1 116 N C Q A L R A K P K −1 173 E G Y I A Y R H D Q −1 124 P K V Y I I Q A C R −2 135 E Q R D P G E T V G −2 221 E G K A R K T N P E −3 4 P R S L E E E K Y D −4 9 E E K Y D M S G A R −4 213P1F11 v.1: HLA-A*0202 decamers 214 A E A E L V Q E G K 4 15 S G A R L A L I L C 3 18 R L A L I L C V T K 3 26 T K A R E G S E E D 3 36 L D A L E H M F R Q 3 56 P T A E Q F Q E E L 3 69 Q Q A I D S R E D P 3 80 S C A F V V L M A H 3 86 L M A H G R E G F L 3 109 F E A L N N K N C Q 3 117 C Q A L R A K P K V 3 120 L R A K P K V Y I I 3 129 I Q A C R G E Q R D 3 163 T D A L H V Y S T V 3 175 Y I A Y R H D Q K G 3 212 R M A E A E L V Q E 3 222 G K A R K T N P E I 3 16 G A R L A L I L C V 2 19 L A L I L C V T K A 2 27 K A R E G S E E D L 2 37 D A L E H M F R Q L 2 57 T A E Q F Q E E L E 2 70 Q A I D S R E D P V 2 81 C A F V V L M A H G 2 87 M A H G R E G F L K 2 110 E A L N N K N C Q A 2 118 Q A L R A K P K V Y 2 121 R A K P K V Y I I Q 2 130 Q A C R G E Q R D P 2 164 D A L H V Y S T V E 2 176 I A Y R H D Q K G S 2 213 E A E L V Q E G K A 2 223 K A R K T N P E I Q 2 17 A A L A L I L C V T 1 20 A L I L C V T K A R 1 28 A R E G S E E D L D 1 38 A L E H M F R Q L R 1 58 A E Q F Q E E L E K 1 71 A I D S R E D P V S 1 82 A F V V L M A H G R 1 88 A H G R E G F L K G 1 111 A L N N K N C Q A L 1 119 A L R A K P K V Y I 1 122 A K P K V Y I I Q A 1 131 A C R G E Q R D P G 1 165 A L H V Y S T V E G 1 177 A Y R H D Q K G S C 1 216 A E L V Q E G K A R 1 224 A R K T N P E I Q S 1 213P1F11 v.1: HLA-A *0203 decamers 207 T E V T R R M A E A 18 8 E E E K Y D M S G A 10 11 K Y D M S G A R L A 10 19 L A L I L C V T K A 10 29 R E G S E E D L D A 10 49 E S T M K R D P T A 10 62 Q E E L E K F Q Q A 10 73 D S R E D P V S C A 10 79 V S C A F V V L M A 10 102 M V K L E N L F E A 10 110 E A L N N K N C Q A 10 113 N N K N C Q A L R A 10 122 A K P K V Y I I Q A 10 156 P Q T I P T Y T D A 10 168 V Y S T V E G Y I A 10 205 L L T E V T R R M A 10 215 E A E L V Q E G K A 10 9 E E K Y D M S G A R 9 12 Y D N S G A R L A L 9 20 A L I L C V T K A R 9 30 E G S E E D L D A L 9 50 S T M K R D P T A E 9 63 E E L E K F Q Q A I 9 74 S R E D P V S C A F 9 80 S C A F V V L M A H 9 103 V K L E N L F E A L 9 111 A L N N K N C Q A L 9 114 N K N C Q A L R A K 9 123 K P K V Y I I Q A C 9 157 Q T I P T Y T D A L 9 169 Y S T V E G Y I A Y 9 206 L T E V T R R M A E 9 208 E V T R R M A E A E 9 216 A E L V Q E G K A R 9 10 E K Y D M S G A R L 8 13 D M S G A R L A L I 8 21 L I L C V T K A R E 8 31 G S E E D L D A L E 8 51 T M K R D P T A E Q 8 64 E L E K F Q Q A I D 8 75 R E D P V S C A F V 8 81 C A F V V L M A H G 8 104 K L E N L F E A L N 8 112 L N N K N C Q A L R 8 115 K N C Q A L R A K P 8 124 P K V Y I I Q A C R 8 158 T I P T Y T D A L H 8 170 S T V E G Y I A Y R 8 209 V T R R M A E A E L 8 217 E L V Q E G K A R K 8 213P1F11 v.1: HLA-A1 decamers 169 Y S T V E G Y I A Y 25 160 P T Y T D A L H V Y 22 153 K D S P Q T I P T Y 19 162 Y T D A L H V Y S T 19 32 S E E D L D A L E H 18 3 N P R S L E E E K Y 17 145 G D E I V M V I K D 17 206 L T E V T R R M A E 17 231 I Q S T L R K R L Y 17 31 G S E E D L D A L E 16 118 Q A L R A K P K V Y 16 166 L H V Y S T V E G Y 16 228 N P E I Q S T L R K 16 38 A L E H M F R Q L R 15 6 S L E E E K Y D M S 14 28 A R E G S E E D L D 14 53 K R D P T A E Q F Q 14 75 R E D P V S C A F V 14 97 G E D G E M V K L E 14 136 Q R D P G E T V G G 14 152 I K D S P Q T I P T 14 62 Q E E L E K F Q Q A 13 79 V S C A F V V L M A 13 104 K L E N L F E A L N 13 195 F T K R K G H I L E 13 219 V Q E G K A R K T N 13 11 K Y D M S G A R L A 12 57 T A E Q F Q E E L E 12 71 A I D S R E D P V S 12 74 S R E D P V S C A F 12 88 A H G R E G F L K G 12 96 K G E D G E M V K L 12 141 E T V G G D E I V M 12 191 L V D V F T K R K G 12 215 E A E L V Q E G K A 12 35 D L D A L E H M F R 11 61 F Q E E L E K F Q Q 11 64 E L E K F Q Q A I D 11 90 G R E G F L K G E D 11 108 L F E A L N N K N C 11 139 P G E T V G G D E I 11 171 T V E G Y I A Y R H 11 184 G S C F I Q T L V D 11 189 Q T L V D V F T K R 11 202 I L E L L T E V T R 11 213 M A E A E L V Q E G 11 232 Q S T L R K R L Y L 11 7 L E E E K Y D M S G 10 8 E E E K Y D M S G A 10 14 M S G A R L A L I L 10 33 E E D L D A L E H M 10 47 R F E S T M K R D P 10 99 D G E M V K L E N L 10 133 R G E Q R D P C E T 10 144 G G D E I V M V I K 10 157 Q T I P T Y T D A L 10 179 R H D Q K G S C F I 10 226 K T N P E I Q S T L 10 233 S T L R K R L Y L Q 10 12 Y D M S G A R L A L 9 1 M S N P R S L E E E 8 15 S G A R L A L I L C 8 25 V T K A R E G S E E 8 50 S T N K R D P T A E 8 121 R A K P K V Y I I Q 8 170 S T V E G Y I A Y R 8 181 D Q K G S C F I Q T 8 198 R K G H I L E L L T 8 58 A E Q F Q E E L E K 7 101 E M V K L E N L F E 7 209 V T R R M A E A E L 7 211 R R M A E A E L V Q 7 16 G A R L A L I L C V 6 29 R E G S E E D L D A 6 39 L E H M F R Q L R F 6 56 P T A E Q F Q E E L 6 98 E D G E M V K L E N 6 105 L E N L F E A L N N 6 113 N N K N C Q A L R A 6 159 I P T Y T D A L H V 6 196 T K R K G H I L E L 6 199 K G H I L E L L T E 6 73 D S R E D P V S C A 5 94 F L K G E D G E M V 5 122 A K P K V Y I I Q A 5 224 A R K T N P E I Q S 10 5 R S L E E E K Y D M 4 49 E S T M K R D P T A 4 65 L E K F Q Q A I D S 4 77 D P V S C A F V V L 4 87 M A H G R E G F L K 4 103 V K L E N L F E A L 4 107 N L F E A L N N K N 4 154 D S P Q T I P T Y T 4 175 Y T A Y R H D Q K G 4 13 D M S G A R L A L I 3 19 L A L I L C V T K A 3 37 D A L E H M F R Q L 3 42 M F R Q L R F E S T 3 45 Q L R F E S T M K R 3 55 D P T A E Q F Q E E 3 78 P V S C A F V V L M 3 85 V L N A H G R E G F 3 115 K N C Q A L R A K P 3 127 Y T I Q A C R G E Q 3 131 A C R G E Q R D P G 3 143 V G G D E I V M V I 3 178 Y R H D Q K G S C F 3 197 K R K G H I L E L L 3 205 L L T E V T R R M A 3 2 S N P R S L E E E K 2 17 A R L A L I L C V T 2 20 A L I L C V T K A R 2 48 F E S T M K R D P T 2 59 E Q F Q E E L E K F 2 80 S C A F V V L M A H 2 84 V V L M A H G R E G 2 100 G E M V K L E N L F 2 111 A L N N K N C Q A L 2 112 L N N K N C Q A L R 2 117 C Q A L R A K P K V 2 119 A L R A K P K V Y I 2 137 R D P G E T V G G D 2 142 T V G G D E I V M V 2 155 S P Q T I P T Y T D 2 158 T T P T Y T D A L H 2 165 A L H V Y S T V E G 2 168 V Y S T V E G Y I A 2 183 K G S C F I Q T L V 2 185 S C F I Q T L V D V 2 186 C F I Q T L V D V F 2 192 V D V F T K R K G H 2 194 V F T K R K G H I L 2 210 T R R M A E A E L V 2 216 A E L V Q E G K A R 2 218 L V Q E G K A R K T 2 227 T N P E I Q S T L R 2 229 P E I Q S T L R K R 2 10 E K Y D M S G A R L 1 18 R L A L I L C V T K 1 22 I L C V T K A R E G 1 23 L C V T K A R E G S 1 34 E D L D A L E H M F 1 41 H M F R Q L R F E S 1 43 F R Q L R F E S T M 1 44 R Q I I R F E S T M 1 68 F Q Q A I D S R E D 1 69 Q Q A I D S R E D P 1 76 E D P V S C A F V V 1 82 A F V V L M A H G R 1 83 F V V L M A H G R E 1 91 R E G F L K G E D G 1 95 L K G E D G E M V K 1 109 F E A L N N K N C Q 1 110 E A L N N K N C Q A 1 120 L R A K P K V Y I I 1 126 V Y I I Q A C R G E 1 128 I I Q A C R G H Q R 1 134 G E Q R D P G E T V 1 135 E Q R D P G E T V G 1 138 D P G E T V G G D E 1 149 V M V I K D S P Q T 1 151 V I K D S P Q T I P 1 164 D A L H V Y S T V E 1 172 V E G Y I A Y R H D 1 173 E G Y I A Y R H D Q 1 177 A Y R H D Q K G S C 1 187 F I Q T L V D V F T 1 188 I L Q T L V D V F T K 190 T L V D V F T K R K 1 201 H I L E L L T E V T 1 203 L E L L T E V T R R 1 204 E L L T E V T R R M 1 214 A E A E L V Q E G K 1 217 E L V Q E G K A R K 1 222 G K A R K T N P E I 1 230 E I Q S T L R K R L 1 2131P1511 v.1: HLA-A26 decamers 141 E T V G G D E I V M 26 230 E T Q S T L R K R L 26 59 E Q F Q E E L E K F 24 160 P T Y T D A L H V Y 24 78 P V S C A F V V L M 23 157 Q T I P T Y T D A L 23 186 C F I Q T L V D V F 23 204 E L L T E V T R R M 23 56 P T A E Q F Q E E L 22 30 E G S E E D L D A L 21 37 D A L E H M F R Q L 21 226 K T N P E I Q S T L 21 33 E E D L D A L E H M 20 77 D P V S C A F V V L 20 209 V T R R M A E A E L 20 85 V L M A H G R E G F 19 99 D G E M V K L E N L 19 193 D V F T K R K G H I 19 34 E D L D A L E H M F 18 111 A L N N K N C Q A L 18 142 T V G G D E I V M V 18 10 E K Y D M S G A R L 17 147 E I V M V I K D S P 17 153 K D S P Q T I P T Y 17 170 S T V E G Y I A Y R 17 208 E V T R R M A E A E 17 217 E L V Q E 0 K A R K 17 35 D L D A L E H M F R 6 64 E L E K F Q Q A I D 16 93 G F L K G E D G E M 16 102 M V K L E N L F E A 16 162 Y T D A L H V Y S T 16 6 S L E E E K Y D M S 15 96 K G E D G E M V K L 15 103 V K L E N L F E A L 15 166 L H V Y S T V E G Y 15 169 Y S T V E G Y I A Y 15 189 Q T L V D V F T K R 15 194 V F T K R K G H I L 15 197 K R K G H I L E L L 15 66 E K F Q Q A I D S R 14 127 Y I I Q A C R G E Q 14 150 M V I K D S P Q T I 14 181 D Q K G S C F I Q T 14 182 Q K G S C F I Q T L 14 196 T K R K G H I L E L 14 233 S T L R K R L Y L Q 14 8 E E E K Y D M S G A 13 13 D M S G A R L A L I 13 25 V T K A R E G S E E 13 71 A I D S R E D P V S 13 73 D S R E D P V S C A 13 107 N L F E A L N N K N 13 171 T V E G Y I A Y R H 13 175 Y I A Y R H D Q K G 13 218 L V Q E G K A R K T 13 3 N P R S L E E E K Y 12 21 L I L C V T K A R E 12 24 C V T K A R E G S E 12 42 M F R Q L R F E S T 12 50 S T M K R D P T A E 12 52 M K R D P T A E Q F 12 55 D P T A E Q F Q E E 12 74 S R E D P V S C A F 12 94 F L K G E D G E M V 12 146 D E I V M V I K D S 12 151 V I K D S P Q T I P 12 158 T I P T Y T D A L H 12 178 Y R H D Q K G S C F 12 191 L V D V F T K R K G 12 201 H I L E L L T E V T 12 206 L T E V T R R M A E 12 20 A L I L C V T K A R 11 39 L E H M F R Q L R F 11 83 F V V L M A H G R E 11 84 V V L M A H G R E G 11 106 E N L F E A L N N K 11 125 K V Y I I Q A C R G 11 128 I I Q A C R G E Q R 11 148 I V M V I K D S P Q 11 167 H V Y S T V E G Y I 11 187 F I Q T L V D V F T 11 195 F T K R K G H I L E 11 205 L L T E V T R R M A 11 5 R S L E E E K Y D M 10 12 Y D M S G A R L A L 10 18 R L A L I L C V T K 10 27 K A R E G S E E D L 10 40 E H M F R Q L R F E 10 45 Q L R F E S T M K R 10 63 E E L E K F Q Q A I 10 100 G E M V K L E N L F 10 118 Q A L R A K P K V Y 10 165 A L H V Y S T V E G 10 190 T L V D V F T K R K 10 231 I Q S T L R K R L Y 10 22 I L C V T K A R E G 9 38 A L E H M F R Q L R 9 43 F R Q L R F E S T M 9 46 L R F E S T M K R D 9 67 K F Q Q A I D S R E 9 86 L M A H G R E G F L 9 92 E G F L K G E D G E 9 98 E D G E M V K L E N 9 104 K L E N L F E A L N 9 119 A L R A K P K V Y I 9 202 I L E L L T E V T R 9 212 R M A E A E L V Q E 9 9 E E K Y D M S G A R 8 14 M S G A R L A L I L 8 60 Q F Q E E L E K F Q 8 81 C A F V V L M A H G 8 137 R D P G E T V G G D 8 138 D P G E T V G G D E 8 144 G G D E I V M V I K 8 154 D S P Q T I P T Y T 8 229 P E I Q S T L R K R 8 232 Q S T L R K R L Y L 8 1 M S N P R S L E E E 7 47 R F E S T M K R D P 7 49 E S T M K R D P T A 7 76 E D P V S C A F V V 7 80 S C A F V V L M A H 7 82 A F V V L M A H G R 7 89 H G R E G F L K G E 7 97 G E D G E M V K L E 7 101 E M V K L E N L F E 7 108 L F E A L N N K N C 7 110 E A L N N K N C Q A 7 121 R A K P K V Y I I Q 7 122 A K P K V Y I I Q A 7 123 K P K V Y I I Q A C 7 135 E Q R D P G E T V G 7 185 S C F I Q T L V D V 7 200 G H I L E L L T E V 7 215 E A E L V Q E G K A 7 221 E G K A R K T N P E 7 36 L D A L E H M F R Q 6 62 Q E E L E K F Q Q A 6 120 L R A K P K V Y I I 6 143 V G G D E I V M V I 6 163 T D A L H V Y S T V 6 164 D A L H V Y S T V E 6 173 E G Y I A Y R H D Q 6 207 T E V T R R M A E A 6 213 M A E A E L V Q E G 6 225 R K T N P E I Q S T 6 15 S G A R L A L I L C 5 19 L A L T L C V T K A 5 79 V S C A F V V L M A 5 88 A H G R E G F L K G 5 114 N K N C Q A L R A K 5 136 Q R D P G E T V G G 5 145 G D E I V M V I K D 5 172 V E G Y I A Y R H D 5 203 L E L L T E V T R R 5 7 L E E E K Y D M S G 4 16 G A R L A L I L C V 4 17 A R L A L I L C V T 4 53 K R D P T A E Q F Q 4 75 R E D P V S C A F V 4 132 C R G E Q R D P G E 4 156 P Q T I P T Y T D A 4 188 I Q T L V D V F T K 4 199 K G H I L E L L T E 4 214 A E A E L V Q E G K 4 31 G S E E D L D A L E 3 41 H M F R Q L R F E S 3 51 T M K R D P T A E Q 3 61 F Q E E L E K F Q Q 3 70 Q A I D S R E D P V 3 90 G R E G F L K G E D 3 95 L K G E D G E M V K 3 115 K N C Q A L R A K P 3 117 C Q A L R A K P K V 3 126 V Y I I Q A C R G E 3 129 I Q A C R G E Q R D 3 174 G Y I A Y R H D Q K 3 222 G K A R K T N P E I 3 227 T N P E I Q S T L R 3 2 S N P R S L E E E K 2 26 T K A R E G S E E D 2 28 A R E G S E E D L D 2 32 S E E D L D A L E H 2 54 R D P T A E Q F Q E 2 72 I D S R E D P V S C 2 87 M A H G R E G F L K 2 112 L N N K N C Q A L R 2 113 N N K N C Q A L R A 2 131 A C R G E Q R D P G 2 133 R G E Q R D P G E T 2 149 V M V I K D S P Q T 2 152 I K D S P Q T I P T 2 161 T Y T D A L H V Y S 2 179 R H D Q K G S C F I 2 219 V Q E G K A R K T N 2 220 Q E G K A R K T N P 2 224 A R K T N P E I Q S 2 11 K Y D M S C A R L A 2 23 L C V T K A R E G S 1 29 R E G S E E D L D A 1 44 R Q L R F E S T M K 1 57 T A E Q F Q E E L E 1 58 A E Q F Q E E L E K 1 65 L E K F Q Q A I D S 1 68 F Q Q A I D S R E D 1 69 Q Q A I D S R E D P 1 105 L E N L F E A L N N 1 109 F E A L N N K N C Q 1 124 P K V Y I I Q A C R 1 13 P G E T V G G D E I 1 159 T P T Y T D A L H V 1 17 I A Y R H D Q K G S 1 177 A Y R H D Q K G S C 1 180 H D Q K G S C F I Q 1 192 V D V F T K R K G H 1 216 A E L V Q E G K A R 1 223 K A R K T N P E I Q 1 228 N P E I Q S T L R K 1 213P1F11 v.1: HLA-A3 decamers 18 R L A L I L C V T K 34 202 I L E L L T E V T R 25 217 E L V Q E G K A R K 23 20 A L I L C V T K A R 22 119 A L R A K P K V Y I 22 128 I I Q A C R G E Q R 22 44 R Q L R F E S T M K 21 125 K V Y T I Q A C R G 21 190 T L V D V F T K R K 21 45 Q L R F E S T M K R 20 171 T V E G Y I A Y R H 19 38 A L E H M F R Q L R 18 118 Q A L R A K P K V Y 18 150 M V I K D S P Q T I 18 174 G Y I A Y R H D Q K 18 35 D L D A L E H M F R 17 85 V L M A H G R E G F 17 94 F L K G E D G E M V 17 104 K L E N L F E A L N 17 142 T V G G D E I V M V 17 165 A L H V Y S T V E G 17 188 I Q T L V D V F T K 17 208 E V T R R M A E A E 17 58 A E Q F Q E E L E K 16 95 L K G E D G E M V K 16 153 K D S P Q T I P T Y 16 22 I L C V T K A R E G 15 71 A I D S R E D P V S 15 84 V V L M A H G R E G 15 87 M A H G R E G F L K 15 111 A L N N K N C Q A L 15 116 N C Q A L R A K P K 15 144 G G D E I V M V I K 15 148 I V M V I K D S P Q 15 158 T I P T Y T D A L H 15 167 H V Y S T V E G Y I 15 201 H I L E L L T E V T 15 214 A E A E L V Q E G K 15 2 S N P R S L E E E K 14 52 M K R D P T A E Q F 14 78 P V S C A F V V L M 14 160 P T Y T D A L H V Y 14 218 L V Q E G K A R K T 14 228 N P E I Q S T L R K 14 6 S L E E E K Y D M S 13 21 L I L C V T K A R E 13 24 C V T K A R E G S E 13 64 E L E K F Q Q A I D 13 127 Y I I Q A C R G E Q 13 205 L L T E V T R R M A 13 211 R R M A E A E L V Q 13 212 R M A E A E L V Q E 13 216 A E L V Q E G K A R 13 226 K T N P E I Q S T L 13 32 S E E D L D A L E H 12 72 I D S R E D P V S C 12 83 F V V L M A H G R E 12 102 M V K L E N L F E A 12 106 E N L F E A L N N K 12 187 F I Q T L V D V F T 12 204 E L L T E V T R R M 12 10 E K Y D M S G A R L 11 17 A R L A L I L C V T 11 107 N L F E A L N N K N 11 114 N K N C Q A L R A K 11 151 V I K D S P Q T I P 11 178 Y R H D Q K G S C F 11 186 C F I Q T L V D V F 11 193 D V F T K R K G H I 11 199 K G H I L E L L T E 11 75 R E D P V S C A F V 10 82 A F V V L M A H G R 10 134 G E Q R D P G E T V 10 147 E I V M V I K D S P 11 191 L V D V F T K R K G 10 3 N P R S L E E E K Y 9 25 V T K A R E G S E E 9 88 A H G R E G F L K G 9 131 A C R G E Q R D P G 9 135 E Q R D P G E T V G 9 136 Q R D P G E T V G G 9 157 Q T I P T Y T D A L 9 163 T D A L H V Y S T V 9 170 S T V E G Y I A Y R 9 175 Y I A Y R H D Q K G 9 176 I A Y R H D Q K G S 9 177 A Y R H D Q K G S C 9 189 Q T L V D V F T K R 9 209 V T R R M A E A E L 9 230 E I Q S T L R K R L 9 14 M S G A R L A L I L 8 34 E D L D A L E H H F 8 43 F R Q L R F E S T M 8 77 D P V S C A F V V L 8 80 S C A F V V L M A H 8 96 K G E D G E M V K L 8 113 N N K N C Q A L R A 8 121 R A K P K V Y I I Q 8 159 I P T Y T D A L H V 8 203 L E L L T E V T R R 8 231 I Q S T L R K R L Y 8 5 R S L E E E K Y D M 7 9 E E K Y D M S G A R 7 16 G A R L A L I L C V 7 27 K A R E G S E E D L 7 29 R E G S E E D L D A 7 39 L E H M F R Q L R F 7 51 T M K R D P T A E Q 7 54 R D P T A E Q F Q E 7 73 D S R E D P V S C A 7 74 S R E D P V S C A F 7 76 E D P V S C A F V V 7 91 R E G F L K G E D G 7 105 L E N L F E A L N N 7 112 L N N K N C Q A L R 7 115 K N C Q A L R A K P 7 124 P K V Y I I Q A C R 7 137 R D P G E T V G G D 7 165 D A L H V Y S T V E 7 169 Y S T V E G Y T A Y 7 184 G S C F I Q T L V D 7 198 R K G H I L E L L T 7 219 V Q E G K A R K T N 7 223 K A R K T N P E I Q 7 224 A R K T N P E I Q S 7 225 R K T N P E I Q S T 7 229 P E I Q S T L R K R 7 232 Q S T L R K R L Y L 7 11 K Y D M S G A R L A 6 37 D A L E H M F R Q L 6 53 K R D P T A E Q F Q 6 62 Q E E L E K F Q Q A 6 66 E K F Q Q A I D S R 6 67 K F Q Q A I D S R E 6 70 Q A I D S R E D P V 6 79 V S C A F V V L M A 6 110 E A L N N K N C Q A 6 122 A K P K V Y T I Q A 6 133 R G E Q R D P G E T 6 141 E T V G G D E I V M 6 143 V G G D E I V M V I 6 166 L H V Y S T V E G Y 6 173 E G Y I A Y R H D Q 6 181 D Q K G S C F I Q T 6 182 Q K G S C F I Q T L 6 185 S C F I Q T L V D V 6 196 T K R K G H I L E I 6 197 K R K G H I L E L L 6 210 T R R M A E A E L V 6 227 T N P E I Q S T L R 6 233 S T L R K R L Y L Q 6 12 Y D M S G A R L A L 5 13 D M S G A R L A L I 5 15 S G A R L A L I L C 5 28 A R E G S E E D L D 5 42 M F R Q L R F E S T 5 69 Q Q A I D S R E D P 5 123 K P K V Y I I Q A C 5 130 Q A C R G E Q R D P 5 138 D P G E T V G G D E S 155 S P Q T I P T Y T D 5 161 T Y T D A L H V Y S 5 192 V D V F T K R K G H 5 200 G H I L E L L T E V 5 7 L E E E K Y D M S G 4 19 L A L I L C V T K A 4 26 T K A R E G S E E D 4 31 G S E E D L D A L E 4 41 H M F R Q L R F E S 4 47 R F E S T M K R D P 4 49 E S T M K R D P T A 4 50 S T M K R D P T A E 4 59 E Q F Q E E L E K F 4 61 F Q E E L E K F Q Q 4 63 E E L E K F Q Q A I 4 65 L E K F Q Q A I D S 4 89 H G R E G F L K G E 4 98 E D G E M V K L E N 4 100 G E M V K L E N L F 4 103 V K L E N L F E A L 4 126 V Y I I Q A C R G E 4 149 V M V I K D S P Q T 3 162 Y T D A L H V Y S T 4 179 R H D Q K G S C F I 4 183 K G S C F I Q T L V 4 195 F T K R K G H I L E 4 222 G K A R K T N P E I 4 1 M S N P R S L E E E 3 8 E E E K Y D M S G A 3 60 Q F Q E E L E K F Q 3 81 C A F V V L M A H G 3 90 G R E G F L K G E D 3 93 G F L K G E D G E M 3 101 E M V K L E N L F E 3 129 I Q A C R G E Q R D 3 139 P G E T V G G D E I 3 146 D E I V M V I K D S 3 168 V Y S T V E G Y I A 3 194 V F T K R K G H I L 3 206 L T E V T R R M A E 3 207 T E V T R R M A E A 3 220 Q E G K A R K T N P 3 221 E G K A R K T N P E 3 30 E G S E E D L D A L 2 36 L D A L E H M F R Q 2 55 D P T A E Q F Q E E 2 56 P T A E Q F Q E E L 2 68 F Q Q A I D S R E D 2 86 L M A H G R E G F L 2 97 G E D G E M V K L E 2 108 L F E A L N N K N C 2 109 F E A L N N K N C Q 2 117 C Q A L R A K P K V 2 152 I K D S P Q T I P T 2 154 D S P Q T I P T Y T 2 156 P Q T I P T Y T D A 2 213 M A E A E L V Q E G 2 215 E A E L V Q E G K A 2 4 P R S L E E E K Y D 1 23 L C V T K A R E G S 1 33 E E D L D A L E H M 1 46 L R F E S T M K R D 1 48 F E S T M K R D P T 1 92 E G F L K G E D G E 1 12 L R A K P K V Y I I 1 132 C R G E Q R D P G E 1 145 G D E T V M V I K D 1 213P1F11 v.1: HLA-B*0702 decamers 77 D P V S C A F V V L 24 +TL,32 159 I P T Y T D A L H V 19 12 Y D M S G A R L A L 15 196 T K R K C H I L E L 15 14 M S G A R L A L I L 14 3 E G S E E D L D A L 14 27 K A R E G S E E D L 13 9 K G E D G E M V K L 13 111 A L N N K N C Q A L 13 119 A L R A K P K V Y I 13 157 Q T I P T Y T D A L 13 197 K R K G H I L E L L 13 209 V T R R M A E A E L 13 228 N P E I Q S T L R K 13 232 Q S T L R K R L Y L 13 3 N P R S L E E E K Y 12 10 E K Y D M S G A R L 12 55 D P T A E Q F Q E E 12 86 L M A H G R E G F L 12 103 V K L E N L F E A L 12 123 K P K V Y I I Q A C 12 226 K T N P E I Q S T L 12 230 E I Q S T L R K R L 12 13 D M S G A R L A L I 11 37 D A L E H M F R Q L 11 56 P T A E Q F Q E E L 11 75 R E D P V S C A F V 11 78 P V S C A F V V L M 11 138 D P G E T V G G D E 11 142 T V G G D E I V M V 11 155 S P Q T I P T Y T D 11 182 Q K G S C F I Q T L 11 194 V F T K R K G H I L 11 16 G A R L A L I L C V 10 17 A R L A L I L C V T 10 29 R E G S E E D L D A 10 48 F E S T M K R D P T 10 79 V S C A F V V L M A 10 99 D G E M V K L E N L 10 141 E T V G G D E I V M 10 152 I K D S P Q T I P T 10 183 K G S C F I Q T L V 10 198 R K G H I L E L L T 10 42 M F R Q L R F E S T 9 73 D S R E D P V S C A 9 85 V L M A H G R E G F 9 120 L R A K P K V Y I I 9 122 A K P K V Y I I Q A 9 143 V G G D E I V M V I 9 162 Y T D A L H V Y S T 9 179 R H D Q K G S C F I 9 181 D Q K G S C F I Q T 9 187 F I Q T L V D V F T 9 8 E E E K Y D M S G A 8 11 K Y D M S G A R L A 8 19 L A L I L C V T K A 8 33 E E D L D A L E H M 8 39 L E H M F R Q L R F 8 49 E S T M K R D P T A 8 52 M K R D P T A E Q F 8 63 E E L E K F Q Q A I 8 76 E D P V S C A F V V 8 94 F L K G E D G E M V 8 113 N N K N C Q A L R A 8 117 C Q A L R A K P K V 8 131 A C R G E Q R D P G 8 168 V Y S T V E G Y I A 8 185 S C F I Q T L V D V 8 186 C F I Q T L V D V F 8 201 H I L E L L T E V T 8 204 E L L T E V T R R M 8 210 T R R M A E A E L V 8 222 G K A R K T N P E I 8 5 R S L E E E K Y D M 7 34 E D L D A L E H M F 7 59 E Q F Q E E L E K F 7 62 Q E E L E K F Q Q A 7 70 Q A I D S R E D P V 7 74 S R E D P V S C A F 7 93 G F L K G E D G E M 7 100 G E M V K L E N L F 7 110 E A L N N K N C Q A 7 133 R G E Q R D P G E T 7 149 V M V I K D S P Q T 7 150 M V I K D S P Q T I 7 154 D S P Q T I P T Y T 7 156 P Q T I P T Y T D A 7 163 T D A L H V Y S T V 7 200 G H I L E L L T E V 7 205 L L T E V T R R H A 7 207 T E V T R R M A E A 7 215 E A E L V Q E G K A 7 225 R K T N P E I Q S T 7 43 F R Q L R F E S T M 6 88 A H G R E G F L K G 6 102 M V K L E N L F E A 6 134 G E Q R D P G E T V 6 139 P G E T V G G D E I 6 140 G E T V G G D E I V 6 167 H V Y S T V E G Y I 6 178 Y R H D Q K G S C F 6 193 D V F T K R K G H I 6 211 R R M A E A E L V Q 6 218 L V Q E G K A R K T 6 71 A I D S R E D P V S 5 72 I D S R E D P V S C 5 101 E H V K L E N L F E 5 135 E Q R D P G E T V G 5 136 Q R D P G E T V G G 5 153 K D S P Q T I P T Y 5 165 A L H V Y S T V E G 5 18 R L A L I L C V T K 4 20 A L I L C V T K A R 4 28 A R E G S E E D L D 4 50 S T M K R D P T A E 4 53 K R D P T A E Q F Q 4 58 A E Q F Q E E L E K 4 97 G E D G E M V K L E 4 98 E P G E M V K L E N 4 137 R P P G E T V G G D 4 177 A Y R H D Q K G S C 4 184 G S C F I Q T L V D 4 212 R M A E A E L V Q E 4 221 E G K A R K T N P E 4 223 K A R K T N P E I Q 4 35 D L D A L E H M F R 3 38 A L E H M F R Q L R 3 40 E H M F R Q L R F E 3 45 Q L R F E S T M K R 3 64 E L E K F Q Q A I D 3 115 K N C Q A L R A K P 3 144 G G D E I V M V I K 3 148 T V M V I K D S P Q 3 199 K G H I L E L L T E 3 202 I L E L L T E V T R 3 214 A E A E L V Q E G K 3 216 A E L V Q E G K A R 3 219 V Q E G K A R K T N 3 220 Q E G K A R K T N P 3 231 I Q S T L R K R L Y 3 4 P R S L E E E K Y D 2 9 E E K Y D M S G A R 2 32 S E E D L D A L E H 2 44 R Q L R F E S T H K 2 51 T H K R D P T A E Q 2 69 Q Q A I D S R E D P 2 80 S C A F V V L M A H 2 82 A F V V L M A H G R 2 87 M A H G R E G F L K 2 89 H G R E G F L K G E 2 90 G R E G F L K G E D 2 91 R E G F L K G E D G 2 95 L K G E D G E M V K 2 104 K L E N L F E A L N 2 105 L E N L F E A L N N 2 112 L N N K N C Q A L R 2 116 N C Q A L R A K P K 2 121 R A K P K V Y I I Q 2 128 I I Q A C R G E Q R 2 129 I Q A C R G E Q R D 2 132 C R G E Q R D P G E 2 161 T Y T D A L H V Y S 2 164 D A L H V Y S T V E 2 171 T V E G Y I A Y R H 2 173 E G Y I A Y R H D Q 2 188 I Q T L V D V F T K 2 189 Q T L V D V F T K R 2 203 L E L L T E V T R R 2 206 L T E V T R R M A E 2 208 E V T R R M A E A E 2 213 M A E A E L V Q E G 2 217 E L V Q E G K A R K 2 224 A R K T N P E I Q S 2 1 M S N P R S L E E E 1 15 S G A R L A L I L C 1 21 L I L C V T K A R E 1 22 I L C V T K A R E G 1 23 L C V T K A R E G S 1 24 C V T K A R E G S E 1 25 V T K A R E G S E E 1 26 T K A R E G S E E D 1 31 G S E E D L D A L E 1 36 L D A L E H M F R Q 1 41 H M F R Q L R F E S 1 47 R F E S T M K R D P 1 54 R D P T A E Q F Q E 1 60 Q F Q E E L E K F Q 1 66 E K F Q Q A I D S R 1 67 K F Q Q A I D S R E 1 68 F Q Q A I D S R E D 1 81 C A F V V L M A H G 1 92 E G F L K G E D G E 1 106 E N L F E A L N N K 1 108 L F E A L N N K N C 1 109 F E A L N N K N C Q 1 114 N K N C Q A L R A K 1 118 Q A L R A K P K V Y 1 124 P K V Y I I Q A C R 1 125 K V Y T I Q A C R G 1 127 Y I I Q A C R G E Q 1 145 G D E I V M V I K D 1 147 E I V M V I K D S P 1 151 V I K D S P Q T I P 1 158 T I P T Y T D A L H 1 160 P T Y T D A L H V Y 1 166 L H V Y S T V E G Y 1 169 Y S T V E G Y I A Y 1 170 S T V E G Y I A Y R 1 172 V E G Y I A Y R H D 1 174 G Y I A Y R H D Q K 1 175 Y I A Y R H D Q K G 1 176 I A Y R H D Q K G S 1 180 H D Q K G S C F I Q 1 190 T L V D V F T K R K 1 191 L V D V F T K R K G 1 192 V D V F T K R K G H 1 227 T N P E I Q S T L R 1 213P1F11 v.1: HLA-B*4402 decamers 63 E E L E K F Q Q A I 23 100 G E M V K L E N L F 23 39 L E H M F R Q L R F 21 153 K D S P Q T I P T Y 19 157 Q T I P T Y T D A L 19 146 D E I V M V I K D S 18 216 A E L V Q E G K A R 18 30 E G S E E D L D A L 16 59 E Q F Q E E L E K F 16 97 G E D G E M V K L E 16 111 A L N N K N C Q A L 16 118 Q A L R A K P K V Y 16 229 P E I Q S T L R K R 16 12 Y D M S G A R L A L 15 33 E E D L D A L E H M 15 34 E D L D A L E H H F 15 186 C F I Q T L V D V F 15 226 K T N P E I Q S T L 15 230 E I Q S T L R K R L 15 9 E E K Y D M S G A R 14 32 S E E D L D A L E H 14 37 D A L E H M F R Q L 14 58 A E Q F Q E E L E K 14 74 S R E D P V S C A F 14 75 R E D P V S C A F V 14 96 K G E D G E M V K L 14 103 V K L E N L F E A L 14 160 P T Y T D A L H V Y 14 182 Q K G S C F I Q T L 14 196 T K R K G H I L E L 14 197 K R K G H I L E L L 14 231 I Q S T L R K R L Y 14 10 E K Y D M S G A R L 13 48 F E S T M K R D P T 13 52 M K R D P T A E Q F 13 62 Q E E L E K F Q Q A 13 77 D P V S C A F V V L 13 105 L E N L F E A L N N 13 150 M V I K D S P Q T I 13 169 Y S T V E G Y I A Y 13 203 L E L L T E V T R R 13 214 A E A E L V Q E G K 13 232 Q S T L R K R L Y L 13 3 N P R S L E E E K Y 12 8 E E E K Y D M S G A 12 13 D M S G A R L A L I 12 14 M S G A R L A L I L 12 20 A L I L C V T K A R 12 65 L E K F Q Q A I D S 12 85 V L M A H G R E G F 12 109 F H A L N N K N C G 12 119 A L R A K P K V Y I i2 134 G E Q R D P G E T V 12 27 K A R E G S E E D L 11 29 R E G S E E D L D A 11 99 D G E M V K L E N L 11 143 V G G D E I V M V I 11 166 L H V Y S T V E G Y 11 172 V E G Y I A Y R H D 11 178 Y R H D Q K G S C F 11 193 D V F T K R K G H I 11 194 V F T K R K C H I L 11 207 T E V T R R M A E A 11 209 V T R R M A E A E L 11 7 L E E E K Y D M S G 10 56 P T A E Q F Q E E L 10 86 L M A H G R E C F L 10 91 R E G F L K G E D G 10 140 G E T V G G D E I V 10 220 Q E G K A R K T N P 10 120 L R A K P K V Y I I 9 122 A K P K V Y I I Q A 9 139 P G E T V G G D E I 9 179 R H D Q K G S C F I 9 222 G K A R K T N P E I 9 167 H V Y S T V E G Y I 8 66 E K F Q Q A I D S R 7 88 A H G R E G F L K G 7 17 A R L A L I L C V T 6 123 K P K V Y I I Q A C 6 136 Q R D P G E T V G G 6 204 E L L T E V T R R M 6 208 E V T R R M A E A E 6 224 A R K T N P E I Q S 6 11 K Y D M S C A R L A 5 15 S G A R L A L I L C 5 16 G A R L A L I L C V 5 28 A R E G S E E D L D 5 38 A L E H M F R Q L R 5 40 E H M F R Q L R F E 5 50 S T M K R D P T A E 5 53 K R D P T A E Q F Q 5 71 A I D S R E D P V S 5 76 E D P V S C A F V V 5 82 A F V V L M A H G R 5 92 E G F L K G E D G E 5 106 E N L F E A L N N K 5 107 N L F E A L N N K N 5 110 E A L N N K N C Q A 5 14 N K N C 0 A L R A K 5 131 A C R G E Q R D P G 5 141 E T V G G D E I V M 5 142 T V G G D E I V M V S 165 A L H V Y S T V E G 5 185 S C F I Q T L V D V 5 200 C H I L E L L T E V 5 219 V Q E G K A R K T N 5 233 S T L R K R L Y L Q 5 1 M S N P R S L E E E 4 4 P R S L E E E K Y D 4 41 H M F R Q L R F E S 4 46 L R F E S T M K R D 4 70 Q A I D S R E D P V 4 72 I D S R E D P V S C 4 78 P V S C A F V V L M 4 80 S C A F V V L M A H 4 113 N N K N C Q A L R A 4 116 N C Q A L R A K P K 4 121 R A K P K V Y I I Q 4 126 V Y I I Q A C R G E 4 127 Y I I Q A C R G E Q 4 135 E Q R D P G E T V G 4 155 S P Q T I P T Y T D 4 177 S T V E G Y I A Y R 4 173 E G Y I A Y R H D Q 4 174 G Y I A Y R H D Q K 4 181 D Q K G S C F I Q T 4 190 T L V D V F T K R K 4 199 K G H I L E L L T E 4 215 E A E L V Q E G K A 4 221 E G K A R K T N P E 4 225 R K T N P E I Q S T 4 2 S N P R S L E E E K 3 18 R L A L I L C V T K 3 19 L A L I L C V T K A 3 49 E S T M K R D P T A 3 54 R D P T A E Q F Q E 3 89 H G R E G F L K G E 3 98 E D G E M V K L E N 3 101 E M V K L E N L F E 3 104 K L E N L F E A L N 3 115 K N C Q A L R A K P 3 137 R D P G E T V G G D 3 145 G D E I V M V I K D 3 152 I K D S P Q T I P T 3 158 T I P T Y T D A L H 3 176 I A Y R H D Q K G S 3 177 A Y R H D Q K G S C 3 183 K G S C F I Q T L V 3 184 G S C F I Q T L V D 3 188 I Q T L V D V F T K 3 189 Q T L V D V F T K R 3 191 L V D V F T K R K G 3 192 V D V F T K R K G H 3 195 F T K R K G H I L E 3 201 H I L E L L T E V T 3 202 I L E L L T E V T R 3 206 L T E V T R R M A E 3 211 R R M A E A E L V Q 3 212 R M A E A E L V Q E 3 218 L V Q E G K A R K T 3 228 N P E I Q S T L R K 3 22 I L C V T K A R E G 2 23 L C V T K A R E G S 2 43 F R Q L R F E S T M 2 44 R Q L R F E S T H K 2 45 Q L R F E S T M K R 2 51 T M K R D P T A E Q 2 60 Q F Q E E L E K F Q 2 64 E L E K F Q Q A I D 2 79 V S C A F V V L M A 2 81 C A F V V L M A H G 2 84 V V L M A H G R E G 2 87 M A H G R E G F L K 2 102 M V K L E N L F E A 2 108 L F E A L N N K N C 2 117 C Q A L R A K P K V 2 125 K V Y I I Q A C R G 2 130 Q A C R G E Q R D P 2 144 G G D E I V M V I K 2 147 E I V M V I K D S P 2 154 D S P Q T I P T Y T 2 159 I P T Y T D A L H V 2 161 T Y T D A L H V Y S 2 162 Y T D A L H V Y S T 2 163 T D A L H V Y S T V 2 164 D A L H V Y S T V E 2 168 V Y S T V E G Y I A 2 171 T V E G Y I A Y R H 2 187 F I Q T L V D V F T 2 198 R K G H I L E L L T 2 205 L L T E V T R R M A 2 210 T R R M A E A E L V 2 217 E L V Q E G K A R K 2 223 K A R K T N P E I Q 2 227 T N P E I Q S T L R 2 5 R S L E E E K Y D M 1 6 S L E E E K Y D M S 1 21 L I L C V T K A R E 1 26 T K A R E G S E E D 1 31 G S E E D L D A L E 1 35 D L D A L E H M F R 1 36 L D A L E H M K R Q 1 42 M F R Q L R F E S T 1 47 R F E S T M K R D P 1 55 D P T A E Q F Q E E 1 57 T A E Q F Q E E L E 1 61 F Q E E L E K F Q Q 1 67 K F Q Q A I D S R E 1 68 F Q Q A I D S R E D 1 69 Q Q A I D S R E D P 1 73 D S R E D P V S C A 1 83 F V V L M A H G R E 1 90 G R E G F L K G E D 1 93 G F L K G E D G E M 1 94 F L K G E D G E M V 1 95 L K G E D G E M V K 1 112 L N N K N C Q A L R 1 128 I I Q A C R G E Q R 1 133 R G E Q R D P G E T 1 148 I V M V I K D S P Q 1 149 V M V I K D S P Q T 1 151 V I K D S P Q T I P 1 175 Y I A Y R H D Q K G 1 213 M A E A TABLE XIXB, part 2: MHC Class I decamer analysis of 213P1F11 v.2 (aa 1-230). 213P1511 v.2: HLA-A*0201 decamers 52 W M C S R R G K D I 15 18 Y L P S E A P P N P 13 38 T D M I R K A H A L 13 46 A L S R P W W M C S 12 10 P T P F Q D P L Y L 11 39 D M I R K A H A L S 11 40 M I R K A H A L S R 11 13 F Q D P L Y L P S E 10 16 P L Y L P S E A P P 10 28 P L W N S Q D T S P 10 32 S Q D T S P T D M I 10 35 T S P T D M I R K A 10 2 H V Y S T V E G P T 9 5 S T V E G P T P F Q 9 44 A H A L S R P W W M 8 55 S R R G K D I S W N 8 8 E G P T P F Q D P L 7 17 L Y L P S E A P P N 7 20 P S E A P P N P P L 7 21 S E A P P N P P L W 7 29 L W N S Q D T S P T 7 37 P T D M I R K A H A 7 1 L H V Y S T V E G P 6 11 T P F Q D P L Y L P 6 4 Q D P L Y L P S E A 6 23 A P P N P P L W N S 6 43 K A H A L S R P W W 6 45 H A L S R P W W M C 6 47 L S R P W W M C S R 6 6 T V E G P T P F Q D 5 19 L P S E A P P N P P 5 31 N S Q D T S P T D M 5 34 D T S P T D M I R K 5 41 I R K A H A L S R P 5 3 V Y S T V E G P T P 4 22 E A P P N P P L W N 4 26 N P P L W N S Q D T 4 7 V E G P T P F Q D P 3 9 G P T P F Q D P L Y 3 24 P P N P P L W N S Q 3 30 W N S Q D T S P T D 3 51 W W M C S R R G K D 3 36 S P T D M I R K A H 2 42 R K A H A L S R P W 2 48 S R P W W M C S R R 2 54 C S R R G K D I S W 2 4 Y S T V E G P T P F 1 15 D P L Y L P S E A P 1 27 P P L W N S Q D T S 1 49 R P W W M C S R R G 1 12 P F Q D P L Y L P S −1 25 P N P P L W N S Q D −1 33 Q D T S P T D M I R −1 213P1F11 v.2: HLA-A*0202 decamers 44 A H A L S R P W W M 4 21 S E A P P N P P L W 3 42 R K A H A L S R P W 3 22 E A P P N P P L W N 2 43 K A H A L S R P W W 2 45 H A L S R P W W M C 2 23 A P P N P P L W N S 1 46 A L S R P W W M C S 1 213P1411 v2: HLA-A*0203 decamers 37 P T D M I R K A H A 18 14 Q D P L Y L P S E A 10 35 T S P T D M I R K A 10 15 D P L Y L P S E A P 9 36 S P T D M I R K A H 9 38 T D M I R K A H A L 9 16 P L Y L P S E A P P 8 39 D M I R K A H A L S 8 213P1F11 v.2: HLA-A1 decamers 9 G P T P F Q D P L Y 21 37 P T D M I R K A H A 16 13 F Q D P L Y L P S E 15 20 P S E A P P N P P L 15 34 D T S P T D M I R K 14 32 S Q D T S P T D M T 13 10 P T P F Q D P L Y L 12 6 T V E G P T P F Q D 11 22 E A P P N P P L W N 10 5 S T V E G P T P F Q 8 7 V E G P T P F Q D P 8 21 S E A P P N P P L W 8 47 L S R P W W M C S R 8 54 C S R R G K D I S W 8 4 Y S T V E G P T P F 6 12 P F Q D P L Y L P S 6 35 T S P T D M I R K A 6 40 M I R K A H A L S R 6 25 P N P P L W N S Q D 5 17 L Y L P S E A P P N 4 24 P P N P P L W N S Q 4 31 N S Q D T S P T D M 4 18 Y L P S E A P P N P 3 36 S P T D M I R K A H 3 51 W W M C S R R G K D 3 55 S R R G K D I S W N 3 3 V Y S T V E G P T P 2 11 T P F Q D P L Y L P 2 39 D M I R K A H A L S 2 46 A L S R P W W M C S 2 48 S R P W W M C S R R 2 1 L H V Y S T V E G P 1 16 P L Y L P S E A P P 1 23 A P P N P P L W N S 1 28 P L W N S Q D T S P 1 43 K A H A L S R P W W 1 44 A H A L S R P W W M 1 45 H A L S R P W W M C 1 50 P W W M C S R R G K 1 52 W M C S R R G K D I 1 213P1F11 v.2: HLA-A26 decamers 34 D T S P T D M I R K 23 10 P T P F Q D P L Y L 19 8 E G P T P F Q D P L 15 5 S T V E G P T P F Q 13 6 T V E G P T P F Q D 13 9 G P T P F Q D P L Y 13 37 P T D M I R K A H A 13 18 Y L P S E A P P N P 12 40 M I R K A H A L S R 12 56 R R G K D I S W N F 12 2 H V Y S T V E G P T 11 12 P F Q D P L Y L P S 11 4 Y S T V E G P T P F 10 13 F Q D P L Y L P S E 9 16 P L Y L P S E A P P 9 20 P S E A P P N P P L 9 22 E A P P N P P L W N 9 28 P L W N S Q D T S P 9 31 N S Q D T S P T D M 9 38 T D M I R K A H A L 9 39 D M I R K A H A L S 9 44 A H A L S R P W W M 9 46 A L S R P W W M C S 9 7 V E G P T P F Q D P 7 11 T P F Q D P L Y L P 7 15 D P L Y L P S E A P 7 41 I R K A H A L S R P 7 47 L S R P W W M C S R 7 55 S R R G K D I S W N 7 23 A P P N P P L W N S 6 35 T S P T D M I R K A 6 1 L H V Y S T V E G P 5 25 P N P P L W N S Q D 5 21 S E A P P N P P L W 4 14 Q D P L Y L P S E A 3 17 L Y L P S E A P P N 3 24 P P N P P L W N S Q 3 48 S R P W W M C S R R 3 19 L P S E A P P N P P 2 26 N P P L W N S Q D T 2 29 L W N S Q D T S P T 2 30 W N S Q D T S P T D 2 32 S Q D T S P T D M I 2 33 Q D T S P T D M I R 2 36 S P T D M I R K A H 2 45 H A L S R P W W M C 2 3 V Y S T V E G P T P 1 42 R K A H A L S R P W 1 43 K A H A L S R P W W 1 49 R P W W M C S R R G 1 50 P W W M C S R R G K 1 51 W W M C S R R G K D 1 52 W M C S R R G K D I 1 54 C S R R G K D I S W 1 213P1F11 v.2: HLA-A3 decamers 40 M I R K A H A L S R 23 16 P L Y L P S E A P P 18 46 A L S R P W W M C S 17 6 T V E G P T P F Q D 16 2 H V Y S T V E G P T 15 28 P L W N S Q D T S P 14 34 D T S P T D M I R K 13 47 L S R P W W M C S R 12 18 Y L P S E A P P N P 11 50 P W W M C S R R G K 11 55 S R R G K D I S W N 10 9 G P T P F Q D P L Y 9 25 P N P P L W N S Q D 9 39 D M I R K A H A L S 9 17 L Y L P S E A P P N 8 22 E A P P N P P L W N 8 36 S P T D M I R K A H 8 41 I R K A H A L S R P 8 48 S R P W W M C S R R 8 54 C S R R C K D I S W 8 56 R R G K D I S W N F 8 3 V Y S T V E G P T P 7 14 Q D P L Y L P S E A 7 33 Q D T S P T D M I R 7 43 K A H A L S R P W W 7 44 A H A L S R P W W M 7 4 Y S T V E G P T P F 6 21 S E A P P N P P L W 6 27 P P L W N S Q D T S 6 13 F Q D P L Y L P S E 5 24 P P N P P L W N S Q 5 38 T D M I R K A H A L 5 42 R K A H A L S R P W 5 10 P T P F Q D P L Y L 4 12 P F Q D P L Y L P S 4 23 A P P N P P L W N S 4 30 W N S Q D T S P T D 4 49 R P W W M C S R R G 4 7 V E G P T P F Q D P 3 15 D P L Y L P S E A P 3 29 L W N S Q D T S P T 3 31 N S Q D T S P T D M 3 37 P T D M I R K A H A 3 45 H A L S R P W W N C 3 52 W M C S R R G K D I 3 53 M C S R R G K D I S 3 5 S T V E G P T P F Q 2 19 L P S E A P P N P P 2 20 P S E A P P N P P L 2 32 S Q D T S P T D M I 2 51 W W M C S R R G K D 2 26 N P P L W N S Q D T 1 213P1F11 v.2 HLA-B*0702 decamers 26 N P P L W N S Q D T 16 19 L P S E A P P N P P 13 20 P S E A P P N P P L 13 8 E G P T P F Q D P L 12 9 G P T P F Q D P L Y 12 10 P T P F Q D P L Y L 12 23 A P P N P P L W N S 12 36 S P T D M I R K A H 12 38 T D M I R K A H A L 12 15 D P L Y L P S E A P 11 24 P P N P P L W N S Q 11 49 R P W W M C S R R C 11 11 T P F Q D P L Y L P 10 27 P P L W N S Q D T S 10 44 A H A L S R P W W M 9 56 R R G K D I S W N F 9 4 Y S T V E G P T P F 8 29 L W N S Q D T S P T 8 32 S Q D T S P T D H I 8 37 P T D M I R K A H A 8 2 H V Y S T V E G P T 7 31 N S Q D T S P T D M 7 14 Q D P L Y L P S E A 6 35 T S P T D M I R K A 6 52 W M C S R R G K D I 6 40 M I R K A H A L S R 5 22 E A P P N P P L W N 4 43 K A H A L S R P W W 4 46 A L S R P W W M C S 4 55 S R R G K D I S W N 4 3 V Y S T V E G P T P 3 5 S T V E G P T P F Q 3 7 V E G P T P F Q D P 3 12 P F Q D P L Y L P S 3 13 F Q D P L Y L P S E 3 17 L Y L P S E A P P N 3 21 S E A P P N P P L W 3 30 W N S Q D T S P T D 3 34 D T S P T D M I R K 3 42 R K A H A L S R P W 3 47 L S R P W W M C S R 3 6 T V E G P T P F Q D 2 16 P L Y L P S E A P P 2 41 T R K A H A L S R P 2 51 W W M C S R R G K D 2 53 M C S R R G K D I S 2 54 C S R R G K D I S W 2 1 L H V Y S T V E G P 1 18 Y L P S E A P P N P 1 25 P N P P L W N S Q D 1 28 P L W N S Q D T S P 1 39 D M I R K A H A L S I 50 P W W M C S R R G K 1 213P1F11 v.2: HLA-B*4402 decamers 21 S E A P P N P P L W 26 38 T D M I R K A H A L 15 7 V E G P T P F Q D P 14 8 E G P T P F Q D P L 14 9 G P T P F Q D P L Y 14 10 P T P F Q D P L Y L 13 32 S Q D T S P T D M I 12 43 K A H A L S R P W W 12 54 C S R R G K D I S W 12 4 Y S T V E G P T P F 11 42 R K A H A L S R P W 11 20 P S E A P P N P P L 10 52 W M C S R R G K D I 10 56 R R G K D I S W N F 10 22 E A P P N P P L W N 8 35 T S P T D M I R K A 7 36 S P T D M I R K A H 7 23 A P P N P P L W N S 6 13 F Q D P L Y L P S E 5 17 L Y L P S E A P P N 5 25 P N P P L W N S Q D 5 34 D T S P T D M I R K 5 39 D M I R K A H A L S 5 44 A H A L S R P W W M 5 46 A L S R P W W M C S 5 55 S R R G K D I S W N 5 11 T P F Q D P L Y L P 4 24 P P N P P L W N S Q 4 3 V Y S T V E G P T P 3 6 T V E G P T P F Q D 3 14 Q D P L Y L P S E A 3 15 D P L Y L P S E A P 3 19 L P S E A P P N P P 3 26 N P P L W M S Q D T 3 31 N S Q D T S P T D M 3 47 L S R P W W M C S R 3 51 W W M C S R R G K D 3 5 S T V E G P T P F Q 2 12 P F Q D P L Y L P S 2 18 Y L P S E A P P N P 2 27 P P L W N S Q D T S 2 30 W N S Q D T S P T D 2 40 M I R K A H A L S R 2 48 S R P W W M C S R R 2 50 P W W M C S R R G K 2 53 M C S R R G K D I S 2 TABLE XIXB, part 3: MHC Class I decamer analysis of 213P1F11 v.3 (aa 1-146). 213P1F11 v.3: HLA-A*0201 decamers 3 I I Q A C R G A T L 24 10 A T L P S P F P Y L 21 2 Y I I Q A C R G A T 17 12 L P S P F P Y L S L 17 6 A C R G A T L P S P 11 11 T L P S P F P Y L S 11 1 V Y I I Q A C R G A 8 5 Q A C R G A T L P S 5 4 I Q A C R G A T L P 4 9 G A T L P S P F P Y 4 8 R G A T L P S P F P 3 213P1F11 v.3: HLA-A*0202 decamers 4 I Q A C R G A T L P 3 8 R G A T L P S P F P 3 5 Q A C R G A T L P S 2 9 G A T L P S P F P Y 2 6 A C R G A T L P S P 1 10 A T L P S P F P Y L 1 213P1511 v.3: HLA-A*0203 decamers 1 V Y I I Q A C R G A 10 2 Y I I Q A C R G A T 9 3 I I Q A C R G A T L 8 213P1511 v.3: HLA-A1 decamers 9 G A T L P S P F P Y 15 10 A T L P S P F P Y L 14 12 L P S P F P Y L S L 10 5 Q A C R G A T L P S 6 2 Y I I Q A C R G A T 3 11 T L P S P F P Y L S 3 4 I Q A C R G A T L P 2 6 A C R G A T L P S P 2 1 V Y I I Q A C R G A 1 3 I I Q A C R C A T L 1 213P1F11 v.3: HLA-A26 decamers 10 A T L P S P F P Y L 25 3 I I Q A C R C A T L 19 12 L P S P F P Y L S L 15 2 Y I I Q A C R C A T 1 7 C R C A T L P S P F 11 9 G A T L P S P F P Y 1 11 T L P S P F P Y L S 1 6 A C R C A T L P S P 8 1 V Y I I Q A C R C A 3 8 R C A T L P S P F P 2 4 I Q A C R G A T L P 1 213P1F11 v.3: HLA-A3 decamers 3 I I Q A C R G A T L 22 2 Y I I Q A C R G A T 14 11 T L P S P F P Y L S 11 6 A C R G A T L P S P 1 7 C R G A T L P S P F 1 5 Q A C R C A T L P S 8 9 G A T L P S P F P Y 8 10 A T L P S P F P Y L 8 12 L P S P F P Y L S L 8 4 I Q A C R G A T L P 7 8 R G A T L P S P F P 5 1 V Y I I Q A C R G A 4 213P1F11 v.3: HLA-B*0702 decamers 12 L P S P F P Y L S L 25 10 A T L P S P F P Y L 15 3 I I Q A C R G A T L 13 7 C R G A T L P S P F 9 2 Y I I Q A C R G A T 8 6 A C R C A T L P S P 7 1 V Y I I Q A C R G A 6 5 Q A C R C A T L P S 4 8 R G A T L P S P F P 4 4 I Q A C R G A T L P 3 TABLE XIXB, part 4: MHC Class I decamer analysis of 213P1F11 v.4 (aa 1-321). 213P1F11 v.4: HLA-A*0201 decamers 12 S V Q P E K R T G L 19 60 E V F G G G V C D I 16 65 G V G D I V G R D L 16 10 S L S V Q P E K R T 15 52 V N D P R E T Q E V 15 57 E T Q E V F G C G V 14 67 G D I V C R D L S I 14 42 F R G S S V H Q K L 13 50 K L V N D P R E T Q 13 61 V F C C C V G D I V 13 68 D I V C R D L S I S 13 84 S A S E E E K Y D M 13 2 G K C Q E Y D K S L 12 20 G L R D E N G E C G 12 33 R L K E E Q C R A F 12 38 Q G R A F R C S S V 12 73 D L S I S F R N S E 12 4 C Q E Y D K S L S V 11 46 S V H Q K L V N D P 11 75 S I S F R N S E T S 11 74 L S I S F R N S E T 10 25 N G E C G Q T F R L 9 41 A F R G S S V H Q K 9 43 R G S S V H Q K L V 9 76 I S F R N S E T S A 9 45 S S V H Q K L V N D 8 49 Q K L V N D P R E T 8 213P1F11 v.4: HLA-A*0201 decamers 51 L V N D P R E T Q E 8 63 G G G V G D I V G R 8 69 I V G R D L S T S F 8 32 F R L K E E Q G R A 7 40 R A F R G S S V H Q 7 64 G G V G D I V G R D 7 5 Q E Y D K S L S V Q 6 18 R T G L R D E N G E 6 70 V G R D L S I S F R 6 78 F R N S E T S A S E 6 7 Y D K S L S V Q P E 5 9 K S L S V Q P E K R 5 21 L R D E N G E C G Q 5 28 C G Q T F R L K E E 5 30 Q T F R L K E E Q G 5 34 L K E E Q G R A F R 5 86 S E E E K Y D M S G 5 11 L S V Q P E K R T G 4 19 T G L R D E N G E C 4 23 D E N G E C G Q T F 4 79 R N S E T S A S E E 4 3 K C Q E Y D K S L S 3 13 V Q P E K R T G L R 3 22 R D E N G E C G Q T 3 26 G E C G Q T F R L K 3 54 D P R E T Q E V F G 3 62 F G G G V G D I V G 3 77 S F R N S E T S A S 3 81 S E T S A S E E E K 3 82 E T S A S E E E K Y 3 85 A S E E E K Y D M S 3 8 D K S L S V Q P E K 2 17 K R T G L R D E N G 2 39 G R A F R G S S V H 2 44 G S S V H Q K L V N 2 59 Q E V F G G G V G D 2 72 R D L S I S F R N S 2 1 M G K C Q E Y D K S 1 29 G Q T F R L K E E Q 1 31 T F R L K E E Q G R 1 47 V H Q K L V N D P R 1 55 P R E T Q E V F G G 1 66 V G D I V G R D L S 1 71 G R D L S I S F R N 1 80 N S E T S A S E E E 1 83 T S A S E E E K Y D 1 6 E Y D K S L S V Q P −1 16 E K R T G L R D E N −1 24 E N G E C G Q T F R −1 37 E Q G R A F R G S S −1 58 T Q E V F G G G V G −1 213P1F11 v.4: HLA-A*0202 decamers 39 G R A F R G S S V H 3 83 T S A S E E E K Y D 3 40 R A F R G S S V H Q 2 84 S A S E E E K Y D M 2 41 A F R G S S V H Q K 1 85 A S E E E K Y D M S 1 213P1F11 v.4: HLA-A*0203 decamers 32 F R L K E E Q G R A 10 76 I S F R N S E T S A 10 33 R L K E E Q G R A F 9 77 S F R N S E T S A S 9 34 L K E E Q G R A F R 8 78 F R N S E T S A S E 8 213P1F11 v.4: HLA-A1 decamers 82 E T S A S E E E K Y 23 14 Q P E K R T G L R D 17 4 C Q E Y D K S L S V 16 85 A S E E E K Y D M S 16 52 V N D P R E T Q E V 15 66 V G D I V G R D L S 14 86 N S E T S A S E E E 14 58 T Q E V F G G G V G 12 86 S E E E K Y D M S G 12 34 L K E E Q G R A F R 11 44 G S S V H Q K L V N 11 55 P R E T Q E V F G G 11 6 E Y D K S L S V Q P 10 21 L R D E N G E C G Q 10 22 R D E N G E C G Q T 10 25 N G E C G Q T F R L 10 35 K E E Q G R A F R G 10 71 G R D L S I S F R N 10 27 E C G Q T F R L K E 9 12 S V Q P E K R T G L 7 57 E T Q E V F G G G V 7 62 F G G G V G D I V G 7 9 K S L S V Q P E K R 6 18 R T G L R D E N G E 6 26 G E C G Q T F R L K 6 30 Q T F R L K E E Q G 6 45 S S V H Q K L V N D 6 67 G D I V G R D L S I 6 74 L S I S F R N S E T 5 11 L S V Q P E K R T G 4 36 E E Q G R A F R G S 4 69 I V G R D L S I S F 4 76 I S F R N S E T S A 4 83 T S A S E E E K Y D 4 7 Y D K S L S V Q P E 3 10 S L S V Q P E K R T 3 13 V Q P E K R T G L R 3 41 A F R G S S V H Q K 3 42 F R G S S V H Q K L 3 46 S V H Q K L V N D P 3 56 R E T Q E V F G G G 3 61 V F G G G V G D I V 3 72 R D L S I S F R N S 3 1 M G K C Q E Y D K S 2 3 K C Q E Y D K S L S 2 20 G L R D E N G E C G 2 23 D E N G E C G Q T F 2 33 R L K E E Q G R A F 2 43 R G S S V H Q K L V 2 47 V H Q K L V N D P R 2 50 K L V N D P R E T Q 2 60 E V F G G G V G D I 2 64 G G V G D I V G R D 2 73 D L S I S F R N S E 2 75 S I S F R N S E T S 2 77 S F R N S E T S A S 2 81 S E T S A S E E E K 2 84 S A S E E E K Y D M 2 5 Q E Y D K S L S V Q 1 16 E K R T G L R D E N 1 17 K R T G L R D E N G 1 19 T G L R D E N G E C 1 32 F R L K E E Q G R A 1 37 E Q G R A F R G S S 1 38 Q G R A F R G S S V 1 39 G R A F R G S S V H 1 53 N D P R E T Q E V F 1 59 Q E V F G G G V G D 1 65 G V G D I V G R D L 1 68 D I V G R D L S I S 1 70 V G R D L S I S F R 1 78 F R N S E T S A S E 1 213P1F11 v.4: HLA-A26 decamers 82 E T S A S E E E K Y 27 60 E V F G G G V G D I 24 68 D I V G R D L S I S 23 33 R L K E E Q G R A F 22 12 S V Q P E K R T G L 21 69 I V G R D L S I S F 21 23 D E N G E C G Q T F 19 57 E T Q E V F G G G V 19 65 G V G D I V G R D L 19 46 S V H Q K L V N D P 15 73 D L S I S F R N S E 15 30 Q T F R L K E E Q G 13 36 E E Q G R A F R G S 13 41 A F R G S S V H Q K 13 51 L V N D P R E T Q E 12 18 R T G L R D E N G E 11 42 F R G S S V H Q K L 11 53 N D P R E T Q E V F 11 2 G K C Q E Y D K S L 10 6 E Y D K S L S V Q P 10 75 S I S F R N S E T S 10 84 S A S E E E K Y D M 10 10 S L S V Q P E K R T 9 20 G L R D E N G E C G 9 25 N G E C G Q T F R L 9 54 D P R E T Q E V F G 9 8 D K S L S V Q P E K 8 24 E N G E C G Q T F R 8 27 E C G Q T F R L K E 8 31 T F R L K E E Q G R 8 50 K L V N D P R E T Q 8 61 V F G G G V G D I V 8 77 S F R N S E T S A S 8 7 Y D K S L S V Q P E 7 16 E K R T G L R D E N 7 37 E Q G R A F R G S S 7 64 G G V G D I V G R D 7 85 A S E E E K Y D M S 7 1 M G K C Q E Y D K S 6 5 Q E Y D K S L S V Q 6 26 G E C G Q T F R L K 6 45 S S V H Q K L V N D 6 56 R E T Q E V F G G G 6 63 G G G V G D I V G R 6 70 V G R D L S I S F R 6 72 R D L S I S F R N S 6 15 P E K R T G L R D E 5 28 C G Q T F R L K E E 5 52 V N D P R E T Q E V 5 55 P R E T Q E V F G G 5 74 L S I S F R N S E T 5 21 L R D E N G E C G Q 4 40 R A F R G S S V H Q 4 79 R N S E T S A S E E 4 86 S E E E K Y D M S G 4 3 K C Q E Y D K S L S 3 13 V Q P E K R T G L R 3 17 K R T G L R D E N G 3 34 L K E E Q G R A F R 3 48 H Q K L V N D P R E 3 67 G D I V G R D L S I 3 76 I S F R N S E T S A 3 78 F R N S E T S A S E 3 9 K S L S V Q P E K R 2 14 Q P E K R T G L R D 2 32 F R L K E E Q G R A 2 35 K E E Q G R A F R G 2 38 Q G R A F R G S S V 2 39 G R A F R G S S V H 2 59 Q E V F G G G V G D 2 62 F G G G V G D I V G 2 71 G R D L S I S F R N 2 81 S E T S A S E E E K 2 83 T S A S E E E K Y D 2 4 C Q E Y D K S L S V 1 11 L S V Q P E K R T G 1 19 T G L R D E N G E C 1 22 R D E N G E C G Q T 1 29 G Q T F R L K E E Q 1 43 R G S S V H Q K L V 1 47 V H Q K L V N D P R 1 49 Q K L V N D P R E T 1 66 V G D T V G R D L S 1 80 N S E T S A S E E E 1 213P1F11 v.4: HLA-A3 decamers 33 R L K E E Q G R A F 24 41 A F R C S S V H Q K 21 69 I V G R D L S I S F 21 50 K L V N D P R E T Q 19 20 G L R D E N G E C G 17 12 S V Q P E K R T G L 16 38 Q G R A F R G S S V 15 51 L V N D P R E T Q E 15 5 Q E Y D K S L S V Q 14 65 G V G D I V G R D L 14 73 D L S I S F R N S E 14 23 D E N G E C G Q T F 13 60 E V F G G G V G D T 13 68 D I V G R D L S I S 13 75 S I S F R N S E T S 13 26 G E C G Q T F R L K 12 39 G R A F R G S S V H 12 8 D K S L S V Q P E K 11 10 S L S V Q P E K R T 11 40 R A F R G S S V H Q 11 46 S V H Q K L V N D P 11 53 N D P R E T Q E V F 11 70 V G R D L S I S F R 11 79 R N S E T S A S E E 11 81 S E T S A S E E E K 11 9 K S L S V Q P E K R 10 22 R D E N G E C G Q T 9 34 L K E E Q G R A F R 9 59 Q E V F G G G V G D 9 77 S F R N S E T S A S 9 14 Q P E K R T G L R D 8 30 Q T F R L K E E Q G 8 31 T F R L K E E Q G R 8 54 D P R E T Q E V F G 8 58 T Q E V F G G G V G 8 63 G G G V G D I V G R 8 67 G D I V G R D L S I 8 76 I S F R N S E T S A 8 82 E T S A S E E E K Y 8 3 K C Q E Y D K S L S 7 6 E Y D K S L S V Q P 7 37 E Q G R A F R G S S 7 44 G S S V H Q K L V N 7 13 V Q P E K R T G L R 6 19 T G L R D E N G E C 6 24 E N G E C G Q T F R 6 35 K E E Q G R A F R G 6 62 F G G G V G D I V G 6 72 R D L S I S F R N S 6 74 L S I S F R N S E T 6 4 C Q E Y D K S L S V 5 17 K R T G L R D E N G 5 18 R T G L R D E N G E 5 45 S S V H Q K L V N D 5 49 Q K L V N D P R E T 5 56 R E T Q E V F G G G 5 57 E T Q E V F G G G V 5 61 V F G G G V G D I V 5 78 F R N S E T S A S E 5 85 A S E E E K Y D M S 5 86 S E E E K Y D M S G 5 11 L S V Q P E K R T G 4 15 P E K R T G L R D E 4 16 E K R T G L R D E N 4 27 E C G Q T F R L K E 4 43 R G S S V H Q K L V 4 47 V H Q K L V N D P R 4 32 F R L K E E Q G R A 3 52 V N D P R E T Q E V 3 64 G G V G D I V G R D 3 2 G K C Q E Y D K S L 2 36 E E Q G R A F R G S 2 48 H Q K L V N D P R E 2 66 V G D I V G R D L S 2 84 S A S E E E K Y D M 2 1 M G K C Q E Y D K S 1 7 Y D K S L S V Q P E 1 21 L R D E N G E C G Q 1 71 G R D L S I S F R N 1 83 T S A S E E E K Y D 1 213P1F11 v.4: HLA-B*0702 decamers 54 D P R E T Q E V F G 15 12 S V Q P E K R T G L 13 14 Q P E K R T G L R D 13 25 N G E C G Q T F R L 11 42 F R G S S V H Q K L 11 65 G V G D I V C R D L 11 2 G K C Q E Y D K S L 10 60 E V F C G G V G D I 10 38 Q G R A F R C S S V 9 43 R G S S V H Q K L V 9 52 V N D P R E T Q E V 9 61 V F G G G V G D I V 9 4 C Q E Y D K S L S V 8 10 S L S V Q P E K R T 8 23 D E N G E C G Q T F 8 33 R L K E E Q G R A F 8 67 G D I V G R D L S I 8 69 I V G R D L S I S F 8 76 I S F R N S E T S A 8 84 S A S E E E K Y D M 8 22 R D E N G E C G Q T 7 32 F R L K E E Q G R A 7 41 A F R G S S V H Q K 7 53 N D P R E T Q E V F 7 57 E T Q E V F C G G V 7 49 Q K L V N D P R E T 6 74 L S I S F R N S E T 6 6 E Y D K S L S V Q P 5 27 E C G Q T F R L K E 5 44 G S S V H Q K L V N 5 16 E K R T G L R D E N 4 24 E N C E C G Q T F R 4 36 E E Q C R A F R G S 4 62 F G G G V G D I V C 4 77 S F R N S E T S A S 4 82 E T S A S E E E K Y 4 8 D K S L S V Q P E K 3 17 K R T G L R D E N G 3 26 C E C G Q T F R L K 3 34 L K E E Q G R A F R 3 37 E Q G R A F R G S S 3 40 R A F R G S S V H Q 3 63 G G G V G D I V G R 3 64 C G V G D I V G R D 3 70 V G R D L S I S F R 3 73 D L S I S F R N S E 3 79 R N S E T S A S E E 3 7 Y D K S L S V Q P E 2 18 R T C L R D E N G E 2 20 C L R D E N G E C C 2 31 T F R L K E E Q G R 2 39 G R A F R G S S V H 2 45 S S V H Q K L V N D 2 47 V H Q K L V N D P R 2 50 K L V N D P R E T Q 2 56 R E T Q E V F G G G 2 59 Q E V F C G G V G D 2 66 V G D I V G R D L S 2 68 D I V C R D L S I S 2 72 R D L S I S F R N S 2 75 S I S F R N S E T S 2 85 A S E E E K Y D M S 2 3 K C Q E Y D K S L S 1 5 Q E Y D K S L S V Q 1 9 K S L S V Q P E K R 1 13 V Q P E K R T G L R 1 21 L R D E N G E C G Q 1 35 K E E Q G R A F R G 1 46 S V H Q K L V N D P 1 48 H Q K L V N D P R E 1 51 L V N D P R E T Q E 1 55 P R E T Q E V F G G 1 58 T Q E V F G G G V G 1 71 G R D L S I S F R N 1 78 F R N S E T S A S E 1 83 T S A S E E E K Y D 1 213P1F11 v.4: HLA-B*4402 decamers 23 D E N G E C G Q T F 22 12 S V Q P E K R T G L 15 36 E E Q G R A F R G S 15 26 G E C G Q T F R L K 14 53 N D P R E T Q E V F 14 60 E V F G G G V G D I 14 82 E T S A S E E E K Y 14 5 Q E Y D K S L S V Q 13 15 P E K R T G L R D E 13 33 R L K E E Q G R A F 13 2 G K C Q E Y D K S L 12 25 N G E C G Q T F R L 12 35 K E E Q G R A F R G 12 65 G V G D I V G R D L 12 67 G D I V C R D L S I 12 69 I V G R D L S I S F 12 42 F R C S S V H Q K L 11 56 R E T Q E V F C G G 11 59 Q E V F C G G V G D 11 81 S E T S A S E E E K 11 86 S E E E K Y D M S G 11 41 A F R C S S V H Q K 7 6 E Y D K S L S V Q P 5 27 E C C Q T F R L K E 5 52 V N D P R E T Q E V 5 77 S F R N S E T S A S 5 9 K S L S V Q P E K R 4 10 S L S V Q P E K R T 4 16 E K R T G L R D E N 4 28 C G Q T F R L K E E 4 30 Q T F R L K E E Q G 4 37 E Q G R A F R G S S 4 40 R A F R G S S V H Q 4 43 R G S S V H Q K L V 4 50 K L V N D P R E T Q 4 63 G G G V G D I V G R 4 70 V G R D L S I S F R 4 13 V Q P E K R T G L R 3 44 G S S V H Q K L V N 3 45 S S V H Q K L V N D 3 46 S V H Q K L V N D P 3 49 Q K L V N D P R E T 3 62 F G G G V G D I V G 3 66 V G D I V G R D L S 3 72 R D L S I S F R N S 3 73 D L S I S F R N S E 3 74 L S I S F R N S E T 3 75 S I S F R N S E T S 3 76 I S F R N S E T S A 3 85 A S E E E K Y D M S 3 3 K C Q E Y D K S L S 2 8 D K S L S V Q P E K 2 11 L S V Q P E K R T G 2 17 K R T G L R D E N G 2 18 R T G L R D E N G E 2 19 T G L R D E N G E C 2 22 R D E N G E C G Q T 2 24 E N G E C G Q T F R 2 51 L V N D P R E T Q E 2 55 P R E T Q E V F G G 2 57 E T Q E V F G& TABLE XIXB, part 5: MHC Class I decamer analysis of 213P1511 v.5 (aa 1-242). 213P1511 v.5: HLA-A*0201 decamers 2 G A R L A L I L R V 20 5 L A L I L R V T K A 18 4 R L A L I L R V T K 16 6 A L I L R V T K A R 14 7 L I L R V T K A R E 14 8 I L R V T K A R E G 14 3 A R L A L I L R V T 12 1 S G A R L A L I L R 8 10 R V T K A R E G S E 5 9 L R V T K A R E G S 3 213P1F11 v.5: HLA-A*0202 decamers 1 S G A R L A L I L R 3 4 R L A L I L R V T K 3 2 G A R L A L I L R V 2 5 L A L I L R V T K A 2 3 A R L A L I L R V T 1 6 A L I L R V T K A R 1 213P1F11 v.5: HLA-A*0203 decamers 5 L A L I L R V T K A 10 6 A L I L R V T K A R 9 7 L I L R V T K A R E 8 213P1F11 v.5: HLA-A1 decamers 1 S G A R L A L I L R 8 2 G A R L A L I L R V 6 5 L A L I L R V T K A 3 3 A R L A L I L R V T 2 4 R L A L I L R V T K 2 6 A L I L R V T K A R 2 8 I L R V T K A R E G 1 9 L R V T K A R E G S 1 213P1F11 v.5: HLA-A26 decamers 7 L I L R V T K A R E 12 10 R V T K A R E G S E 12 6 A L I L R V T K A R 11 4 R L A L I L R V T K 10 8 I L R V T K A R E G 9 1 S G A R L A L I L R 5 2 G A R L A L I L R V 5 3 A R L A L I L R V T 4 9 L R V T K A R E G S 1 213P1F11 v.5: HLA-A3 decamers 4 R L A L I L R V T K 35 6 A L I L R V T K A R 22 8 I L R V T K A R E G 18 10 R V T K A R E G S E 17 7 L I L R V T K A R E 16 3 A R L A L I L R V T 11 1 S G A R L A L I L R 9 2 G A R L A L I L R V 7 5 L A L I L R V T K A 4 9 L R V T K A R E G S 1 213P1F11 v.5: HLA-B*0702 decamers 2 G A R L A L I L R V 10 3 A R L A L I L R V T 10 5 L A L I L R V T K A 8 4 R L A L I L R V T K 5 6 A L I L R V T K A R 4 8 I L R V T K A R E G 3 10 R V T K A R E G S E 2 1 S G A R L A L I L R 1 7 L I L R V T K A R E 1 9 L R V T K A R E G S 1 213P1F11 v.5: HLA-B*4402 decamers 6 A L I L R V T K A R 12 3 A R L A L I L R V T 7 1 S G A R L A L I L R 5 2 G A R L A L T L R V 5 4 TABLE XIXB, part 6: MHC Class I decamer analysis of 213P1F11 v.6 (aa 1-242). 213P1F11 v.6: HLA-A*0201 decamers 9 A M N N K N C Q A L 22 5 N L F E A M N N K N 16 2 K L E N L F E A M N 12 1 V K L E N L F E A M 10 8 E A M N N K N C Q A 6 7 F E A M N N K N C Q 4 3 L E N L F E A M N N 3 10 M N N K N C Q A L R 3 4 E N L F E A M N N K 1 213P1F11 v.6: HLA-A*0202 decamers 7 F E A M N N K N C Q 3 8 E A M N N K N C Q A 2 9 A M N N K N C Q A L 1 213P1F11 v.6: HLA-A*0203 decamers 8 E A M N N K N C Q A 10 1 V K L E N L F E A M 9 9 A M N N K N C Q A L 9 2 K L E N L F E A M N 8 10 M N N K N C Q A L R 8 213P1F11 v.6: HLA-A1 decamers 2 K L E N L F E A M N 12 6 L F E A M N N K N C 11 1 V K L E N L F E A M 4 5 N L F E A M N N K N 4 10 M N N K N C Q A L R 2 7 F E A M N N K N C Q 1 8 E A M N N K N C Q A 1 9 A M N N K N C Q A L 1 213P1F11 v.6: HLA-A26 decamers 1 V K L E N L F E A M 15 5 N L F E A M N N K N 13 4 E N L F E A M N N K 11 2 K L E N L F E A M N 10 9 A M N N K N C Q A L 10 6 L F E A M N N K N C 7 8 E A M N N K N C Q A 7 3 L E N L F E A M N N 2 10 M N N K N C Q A L R 2 7 F E A M N N K N C Q 1 213P1F11 v.6: HLA-A3 decamers 2 K L E N L F E A M N 18 4 E N L F E A M N N K 12 5 N L F E A M N N K N 11 10 M N N K N C Q A L R 7 3 L E N L F E A M N N 5 9 A M N N K N C Q A L 5 1 V K L E N L F E A M 4 8 E A M N N K N C Q A 4 6 L F E A M N N K N C 2 7 F E A M N N K N C Q 1 213P1F11 v.6: HLA-B*0702 decamers 9 A M N N K N C Q A L 13 1 V K L E N L F E A M 8 8 E A M N N K N C Q A 8 2 K L E N L F E A M N 2 10 M N N K N C Q A L R 2 4 E N L F E A M N N K 1 6 L F E A M N N K N C 1 7 F E A M N N K N C Q 1 213P1F11 v.6: HLA-B*4402 decamers 9 A M N N K N C Q A L 16 3 L E N L F E A M N N 12 7 F E A M N N K N C Q 11 5 N L F E A M N N K N 5 8 E A M N N K N C Q A 5 1 V K L E N L F E A M 4 4 E N L F E A M N N K 4 2 K L E N L F E A M N 2 Listed are scores which correlate with the ligation strength to a defined HLA type for a sequence of amino acids. The algorithms used are based on the book “MHC Ligands and Peptide Motifs” by H. G. Rammensee, J. Bachmann and S. Stevanovic. The probability of being processed and presented is given in order to predict T-cell epitopes.
[0771] 24 TABLE XIXC MHC Class II Analysis of 213P1F11. Listed are scores which correlate with the ligation strength to a defined HLA type for a sequence of amino acids. The algorithms used are based on the book “MHC Ligands and Peptide Motifs” by H. G. Rammensee, J. Bachmann and S. Stevanovic. The probability of being processed and presented is given in order to predict T-cell epitopes. Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score SEQ. ID NO. Table XIXC, part 1: MHC Class II 15-mer analysis of 213P1F11 v.1 (aa 1-242). 213P1F11 v.1: HLA-DR B1*0101 15-mers 166 L H V Y S T V E G Y I A Y R H 33 9 E E K Y D M S G A R L A I L I L 29 80 S C A F V V L M A H G R E G F 29 192 V D V F T K R K G H I L E L L 29 92 E G F L K G E D G E M V K L E 25 123 K P K V Y I I Q A C R G E Q R 25 63 E E L E K F Q Q A I D S R E D 24 145 G D E I V M V I K D S P Q T I 24 148 I V M V I K D S P Q T I P T Y 24 203 L E L L T E V T R R M A E A E 24 137 R D P G E T V G G D E I V M V 23 202 I L E L L T E V T R R M A E A 23 8 E E E K Y D M S G A R L A L I 22 39 L E H M F R Q L R F E S T M K 22 124 P K V Y I I Q A C R G E Q R D 22 176 I A Y R D Q K G S C F I Q T 22 188 I Q T L V D V F T K R K G H I 22 199 K G H I L E L L T E V T R R M 22 17 A R L A L I L C V T K A R E G 21 91 R E G F L K G E D G E M V K L 21 40 E H M F R Q L R F E S T M K R 20 99 D G E M V K L E N L F E A L N 20 208 E V T R R M A E A E L V Q E G 20 45 Q L R F E S T M K R D P T A E 19 83 F V V L M A H G R E G F L K G 19 106 E N L F E A L N N K N C Q A L 19 159 I P T Y T D A L H V Y S T V E 19 43 F R Q L R F E S T M K R D P T 18 50 S T M K R D P T A E Q F Q E E 18 82 A F V V L M A H G R E G F L K 18 100 G E M V K L E N L F E A L N N 18 117 C Q A L R A K P K V Y I I Q A 18 147 E I V M V I K D S P Q T I P T 18 149 V M V I K D S P Q T I P T Y T 18 184 G S C F I Q T L V D V F T K R 18 216 A E L V Q E G K A R K T N P E 18 20 A L I L C V T K A R E G S E E 17 33 E E D L D A L E H M F R Q L R 17 69 Q Q A I D S R E D P V S C A F 17 89 H G R E G F L K G E D G E M V 17 109 F E A L N N K N C Q A L R A K 17 111 A L N N K N C Q A L R A K P K 17 114 N K N C Q A L R A K P K V I 17 140 G E T V G G D E I V M V I K D 17 157 Q T I P T Y T D A L H V Y S T 17 183 K G S C F I Q T L V D V F T K 17 200 G H I L E L L T E V T R R M A 17 206 L T E V T R R M A E A E L V Q 17 228 N P E I Q S T L R K R L Y L Q 17 4 P R S L E E E K Y D M S G A R 16 11 K Y D M S G A R L A L T L C V 16 12 Y D M S G A R L A L I L C V T 16 18 R L A L I L C V T K A R E G S 16 30 E G S E E D L D A L E H M F R 16 74 S R E D P V S C A F V V L M A 16 76 E D P V S C A F V V L M A H G 16 81 C A F V V L M A H G R E G F L 16 84 V V L M A H G R E G F L K G E 16 102 M V K L E N L F E A L N N K N 16 120 L R A K P K V Y I I Q A C R G 16 126 V Y I I Q A C R G E Q R D P G 16 132 C R G E Q R D P G E T V G G D 16 165 A L H V Y S T V E G Y I A Y R 16 169 Y S T V E G Y I A Y R H D Q K 16 185 S C F I Q T L V D V F T K R K 16 191 L V D V F T K R K G H I L E L 16 207 T E V T R R M A E A E L V Q E 16 213 M A E A E L V Q E G K A R K T 16 214 A E A E L V Q E G K A R K T N 16 16 G A R L A L I L C V T K A R E 15 49 E S T M K R D P T A E Q F Q E 15 97 G E D G E M V K L E N L F E A 15 144 G G D E I V M V I K D S P Q T 15 177 A Y R H D Q K G S C F I Q T L 15 226 K T N P E I Q S T L R K R L Y 15 19 L A L I L C V T K A R E G S E 14 36 L D A L E H M F R Q L R F E S 14 54 R D P T A E Q F Q E E L E K F 14 72 I D S R E D P V S C A F V V L 14 73 D S R E D P V S C A F V V L M 14 146 D E I V M V I K D S P Q T I P 14 155 S P Q T I P T Y T D A L H V Y 14 163 T D A L H V Y S T V E G Y I A 14 66 E K F Q Q A I D S R E D P V S 13 154 D S P Q T I P T Y T D A L H V 13 162 Y T D A L H V Y S T V E G Y T 13 182 Q K G S C F I Q T L V D V F T 13 225 R K T N P E I Q S T L R K R L 13 1 M S N P R S L E E E K Y D M S 12 6 S L E E E K Y D M S G A R L A 12 153 K D S P Q T I P T Y T D A L H 12 175 Y T A Y R H D Q K G S C F I Q 12 194 V F T K R K G H I L E L L T E 12 197 K R K G H I L E L L T E V T R 12 14 M S G A R L A L I L C V T K A 11 21 L I L C V T K A R E G S E E D 11 22 I L C V T K A R E G S E E D L 11 58 A E Q F Q E E L E K F Q Q A I 11 67 K F Q Q A I D S R E D P V S C 11 103 V K L E N L F E A L N N K N C 11 174 G Y I A Y R H D Q K G S C F I 11 218 L V Q E G K A R K T N P E I Q 11 23 L C V T K A R E G S E E D L D 10 25 V T K A R E G S E E D L D A L 10 47 R F E S T M K R D P T A E Q F 10 57 T A E Q F Q E E L E K F Q Q A 10 62 Q E E L E K F Q Q A I D S R E 10 65 L E K F Q Q A I D S R E D P V 10 71 A I D S R E D P V S C A F V V 10 94 F L K G E D G E M V K L E N L 10 115 K N C Q A L R A K P K V Y I I 10 118 Q A L R A K P K V Y I I Q A C 10 128 I I Q A C R G E Q R D P G E T 10 138 D P G E T V G G D E I V M V I 10 143 V G G D E I V M V I K D S P Q 10 172 V E G Y I A Y R H D Q K G S C 10 189 Q T L V D V F T K R K G H I L 10 198 R K G H I L E L L T E V T R R 10 212 R M A E A E L V Q E G K A R K 10 215 E A E L V Q E G K A R K T N P 10 221 E G K A R K T N P E I Q S T L 10 3 N P R S L E E E K Y D M S G A 9 27 K A R E G S E E D L D A L E H 9 31 G S E E D L D A L E H M F R Q 9 35 D L D A L E H M F R Q L R F E 9 37 D A L E H M F R Q L R F E S T 9 41 H M F R Q L R F E S T M K R D 9 48 F E S T M K R D P T A E Q F Q 9 59 E Q F Q E E L E K F Q Q A I D 9 60 Q F Q E E L E K F Q Q A I D S 9 75 R E D P V S C A F V V L M A H 9 85 V L M A H G R E G F L K G E D 9 105 L E N L F E A L N N K N C Q A 9 113 N N K N C Q A L R A K P K V Y 9 134 G E Q R D P G E T V G G D E I 9 139 P G E T V G G D E I V M V I K 9 161 T Y T D A L H V Y S T V E G Y 9 173 E G Y I A Y R H D Q K G S C F 9 181 D Q K G S C F I Q T L V D F 9 195 F T K R K G H I L E L L T E V 9 205 L L T E V T R R M A E A E L V 9 210 T R R M A E A E L V Q E G K A 9 222 G K A R K T N P E I Q S T L R 9 224 A R K T N P E I Q S T L R K R 9 10 E K Y D M S G A R L A L I L C 8 13 D M S G A R L A L I L C V T K 8 15 S G A R L A L I L C V T K A R 8 28 A R E G S E E D L D A L E H M 8 32 S E E D L D A L E H M F R Q L 8 42 M F R Q L R F E S T M K R D P 8 56 P T A E Q F Q E E L E K F Q Q 8 61 F Q E E L E K F Q Q A I D S R 8 68 F Q Q A I D S R E D P V S C A 8 77 D P V S C A F V V L M A H G R 8 98 E D G E M V K L E N L F E A L 8 101 E M V K L E N L F E A L N N K 8 108 L F E A L N N K N C Q A L R A 8 112 L N N K N C Q A L R A K P K V 8 125 K V Y I I Q A C R G E Q R D P 8 130 Q A C R G E Q R D P G E T V G 8 136 Q R D P G E T V G G D E I V M 8 141 E T V G G D E I V M V I K D S 8 156 P Q T I P T Y T D A L H V Y S 8 160 P T Y T D A L H V Y S T V E G 8 180 H D Q K G S C F I Q T L V D V 8 186 C F I Q T L V D V F T K R K G 8 220 Q E G K A R K T N P E I Q S T 8 7 L E E E K Y D M S G A R L A L 7 24 C V T K A R E G S E E D L D A 7 78 P V S C A F V V L M A H G R E 7 116 N C Q A L R A K P K V Y I I Q 7 122 A K P K V Y T I Q A C R G E Q 7 152 I K D S P Q T I L P T Y T D A L 7 170 S T V E G Y I A Y R H D Q K G 7 219 V Q E G K A R K T N P E I Q S 7 46 L R F E S T M K R D P T A E Q 6 64 E L E K F Q Q A I D S R E D P 6 70 Q A I D S R E D P V S C A F V 6 79 V S C A F V V L M A H G R E G 6 96 K G E D G E M V K L E N L F E 6 131 A C R G E Q R D P G E T V G G 6 142 T V G G D E I V M V I K D S P 6 150 M V I K D S P Q T I P T Y T D 6 151 V I K D S P Q T I P T Y T D A 6 196 T K R K G H I L E L L T E V T 6 87 M A H G R E G F L K G E D G E 5 190 T L V D V F T K R K G H I L E 5 90 G R E G F L K G E D G E M V K 4 133 R G E Q R D P G E T V G G D E 4 2 S N P R S L E E E K Y D M S G 3 110 E A L N N K N C Q A L R A K P 3 164 D A L H V Y S T V E G Y lA Y 3 171 T V E G Y I A Y R H D Q K G S 3 201 H I L E L L T E V T R R M A E 3 38 A L E H M F R Q L R F E S T M 2 86 L M A H G R E G F L K G E D G 2 129 I Q A C R G E Q R D P G E T V 2 135 E Q R D P G E T V G G D E T V 2 167 H V Y S T V E G Y I A Y R H D 2 178 Y R H D Q K G S C F I Q T L V 2 187 F I Q T L V D V F T K R K G H 2 193 D V F T K R K G H I L E L L T 2 223 I C A R K T N P E I Q S T L R K 2 227 T N P E I Q S T L R K R L Y L 2 26 T K A R E G S E E D L D A L E 1 34 E D L D A L E H M F R Q L R F 1 44 R Q L R F E S T M K R D P T A 1 52 H K R D P T A E Q F Q E E L E 1 53 K R D P T A E Q F Q E E L E K 1 93 G F L K G E D G E M V K L E M 1 95 L K G E D G E M V K L E N L F 1 104 K L E N L F E A L N N K N C Q 1 107 N L F E A L N N K N C Q A L R 1 119 A L R A K P K V Y I I Q A C R 1 121 R A K P K V Y I I Q A C R G E 1 127 Y I I Q A C R G E Q R D P G E 1 168 V Y S T V E G Y I A Y R H D Q 1 179 R H D Q K G S C F I Q T L V D 1 217 E L V Q E G K A R K T N P E I 1 213P1F11 v.1: HLA-DRB1*0301 (DR17) 15-mers 228 N P E I Q S T L R K R L Y L Q 26 83 F V V L M A H G R E G F L K G 25 84 V V L M A H G R E G F L K G E 24 49 E S T M K R D P T A E Q F Q E 23 148 I V M V I L K D S P Q T I P T Y 23 140 G E T V G G D E I V M V I K D 22 105 L E N L F E A L N N K C Q A 20 109 F E A L N N K N C Q A L R A K 20 19 L A L I L C V T K A R E G S E 19 99 D G E M V K L E N L F E A L N 19 117 C Q A L R A K P K V Y I I Q A 19 215 E A E L V Q E G K A R K T P 19 1 M S N P R S L E E E K Y D M S 18 36 L D A L E H M F R Q L R F E S 18 93 G F L K G E D G E M V K L E N 18 102 M V K L E N L F E A L N N K N 18 145 G D E I V M V I K D S P Q T I 18 149 V M V I K D S P Q T I P T Y T 18 158 T I P T Y T D A L H V Y S T V 18 184 G S C F I Q T L V D V F T K R 18 192 V D V F T K R K G H I L E L L 18 200 G H I L E L L T E V T R R M A 18 203 L E L L T E V T R R M A E A E 18 10 E K Y D M S G A R L A L T L C 17 22 I L C V T K A R E G S E E D L 17 25 V T K A R E G S E E D L D A L 17 32 S E E D L D A L E H M F R Q L 17 39 L E H M F R Q L R F E S T M K 17 62 Q E E L E K F Q Q A I D S R E 17 67 K F Q Q A I D S R E D P V S C 17 98 E D G E M V K L E N L F E A L 17 125 K V Y I I Q A C R G E Q R D P 17 165 A L H V Y S T V E G Y I A Y R 17 175 Y I A Y R H D Q K G S C F I Q 17 189 Q T L V D V F T K R K G H I L 17 37 D A L E H M F R Q L R F E S T 16 58 A E Q F Q E E L E K F Q Q A I 16 191 L V D V F T K R K G H I L E L 16 207 T E V T R R M A E A E L V Q E 16 45 Q L R F E S T M K R D P T A E 15 54 R D P T A E Q F Q E E L E K F 15 106 E N L F E A L N N K N C Q A L 15 224 A R K T N P E I Q S T L R K R 15 29 R E G S E E D L D A L E H M F 14 199 K G H I L E L L T E V T R R M 14 16 G A R L A L T L C V T K A R E 13 18 R L A L I L C V T K A R E G S 13 43 F R Q L R F E S T M K R D P T 13 71 A T D S R E D P V S C A F V V 13 82 A F V V L M A H G R E G F L K 13 101 E M V K L E N L F E A L N N K 13 187 F I Q T L V D V F T K R K G H 13 210 T R R M A E A E L V Q E G K A 13 216 A E L V Q E G K A R K T N P E 13 4 P R S L E E E K Y D M S G A R 12 7 L E E E K Y D M S G A R L A L 12 11 K Y D M S G A R L A L I L C V 12 69 Q Q A I D S R E D P V S C A F 12 76 E D P V S C A F V V L M A H G 12 81 C A F V V L M A H G R E G F L 12 100 G E M V K L E N L F E A L N N 12 156 P Q T I P T Y T D A L H V Y S 12 163 T D A L H V Y S T V E G Y T A 12 173 E G Y I A Y R H D Q K G S C F 12 202 I L E L L T E V T R R M A E A 12 206 L T E V T R R M A E A E L V Q 12 20 A L I L C V T K A R E G S E E 11 31 G S E E D L D A L E H M F R Q 11 33 E E D L D A L E H M F R Q L R 11 35 D L D A L E H M F R Q L R F E 11 61 F Q E E L E K F Q Q A I D S R 11 75 R E D P V S C A F V V L M A H 11 91 R E G F L K G E D G E M V K L 11 92 E G F L K G E D G E M V K L E 11 94 F L K G E D G E M V K L E N L 11 97 G E D G E M V K L E N L F E A 11 116 N C Q A L R A K P K V Y I I Q 11 132 C R G E Q R D P G E T V G G D 11 146 D E I V M V I K D S P Q T I P 11 147 E T V M V I K D S P Q T I P T 11 155 S P Q T I P T Y T D A L H V Y 11 167 H V Y S T V E G Y I A Y R H D 11 185 S C F I Q T L V D V F T K R K 11 188 I Q T L V D V F T K R K G H I 11 3 N P R S L E E E K Y D M S G A 10 8 E E E K Y D M S G A R L A L I 10 12 Y D M S G A R L A L I L C V T 10 28 A R E G S E E D L D A L E H M 10 57 T A E Q F Q E E L E K F Q Q A 10 123 K P K V Y I I Q A C R G E Q R 10 126 V Y I I Q A C R G E Q R D P G 10 169 Y S T V E G Y I A Y R H D Q K 10 180 H D Q K G S C F I Q T L V D V 10 194 V F T K R K G H I L E L L T E 10 195 F T K R K G H I L E L L T E V 10 40 E H M F R Q L R F E S T M K R 9 50 S T M K R D P T A E Q F Q E E 9 72 I D S R E D P V S C A F V V L 9 80 S C A F V V L M A H G R E G F 9 108 L F E A L N N K N C Q A L R A 9 113 N N K N C Q A L R A K P K V Y 9 151 V I K D S P Q T I P T Y T D A 9 164 D A L H V Y S T V E G Y lA Y 9 176 I A Y R H D Q K G S C F I Q T 9 196 T K R K G I I I L E L L T E V T 9 2 S N P R S L E E E K Y D M S G 8 26 T K A R E G S E E D L D A L E 8 51 T M K R D P T A E Q F Q E E L 8 56 P T A E Q F Q E E L E K F Q Q 8 59 E Q F Q E E L E K F Q Q A I D 8 65 L E K F Q Q A I D S R E D P V 8 90 G R E G F L K G E D G E M V K 8 96 K G E D G E M V K L E N L F E 8 107 N L F E A L N N K N C Q A L R 8 110 E A L N N K N C Q A L R A K P 8 115 K N C Q A L R A K P K V Y I I 8 122 A K P K V Y I I Q A C R G E Q 8 127 Y I I Q A C R G E Q R D P G E 8 133 R G E Q R D P G E T V G G D E 8 190 T L V D V F T K R K G H I L E 8 213 M A E A E L V Q E G K A R K T 8 222 G K A R K T N P E I Q S T L R 8 21 L I L C V T K A R E G S E E D 7 41 H M F R Q L R F E S T M K R D 7 46 L R F E S T M K R D P T A E Q 7 52 M K R D P T A E Q F Q E E L E 7 55 D P T A E Q F Q E E L E K F Q 7 68 F Q Q A I D S R E D P V S C A 7 88 A H G R E G F L K G E D G E M 7 128 I I Q A C R G E Q R D P G E T 7 129 I Q A C R G E Q R D P G E T V 7 139 P G E T V G G D E I V M V T K 7 181 D Q K G S C F I Q T L V D V F 7 204 E L L T E V T R R M A E A E L 7 209 V T R R M A E A E L V Q E G K 7 212 R M A E A E L V Q E G K A R K 7 218 L V Q E G K A R K T N P E I Q 7 171 T V E G Y I A Y R H D Q K G S 6 174 G Y I A Y R H D Q K G S C F I 6 217 E L V Q E G K A R K T N P E I 6 220 Q E G K A R K T N P E I Q S T 6 14 M S G A R L A L I L C V T K A 4 103 V K L E N L F E A L N N K N C 4 104 K L E N L F E A L N N K N C Q 4 201 H I L E L L T E V T R R M A E 4 208 E V T R R H A E A E L V Q E G 4 214 A E A E L V Q E G K A R K T N 4 9 E E K Y D M S G A R L A L I L 3 17 A R L A L I L C V T K A R E G 3 27 K A R E G S E E D L D A L E H 3 42 M F R Q L R F E S T M K R D P 3 60 Q F Q E E L E K F Q Q A I D S 3 73 D S R E D P V S C A F V V L M 3 86 L M A H G R E G F L K G E D G 3 121 R A K P K V Y I I Q A C R G E 3 134 G E Q R D P G E T V G G D E I 3 159 I P T Y T D A L H V Y S T V E 3 168 V Y S T V E G Y I A Y R H D Q 3 193 D V F T K R K G H T L E L L T 3 198 R K G H I L E L L T E V T R R 3 219 V Q E G K A R K T N P E I Q S 3 223 K A R K T N P E I Q S T L R K 3 15 S G A R L A L I L C V T K A R 2 23 L C V T K A R E G S E E D L D 2 47 R F E S T M K R D P T A E Q F 2 53 K R D P T A E Q F Q E E L E K 2 79 V S C A F V V L M A H G R E G 2 85 V L M A H G R E G F L K G E D 2 87 M A H G R E G F L K G E D G E 2 111 A L N N K N C Q A L R A K P K 2 119 A L R A K P K V Y I I Q A C R 2 138 D P G E T V G G D E I V M V I 2 142 T V G G D E T V M V I K D S P 2 157 Q T I P T Y T D A L H V Y S T 2 162 Y T D A L H V Y S T V E G Y I 2 186 C F T Q T L V D V F T K R K G 2 197 K R K G H I L E L L T E V T R 2 205 L L T E V T R R M A E A E L V 2 211 R R M A E A E L V Q E G K A R 2 225 R K T N P E I Q S T L R K R L 2 226 K T N P E I Q S T L R K R L Y 2 227 T N P E I Q S T L R K R L Y L 2 5 R S L E E E K Y D M S G A R L 1 13 D M S G A R L A L I L C V T K 1 24 C V T K A R E G S E E D L D A 1 30 E G S E E D L D A L E H M F R 1 34 E D L D A L E H M F R Q L R F 1 44 R Q L R F E S T M K R D P T A 1 48 F E S T M K R D P T A E Q F Q 1 63 E E L E K F Q Q A I D S R E D 1 70 Q A T D S R E D P V S C A F V 1 77 D P V S C A F V V L M A H G R 1 78 P V S C A F V V L M A H G R E 1 89 H G R E G F L K G E D G E M V 1 95 L K G E D G E M V K L E N L F 1 112 L N N K N C Q A L R A K P K V 1 118 Q A L R A K P K V Y I I Q A C 1 124 P K V Y I I Q A C R G E Q R D I 130 Q A C R G E Q R D P G E T V G I 136 Q R D P G E T V G G D E I V M 1 137 R D P G E T V G G D E I V M V 1 143 V G G D E I V M V I K D S P Q 1 144 G G D E I V M V I K D S P Q T 1 150 M V T K D S P Q T I P T Y T D 1 153 K D S P Q T I P T Y T D A L H 1 154 D S P Q T I P T Y T D A L H V 1 161 T Y T D A L H V Y S T V E G Y 1 166 L H V Y S T V E G Y I A Y R H 1 172 V E G Y I A Y R H D Q K G S C 1 177 A Y R H D Q K G S C F I Q T L I 178 Y R H D Q K G S C F I Q T L V 1 179 R H D Q K G S C F I Q T L V D 1 182 Q K G S C F I Q T L V D V F T 1 183 K G S C F I Q T L V D V F T K I 221 E G K A R K T N P E I Q S T L 1 213P1F11 v.1:HLA-DRB1*0401 (DR4Dw4) 15-mers 159 I P T Y T D A L H V Y S T V E 28 49 E S T H K R D P T A E Q F Q E 26 105 L E N L F E A L N N K N C Q A 26 125 K V Y I I Q A C R G E Q R D P 26 188 I Q T L V D V F T K R K G H I 26 199 K G H I L E L L T E V T R R M 26 202 I L E L L T E V T R R M A E A 26 203 L E L L T E V T R R M A E A E 26 40 E H M F R Q L R F E S T M K R 22 58 A E Q F Q E E L E K F Q Q A I 22 106 E N L F E A L N N K N C Q A L 22 166 L H V Y S T V E G Y I A Y R H 22 172 V E G Y I A Y R H D Q K G S C 22 4 P R S L E E E K Y D M S G A R 20 11 K Y D H S G A R L A L I L C V 20 16 G A R L A L I L C V T K A R E 20 33 E E D L D A L E H M F R Q L R 20 39 L E H N F R Q L R F E S T M K 20 43 F R Q L R F E S T M K R D P T 20 62 Q E E L E K F Q Q A T D S R E 20 76 E D P V S C A F V V L M A H G 20 92 E G F L K G E D G E M V K L E 20 99 D G E M V K L E N L F E A L N 20 100 G E M V K L E N L F E A L N N 20 102 M V K L E N L F E A L N N K N 20 140 G E T V G G D E I V M V I K D 20 146 D E I V M V I K D S P Q T I P 20 148 I V M V I K D S P Q T I P T Y 20 185 S C F I Q T L V D V F T K R K 20 215 E A E L V Q E G K A R K T N P 20 228 N P E I Q S T L R K R L Y L Q 20 42 M F R Q L R F E S T M K R D P 18 171 T V E G Y I A Y R H D Q K G S 18 181 D Q K G S C F I Q T L V D V F 18 212 R M A E A E L V Q E G K A R K 18 224 A R K T N P E I Q S T L R K R 18 9 E E K Y D M S G A R L A L I L 16 65 L E K F Q Q A I D S R E D P V 16 80 S C A F V V L M A H G R E G F 16 91 R E G F L K G E D G E M V K L 16 175 Y I A Y R H D Q K G S C F I Q 16 147 E T V M V I K D S P Q T I P T 15 18 R L A L I L C V T K A R E G S 14 19 L A L I L C V T K A R E G S E 14 22 I L C V T K A R E G S E E D L 14 36 L D A L E H M F R Q L R F E S 14 81 C A F V V L H A H G R E G F L 14 82 A F V V L M A H G R E G F L K 14 83 F V V L M A H G R E G F L K G 14 84 V V L M A H G R E G F L K G E 14 123 K P K V Y I I Q A C R G E Q R 14 145 G D E I V M V I K D S P Q T I 14 149 V M V I K D S P Q T I P T Y T 14 163 T D A L H V Y S T V E G Y I A 14 165 A L H V Y S T V E G Y I A Y R 14 189 Q T L V D V F T K R K G H I L 14 200 G H I L E L L T E V T R R M A 14 210 T R R H A E A E L V Q E G K A 14 216 A E L V Q E G K A R K T N P E 14 3 N P R S L E E E K Y D M S G A 12 7 L E E E K Y D M S G A R L A L 12 10 E K Y D M S G A R L A L I L C 12 14 M S G A R L A L I L C V T K A 12 15 S G A R L A L I L C V T K A R 12 17 A R L A L I L C V T K A R E G 12 24 C V T K A R E G S E E D L D A 12 27 K A R E G S E E D L D A L E H 12 28 A R E G S E E D L D A L E H M 12 31 G S E E D L D A L E H M F R Q 12 34 E D L D A L E H M F R Q L R F 12 37 D A L E H M F R Q L R F E S T 12 46 L R F E S T M K R D P T A E Q 12 52 M K R D P T A E Q F Q E E L E 12 54 R D P T A E Q F Q E E L E K F 12 59 E Q F Q E E L E K F Q Q A I D 12 60 Q F Q E E L E K F Q Q A I D S 12 66 E K F Q Q A I D S R E D P V S 12 67 K F Q Q A I D S R E D P V S C 12 71 A I D S R E D P V S C A F V V 12 73 D S R E D P V S C A F V V L M 12 77 D P V S C A F V V L M A H G R 12 93 G F L K G E D G E M V K L E N 12 97 G E D G E M V K L E N L F E A 12 103 V K L E N L F E A L N N K N C 12 104 K L E N L F E A L N N K N C Q 12 108 L F E A L N N K N C Q A L R A 12 113 N N K N C Q A L R A K P K V Y 12 114 N K N C Q A L R A K P K V Y T 12 120 L R A K P K V Y I I Q A C R G 12 122 A K P K V Y I I Q A C R G E Q 12 129 I Q A C R G E Q R D P G E T V 12 132 C R G E Q R D P G E T V G G D 12 137 R D P G E T V G G D E I V M V 12 141 E T V G G D E I V M V I K D S 12 142 T V G G D E I V M V I K D S P 12 150 M V I K D S P Q T I P T Y T D 12 153 K D S P Q T I P T Y T D A L H 12 155 S P Q T I P T Y T D A L H V Y 12 160 P T Y T D A L H V Y S T V E G 12 162 Y T D A L H V Y S T V E G Y I 12 174 G Y I A Y R H D Q K G S C F I 12 182 Q K G S C F I Q T L V D V F T 12 186 C F I Q T L V D V F T K R K G 12 196 T K R K G H I L E L L T E V T 12 198 R K G H I L E L L T E V T R R 12 208 E V T R R M A E A E L V Q E G 12 209 V T R R M A E A E L V Q E G K 12 213 H A E A E L V Q E G K A R K T 12 225 R K T N P E I Q S T L R K R L 12 226 K T N P E I Q S T L R K R L Y 12 192 V D V F T K R K G H I L E L L 11 45 Q L R F E S T M K R D P T A E 10 124 P K V Y I I Q A C R G E Q R D 10 184 G S C F I Q T L V D V F T K R 10 69 Q Q A I D S R E D P V S C A F 9 109 F E A L N N K N C Q A L R A K 9 117 C Q A L R A K P K V Y T I Q A 9 191 L V D V F T K R K G H I L E L 9 20 A L I L C V T K A R E G S E E 8 126 V Y I I Q A C R G E Q R D P G 8 156 P Q T I P T Y T D A L H V Y S 8 169 Y S T V E G Y I A Y R H D Q K 8 21 L I L C V T K A R E G S E E D 7 61 F Q E E L E K F Q Q A I D S R 7 177 A Y R H D Q K G S C F I Q T L 7 217 E L V Q E G K A R K T N P E I 7 220 Q E G K A R K T N P E I Q S T 7 1 M S N P R S L E E E K Y D M S 6 2 S N P R S L E E E K Y D M S G 6 6 S L E E E K Y D M S G A R L A 6 8 E E E K Y D M S G A R L A L I 6 13 D M S G A R L A L I L C V T K 6 25 V T K A R E G S E E D L D A L 6 29 R E G S E E D L D A L E H M F 6 30 E G S E E D L D A L E H M F R 6 32 S E E D L D A L E H M F R Q L 6 35 D L D A L E H M F R Q L R F E 6 51 T M K R D P T A E Q F Q E E L 6 53 K R D P T A E Q F Q E E L E K 6 55 D P T A E Q F Q E E L E K F Q 6 56 P T A E Q F Q E E L E K F Q Q 6 57 T A E Q F Q E E L E K F Q Q A 6 63 E E L E K F Q Q A I D S R E D 6 68 F Q Q A I D S R E D P V S C A 6 70 Q A I D S R E D P V S C A F V 6 72 I D S R E D P V S C A F V V L 6 74 S R E D P V S C A F V V L M A 6 75 R E D P V S C A F V V L M A H 6 78 P V S C A F V V L M A H G R E 6 79 V S C A F V V L M A H G R E G 6 86 L M A H G R E G F L K G E D G 6 88 A H G R E G F L K G E D G E M 6 89 H G R E G F L K G E D G E M V 6 94 F L K G E D G E H V K L E N L 6 95 L K G E D G E M V K L E N L F 6 96 K G E D G E M V K L E N L F E 6 101 E M V K L E N L F E A L N N K 6 107 N L F E A L N N K N C Q A L R 6 110 E A L N N K N C Q A L R A K P 6 111 A L N N K N C Q A L R A K P K 6 112 L N N K N C Q A L R A K P K V 6 116 N C Q A L R A K P K V Y I I Q 6 118 Q A L R A K P K V Y I I Q A C 6 128 I I Q A C R G E Q R D P G E T 6 134 G E Q R D P G E T V G G D E I 6 135 E Q R D P G E T V G G D E I V 6 138 D P G E T V G G D E I V M V I 6 139 P G E T V G G D E I V M V I K 6 143 V G G D E I V M V I K D S P Q 6 144 G G D E I V M V I K D S P Q T 6 152 I K D S P Q T I P T Y T D A L 6 154 D S P Q T I P T Y T D A L H V 6 157 Q T I P T Y T D A L H V Y S T 6 158 T I P T Y T D A L H V Y S T V 6 161 T Y T D A L H V Y S T V E G Y 6 167 H V Y S T V E G Y I A Y R H D 6 170 S T V E G Y I A Y R H D Q K G 6 178 Y R H D Q K G S C F I Q T L V 6 179 R H D Q K G S C F I Q T L V D 6 180 H D Q K G S C F I Q T L V D V 6 183 K G S C F I Q T L V D V F T K 6 187 F I Q T L V D V F T K R K G H 6 190 T L V D V F T K R K G H I L E 6 194 V F T K R K G H I L E L L T E 6 195 F T K R K G H I L E L L T E V 6 197 K R K G H I L E L L T E V T R 6 204 E L L T E V T R R M A E A E L 6 207 T E V T R R M A E A E L V Q E 6 211 R R M A E A E L V Q E G K A R 6 218 L V Q E G K A R K T N P E I Q 6 221 E G K A R K T N P E I Q S T L 6 222 G K A R K T N P E T Q S T L R 6 223 K A R K T N P E I Q S T L R K 6 173 E G Y I A Y R H D Q K G S C F 3 206 L T E V T R R M A E A E L V Q 3 12 Y D M S G A R L A L I L C V T 1 41 H M F R Q L R F E S T M K R D 1 47 R F E S T M K R D P T A E Q F 1 85 V L M A H G R E G F L K G E D 1 115 K N C Q A L R A K P K V Y I I I 119 A L R A K P K V Y I I Q A C R 1 131 A C R G E Q R D P G E T V G G 1 193 D V F T K R K G H I L E L L T 1 205 L L T E V T R R M A E A E L V 1 219 V Q E G K A R K T N P E I Q S 1 5 R S L E E E K Y D M S G A R L -5 23 L C V T K A R E G S E E D L D -5 38 A L E H M F R Q L R F E S T M -5 48 F E S T M K R D P T A E Q F Q -5 90 G R E G F L K G E D G E M V K -5 98 E D G E M V K L E N L F E A L -5 127 Y I I Q A C R G E Q R D P G E -5 213P1F11 v.1: HLA-DRB1*1101 15-mers 203 L E L L T E V T R R M A E A E 29 145 G D E I V M V I K D S P Q T I 26 159 I P T Y T D A L H V Y S T V E 26 45 Q L R F E S T M K R D P T A E 25 166 L H V Y S T V E G Y I A Y R H 23 189 Q T L V D V F T K R K G H I L 23 19 L A L I L C V T K A R E G S E 22 81 C A F V V L M A H G R E G F L 22 36 L D A L E H M F R Q L R F E S 21 39 L E H M F R Q L R F E S T M K 21 33 E E D L D A L E I I M F R Q L R 20 123 K P K V Y I I Q A C R G E Q R 20 9 E E K Y D M S G A R L A L T L 18 172 V E G Y I A Y R H D Q K G S C 18 175 Y I A Y R H D Q K G S C F I Q 18 80 S C A F V V L M A I I G R E G F 17 40 E H M F R Q L R F E S T M K R 16 96 K G E D G E M V K L E N L F E 16 106 E N L F E A L N N K N C Q A L 16 215 E A E L V Q E G K A R K T N P 16 228 N P E I Q S T L R K R L Y L Q 16 113 N N K N C Q A L R A K P K V Y 15 144 G G D E I V M V I K D S P Q T 15 191 L V D V F T K R K G H I L E L 15 20 A L I L C V T K A R E G S E E 14 21 L I L C V T K A R E G S E E D 14 46 L R F E S T M K R D P T A E Q 14 59 E Q F Q E E L E K F Q Q A I D 14 67 K F Q Q A I D S R E D P V S C 14 79 V S C A F V V L M A H G R E G 14 83 F V V L M A H G R E G F L K G 14 117 C Q A L R A K P K V Y I I Q A 14 125 K V Y I I Q A C R G E Q R D P 14 129 I Q A C R G E Q R D P G E T V 14 185 S C F I Q T L V D V F T K R K 14 188 I Q T L V D V F T K R K G H I 14 218 L V Q E G K A R K T N P E I Q 14 17 A R L A L I L C V T K A R E G 13 69 Q Q A I D S R E D P V S C A F 13 82 A F V V L M A H G R E G F L K 13 102 H V K L E N L F E A L N N K N i3 146 D E I V M V I K D S P Q T I P 13 149 V M V I K D S P Q T I P T Y T 13 163 T D A L H V Y S T V E G Y I A 13 173 E G Y I A Y R H D Q K G S C F 13 192 V D V F T K R K G H I L E L L 13 199 K G H I L E L L T E V T R R M 13 213 M A E A E L V Q E G K A R K T 13 4 P R S L E E E K Y D M S G A R 12 16 G A R L A L I L C V T K A R E 12 65 L E K F Q Q A I D S R E D P V 12 89 H G R E G F L K G E D G E M V 12 99 D G E M V K L E N L F E A L N 12 100 G E M V K L E N L F E A L N N 12 120 L R A K P K V Y I I Q A C R G 12 200 G H I L E L L T E V T R R M A 12 124 P K V Y I I Q A C R G E Q R D 11 184 G S C F I Q T L V D V F T K R 11 202 I L E L L T E V T R R M A E A 11 58 A E Q F Q E E L E K F Q Q A I 10 91 R E G F L K G E D G E M V K L 10 115 K N C Q A L R A K P K V Y I I 9 142 T V G G D E I V M V I K D S P 9 193 D V F T K R K G H I L E L L T 9 204 E L L T E V T R R M A E A E L 9 3 N P R S L E E E I C Y D M S G A 8 8 E E E K Y D M S G A R L A L I 8 10 E K Y D M S G A R L A L I L C 8 18 R L A L I L C V T K A R E G S 8 22 I L C V T K A R E G S E E D L 8 47 R F E S T M K R D P T A E Q F 8 77 D P V S C A F V V L M A H G R 8 78 P V S C A F V V L M A H G R E 8 88 A H G R E G F L K G E D G E M 8 105 L E N L F E A L N N K N C Q A 8 107 N L F E A L N N K N C Q A L R 8 111 A L N N K N C Q A L R A K P K 8 154 D S P Q T I P T Y T D A L H V 8 165 A L H V Y S T V E G Y I A Y R 8 169 Y S T V E G Y I A Y R H D Q K 8 171 T V E G Y I A Y R H D Q K G S 8 186 C F I Q T L V D V F T K R K G 8 190 T L V D V F T K R K G H I L E 8 206 L T E V T R R M A E A E L V Q 8 212 R M A E A E L V Q E G K A R K 8 216 A E L V Q E G K A R K T N P E 8 217 E L V Q E G K A R K T N P E I 8 1 M S N P R S L E E E K Y D M S 7 11 K Y D M S G A R L A L I L C V 7 13 D M S G A R L A L I L C V T K 7 15 S G A R L A L I L C V T K A R 7 43 F R Q L R F E S T M K R D P T 7 62 Q E E L E K F Q Q A I D S R E 7 66 E K F Q Q A I D S R E D P V S 7 76 E D P V S C A F V V L M A H G 7 86 L M A H G R E G F L K G E D G 7 128 I I Q A C R G E Q R D P G E T 7 133 R G E Q R D P G E T V G G D E 7 143 V G G D E I V M V I K D S P Q 7 156 P Q T I P T Y T D A L H V Y S 7 162 Y T D A L H V Y S T V E G Y I 7 170 S T V E G Y I A Y R H D Q K G 7 182 Q K G S C F I Q T L V D V F T 7 196 T K R K G H I L E L L T E V T 7 225 R K T N P E I Q S T L R K R L 7 5 R S L E E E K Y D M S G A R L 6 6 S L E E E K Y D M S G A R L A 6 27 K A R E G S E E D L D A L E H 6 30 E G S E E D L D A L E H M F R 6 49 E S T M K R D P T A E Q F Q E 6 60 Q F Q E E L E K F Q Q A I D S 6 63 E E L E K F Q Q A I D S R E D 6 71 A I D S R E D P V S C A F V V 6 73 D S R E D P V S C A F V V L M 6 84 V V L M A H G R E G F L K G E 6 92 E G F L K G E D G E M V K L E 6 97 G E D G E M V K L E N L F E A 6 108 L F E A L N N K N C Q A L R A 6 109 F E A L N N K N C Q A L R A K 6 114 N K N C Q A L R A K P K V Y I 6 122 A K P K V Y I I Q A C R G E Q 6 126 V Y I I Q A C R G E Q R D P G 6 134 G E Q R D P G E T V G G D E I 6 137 R D P G E T V G G D E I V M V 6 140 G E T V G G D E I V M V I K D 6 147 E I V M V I K D S P Q T I P T 6 148 I V M V I K D S P Q T I P T Y 6 153 K D S P Q T I P T Y T D A L H 6 160 P T Y T D A L H V Y S T V E G 6 174 G Y I A Y R H D Q K G S C F I 6 197 K R K G H I L E L L T E V T R 6 205 L L T E V T R R M A E A E L V 6 207 T E V T R R M A E A E L V Q E 6 210 T R R M A E A E L V Q E G K A 6 211 R R M A E A E L V Q E G K A R 6 222 G K A R K T N P E I Q S T L R 6 138 D P G E T V G G D E I V M V I 4 187 F I Q T L V D V F T K R K G H 4 44 R Q L R F E S T M K R D P T A 3 119 A L R A K P K V Y I T Q A C R 3 167 H V Y S T V E G Y I A Y R H D 3 34 E D L D A L E H M F R Q L R F 2 37 D A L E H M F R Q L R F E S T 2 53 K R D P T A E Q F Q E E L E K 2 57 T A E Q F Q E E L E K F Q Q A 2 72 I D S R E D P V S C A F V V L 2 74 S R E D P V S C A F V V L M A 2 98 E D G E M V K L E N L F E A L 2 127 Y I I Q A C R G E Q R D P G E 2 136 Q R D P G E T V G G D E I V M 2 157 Q T I P T Y T D A L H V Y S T 2 161 T Y T D A L H V Y S T V E G Y 2 201 H I L E L L T E V T R R M A E 2 223 K A R K T N P E I Q S T L R K 2 226 K T N P E I Q S T L R K R L Y 2 227 T N P E I Q S T L R K R L Y L 2 12 Y D M S G A R L A L I L C V T 1 26 T K A R E G S E E D L D A L E 1 29 R E G S E E D L D A L E H M F 1 35 D L D A L E H M F R Q L R F E 1 38 A L E H M F R Q L R F E S T M 1 55 D P T A E Q F Q E E L E K F Q 1 64 E L E K F Q Q A I D S R E D P 1 75 R E D P V S C A F V V L M A H 1 85 V L M A H G R E G F L K G E D 1 90 G R E G F L K G E D G E M V K 1 93 G F L K G E D G E M V K L E N 1 94 F L K G E D C E M V K L E N L 1 95 L K G E D G E M V K L E N L F 1 110 E A L N N K N C Q A L R A K P 1 112 L N N K N C Q A L R A K P K V 1 116 N C Q A L R A K P K V Y I I Q 1 118 Q A L R A K P K V Y I I Q A C 1 121 R A K P K V Y I I Q A C R G E 1 139 P G E T V G G D E I V M V I K 1 TABLE XIXC, part 2: MHC Class II 15-mer analysis of 213P1F11 v.2 (aa 1-230). 213P1F11 v.2: HLA-DRB1*0101 15-mers 6 L H V Y S T V E G P T P F Q D 33 55 P W W M C S R R G K D I S W N 25 32 P P L W N S Q D T S P T D M I 23 18 F Q D P L Y L P S E A P P N P 22 21 P L Y L P S E A P P N P P L W 22 13 E G P T P F Q D P L Y L P S E 21 43 T D M I R K A H A L S R P W W 20 20 D P L Y L P S E A P P N P P L 19 41 S P T D M I R K A H A L S R P 19 15 P T P F Q D P L Y L P S E A P 18 7 H V Y S T V E G P T P F Q D P 17 19 Q D P L Y L P S E A P P N P P 17 31 N P P L W N S Q D T S P T D M 17 42 P T D M I R K A H A L S R P W 16 49 A H A L S R P W W M C S R R G 16 17 P F Q D P L Y L P S E A P P N 15 34 L W N S Q D T S P T D M I R K 15 40 T S P T D M I R K A H A L S R 15 47 R K A H A L S R P W W M C S R 15 3 T D A L H V Y S T V E G P T P 14 9 Y S T V E G P T P F Q D P L Y 14 22 L Y L P S E A P P N P P L W N 14 35 W N S Q D T S P T D M I R K A 14 53 S R P W W M C S R R G K D I S 14 2 Y T D A L H V Y S T V E G P T 13 52 L S R P W W M C S R R G K D I 13 4 D A L H V Y S T V E G P T P F 11 54 R P W W M C S R R G K D I S W 11 38 Q D T S P T D M I R K A H A L 10 1 T Y T D A L H V Y S T V E G P 9 44 D M I R K A H A L S R P W W M 9 56 W W M C S R R G K D I S W N F 9 5 A L H V Y S T V E G P T P F Q 8 10 S T V E G P T P F Q D P L Y L 8 11 T V E G P T P F Q D P L Y L P 8 14 G P T P F Q D P L Y L P S E A 8 16 T P F Q D P L Y L P S E A P P 8 23 Y L P S E A P P N P P L wN S 8 26 S E A P P N P P L W N S Q D T 8 28 A P P N P P L W N S Q D T S P 8 30 P N P P L W N S Q D T S P T D 8 39 D T S P T D M I R K A H A L S 8 46 I R K A H A L S R P W W M C S 8 8 V Y S T V E G P T P F Q D P L 7 29 P P N P P L W N S Q D T S P T 7 45 M I R K A H A L S R P W W M C 7 24 L P S E A P P N P P L W N S Q 6 25 P S E A P P N P P L W N S Q D 6 33 P L W N S Q D T S P T D M I R 6 50 H A L S R P W W M C S R R G K 2 12 V E G P T P F Q D P L Y L P S 1 27 E A P P N P P L W N S Q D T S 1 36 N S Q D T S P T D M I R K A H 1 213P1F11 v.2: HLA-DRB1*0301 (DR17) 15-mers 19 Q D P L Y L P S E A P P N P P 19 5 A L H V Y S T V E G P T P F Q 17 23 Y L P S E A P P N P P L W N S 15 11 T V E G P T P F Q D P L Y L P 14 42 P T D H I R K A H A L S R P W 13 3 T D A L H V Y S T V E G P T P 12 14 G P T P F Q D P L Y L P S E A 12 21 P L Y L P S E A P P N P P L W 12 43 T D M I R K A H A L S R P W W 12 12 V E G P T P F Q D P L Y L P S 11 38 Q D T S P T D M I R K A H A L 11 55 P W W M C S R R G K D I S W N 11 7 H V Y S T V E G P T P F Q D P 10 9 Y S T V E G P T P F Q D P L Y 10 15 P T P F Q D P L Y L P S E A P 10 31 N P P L W N S Q D T S P T D M 10 33 P L W N S Q D T S P T D M I R 10 41 S P T D M I R K A H A L S R P 10 49 A H A L S R P W W M C S R R G 10 13 E G P T P F Q D P L Y L P S E 9 30 P N P P L W N S Q D T S P T D 9 39 D T S P T D M I R K A H A L S 8 40 T S P T D M I R K A H A L S R 8 53 S R P W W M C S R R G K D I S 8 28 A P P N P P L W N S Q D T S P 7 46 I R K A H A L S R P W W M C S 7 54 R P W W M C S R R G K D I S W 7 56 W W M C S R R G K D I S W N F 7 18 F Q D P L Y L P S E A P P N P 4 20 D P L Y L P S E A P P N P P L 4 48 K A H A L S R P W W M C S R R 4 8 V Y S T V E G P T P F Q D P L 3 10 S T V E G P T P F Q D P L Y L 3 22 L Y L P S E A P P N P P L W N 3 25 P S E A P P N P P L W N S Q D 3 27 E A P P N P P L W N S Q D T S 3 2 Y T D A L H V Y S T V E G P T 2 17 P F Q D P L Y L P S E A P P N 2 24 L P S E A P P N P P L W N S Q 2 26 S E A P P N P P L W N S Q D T 2 29 P P N P P L W N S Q D T S P T 2 36 N S Q D T S P T D M I R K A H 2 50 H A L S R P W W M C S R R G K 2 1 T Y T D A L H V Y S T V E G P I 4 D A L H V Y S T V E G P T P F 1 6 L H V Y S T V E G P T P F Q D I 16 T P F Q D P L Y L P S E A P P I 32 P P L W N S Q D T S P T D M I 1 34 L W U S Q D T S P T D M I R K 1 35 W N S Q D T S P T D M I R K A 1 37 S Q D T S P T D M I R K A H A 1 44 D M I R K A H A L S R P W W M 1 45 M I R K A H A L S R P W W M C I 47 R K A H A L S R P W W M C S R 1 213P1F11 v.2: HLA-DRB1*0401 (DR4Dw4) 15-mers 32 P P L W N S Q D T S P T D M I 22 53 S R P W W M C S R R G K D I S 22 6 L H V Y S T V E G P T P F Q D 16 15 P T P F Q D P L Y L P S E A P 16 20 D P L Y L P S E A P P N P P L 16 54 R P W W M C S R R G K D I S W 16 42 P T D M I R K A H A L S R P W 15 3 T D A L H V Y S T V E G P T P 14 5 A L H V Y S T V E G P T P F Q 14 21 P L Y L P S E A P P N P P L W 14 43 T D M I R K A H A L S R P W W 14 49 A H A L S R P W W M C S R R G 14 2 Y T D A L H V Y S T V E G P T 12 8 V Y S T V E G P T P F Q D P L 12 11 T V E G P T P F Q D P L Y L P 12 18 F Q D P L Y L P S E A P P N P 12 23 Y L P S E A P P N P P L W N S 12 28 A P P N P P L W N S Q D T S P 12 29 P P N P P L W N S Q D T S P T 12 30 P N P P L W N S Q D T S P T D 12 33 P L W N S Q D T S P T D M T R 12 35 W N S Q D T S P T D M I R K A 12 38 Q D T S P T D M I R K A H A L 12 40 T S P T D M I R K A H A L S R 12 45 M T R K A H A L S R P W W M C 12 46 I R K A H A L S R P W W M C S 12 55 P W W M C S R R G K D I S W N 9 9 Y S T V E G P T P F Q D P L Y 8 19 Q D P L Y L P S E A P P N P P 8 31 N P P L W N S Q D T S P T D M 8 1 T Y T D A L H V Y S T V E G P 6 7 H V Y S T V E G P T P F Q D P 6 12 V E G P T P F Q D P L Y L P S 6 13 E G P T P F Q D P L Y L P S E 6 14 G P T P F Q D P L Y L P S E A 6 16 T P F Q D P L Y L P S E A P P 6 17 P F Q D P L Y L P S E A P P N 6 22 L Y L P S E A P P N P P L W N 6 25 P S E A P P N P P L W N S Q D 6 26 S E A P P N P P L W N S Q D T 6 27 E A P P N P P L W N S Q D T S 6 34 L W N S Q D T S P T D M T R K 6 37 S Q D T S P T D M I R K A H A 6 39 D T S P T D H I R K A H A L S 6 47 R K A H A L S R P W W M C S R 6 52 L S R P W W M C S R R G K D I 6 41 S P T D M I R K A H A L S R P 1 56 W W M C S R R G K D I S W N F 1 48 K A H A L S R P W W M C S R R −5 213P1F11 v.2: HLA-DRB1*1101 15-mers 6 L H V Y S T V E G P T P F Q D 22 42 P T D M I R K A H A L S R P W 21 40 T S P T D M I R K A H A L S R 20 53 S R P W W M C S R R G K D I S 19 54 R P W W M C S R R G K D I S W 19 15 P T P F Q D P L Y L P S E A P 16 39 D T S P T D M I R K A H A L S 16 52 L S R P W W M C S R R G K D T 15 56 W W M C S R R G K D I S W N F 15 46 I R K A H A L S R P W W M C S 14 3 T D A L H V Y S T V E G P T P 13 18 F Q D P L Y L P S E A P P N P 12 19 Q D P L Y L P S E A P P N P P 12 21 P L Y L P S E A P P N P P L W 12 31 N P P L W N S Q D T S P T D M 12 49 A H A L S R P W W M C S R R G 12 20 D P L Y L P S E A P P N P P L 10 32 P P L W N S Q D T S P T D M I 10 5 A L H V Y S T V E G P T P F Q 8 14 G P T P F Q D P L Y L P S E A 8 38 Q D T S P T D M I R K A H A L 8 44 D M I R K A H A L S R P W W M 8 2 Y T D A L H V Y S T V E G P T 7 4 D A L H V Y S T V E G P T P F 6 9 Y S T V E G P T P F Q D P L Y 6 10 S T V E G P T P F Q D P L Y L 6 16 T P F Q D P L Y L P S E A P P 6 17 P F Q D P L Y L P S E A P P N 6 22 L Y L P S E A P P N P P L W N 6 26 S E A P P N P P L W N S Q D T 6 28 A P P N P P L W N S Q D T S P 6 30 P N P P L W N S Q D T S P T D 6 43 T D M I R K A H A L S R P W W 6 55 P W W M C S R R G K D I S W N 6 7 H V Y S T V E G P T P F Q D P 3 12 V E G P T P F Q D P L Y L P S 3 36 N S Q D T S P T D M I R K A H TABLE XIXC, part 3: MHC Class II 15-mer analysis of 213P1F11 v.3 (aa 1-146). 213P1F11 v.3: HLA-DRB1*0101 15-mers 4 P K V Y T I Q A C R G A T L P 30 3 K P K V Y I I Q A C R G A T L 25 6 V Y I I Q A C R G A T L P S P 24 11 A C R G A T L P S P F P Y L S 22 7 Y T I Q A C R G A T L P S P F 18 8 I I Q A C R G A T L P S P F P 14 10 Q A C R G A T L P S P F P Y L 14 9 I Q A C R G A T L P S P F P Y 10 5 K V Y I I Q A C R G A T L P S 8 12 C R G A T L P S P F P Y L S L 8 2 A K P K V Y I I Q A C R G A T 7 1 R A K P K V Y I I Q A C R G A 1 213P1F11 v.3: HLA-DRB1*0301 (DR17) 15-mers 6 V Y I I Q A C R G A T L P S P 19 5 K V Y I I Q A C R G A T L P S 18 12 C R G A T L P S P F P Y L S L 11 3 K P K V Y I I Q A C R G A T L 10 10 Q A C R G A T L P S P F P Y L 9 2 A K P K V Y I I Q A C R G A T 8 1 R A K P K V Y I I Q A C R G A 3 7 Y I I Q A C R G A T L P S P F 2 4 P K V Y I I Q A C R G A T L P 1 8 I I Q A C R G A T L P S P F P 1 9 I Q A C R G A T L P S P F P Y 1 11 A C R G A T L P S P F P Y L S 1 213P1F11 v.3: HLA-DRB1*0401 (DR4Dw4) 15-mers 5 K V Y I I Q A C R G A T L P S 26 4 P K V Y I I Q A C R G A T L P 16 3 K P K V Y I I Q A C R G A T L 14 6 V Y I I Q A C R G A T L P S P 14 2 A K P K V Y I I Q A C R G A T 12 8 I I Q A C R G A T L P S P F P 12 11 A C R G A T L P S P F P Y L S 12 9 I Q A C R G A T L P S P F P Y 6 7 Y I I Q A C R G A T L P S P F -5 213P1F11 v.3: HLA-DRB1*1101 15-mers 3 K P K V Y I T Q A C R G A T L 20 4 P K V Y I I Q A C R G A T L P 17 5 K V Y I I Q A C R G A T L P S 14 11 A C R G A T L P S P F P Y L S 12 7 Y I I Q A C R G A T L P S P F 7 2 A K P K V Y I I Q A C R G A T 6 TABLE XIXC, part 4: MHC Class II 15-mer analysis of 213P1F11 v.4 (aa 1-321). 213P1F11 v.4: HLA-DRB1*0101 15-mers 63 G G G V G D I V G R D L S I S 31 75 S I S F R N S E T S A S E E E 27 4 C Q E Y D K S L S V Q P E K R 26 31 T F R L K E E Q G R A F R G S 26 59 Q E V F G G G V G D I V G R D 26 55 P R E T Q E V F G G G V G D I 24 73 D L S I S F R N S E T S A S E 23 67 G D I V G R D L S I S F R N S 22 10 S L S V Q P E K R T G L R D E 19 18 R T G L R D E N G E C G Q T F 18 36 E E Q G R A F R G S S V H Q K 18 64 G G V G D I V G R D L S I S F 18 21 L R D E N G E C G Q T F R L K 17 58 T Q E V F G G G V G D I V G R 17 66 V G D I V G R D L S I S F R N 17 29 G Q T F R L K E E Q G R A F R 16 44 G S S V H Q K L V N D P R E T 16 8 D K S L S V Q P E K R T G L R 15 41 A F R G S S V H Q K L V N D P 15 50 K L V N D P R E T Q E V F G G 15 70 V G R D L S I S F R N S E T S 15 2 G K C Q E Y D K S L S V Q P E 14 23 D E N G E C G Q T F R L K E E 14 7 Y D K S L S V Q P E K R T G L 13 57 E T Q E V F G G G V G D I V G 13 39 G R A F R G S S V H Q K L V N 12 85 A S E E E K Y D M S G A R L A 12 30 Q T F R L K E E Q G R A F R G 11 34 L K E E Q G R A F R G S S V H 11 71 G R D L S I S F R N S E T S A 11 12 S V Q P E K R T G L R D E N G 10 45 S S V H Q K L V N D P R E T Q 10 49 Q K L V N D P R E T Q E V F G 10 68 D T V G R D L S I S F R N S E 10 5 Q E Y D K S L S V Q P E K R T 9 15 P E K R T G L R D E N G E C G 9 40 R A F R G S S V H Q K L V N D 9 48 H Q K L V N D P R E T Q E V F 9 53 N D P R E T Q E V F G G G V G 9 56 R E T Q E V F G G G V G D I V 9 74 L S I S F R N S E T S A S E E 9 82 E T S A S E E E K Y D M S G A 9 1 M G K C Q E Y D K S L S V Q P 8 28 C G Q T F R L K E E Q G R A F 8 33 R L K E E Q G R A F R G S S V 8 35 K E E Q G R A F R G S S V H Q 8 37 E Q G R A F R G S S V H Q K L 8 46 S V H Q K L V N D P R E T Q E 8 51 L V N D P R E T Q E V F G G G 8 65 G V G D I V G R D L S I S F R 8 77 S F R N S E T S A S E E E K Y 8 83 T S A S E E E K Y D M S G A R 8 24 E N G E C G Q T F R L K E E Q 7 32 F R L K E E Q G R A F R G S S 7 43 R G S S V H Q K L V N D P R E 7 47 V H Q K L V N D P R E T Q E V 7 60 E V F G G G V G D I V G R D L 7 69 I V G R D L S I S F R N S E T 7 76 I S F R N S E T S A S E E E K 7 86 S E E E K Y D M S G A R L A L 7 11 L S V Q P E K R T G L R D E N 6 38 Q G R A F R G S S V H Q K L v 6 78 F R N S E T S A S E E E K Y D 6 9 K S L S V Q P E K R T G L R D 3 13 V Q P E K R T G L R D E N G E 3 16 E K R T G L R D E N G E C G Q 3 19 T G L R D E N G E C G Q T F R 3 54 D P R E T Q E V F G G G V G D 3 14 Q P E K R T G L R D E N G E C 2 22 R D E N G E C G Q T F R L K E 2 27 E C G Q T F R L K E E Q G R A 2 52 V N D P R E T Q E V F G G G v 2 61 V F G G G V G D I V G R D L S 2 80 N S E T S A S E E E K Y D M S 2 81 S E T S A S E E E K Y D M S G 2 6 E Y D K S L S V Q P E K R T G 1 17 K R T G L R D E N G E C G Q T 1 20 G L R D E N G E C G Q T F R L 1 25 N G E C G Q T F R L K E E Q G 1 26 G E C G Q T F R L K E E Q G R 1 62 F G G G V G D I V G R D L S I 1 79 R N S E T S A S E E E K Y D M 1 213P1F11 v.4: HLA-DRB1*0301 (DR17) 15-mers 67 G D I V G R D L S I S F R N S 29 48 H Q K L V N D P R E T Q E V F 28 10 S L S V Q P E K R T G L R D E 26 31 T F R L K E E Q G R A F R G S 21 17 K R T G L R D E N G E C G Q T 19 29 G Q T F R L K E E Q G R A F R 18 63 G G G V G D I V G R D L S I S 18 8 D K S L S V Q P E K R T G L R 17 71 G R D L S I S F R N S E T S A 17 49 Q K L V N D P R E T Q E V F G 16 80 N S E T S A S E E E K Y D M S 16 51 L V N D P R E T Q E V F G G G 14 2 G K C Q E Y D K S L S V Q P E 12 44 G S S V H Q K L V N D P R E T 12 86 S E E E K Y D M S G A R L A L 12 18 R T G L R D E N G E C G Q T F 11 40 R A F R G S S V H Q K L V N D 11 58 T Q E V F G G G V G D I V G R 11 62 F G G G V G D I V G R D L S I 11 66 V G D I V G R D L S I S F R N 11 75 S I S F R N S E T S A S E E E 11 39 G R A F R G S S V H Q K L V N 10 73 D L S I S F R N S E T S A S E 10 21 L R D E N G E C G Q T F R L K 9 30 Q T F R L K E E Q G R A F R G 9 32 F R L K E E Q G R A F R G S S 9 52 V N D P R E T Q E V F G G G V 9 79 R N S E T S A S E E E K Y D M 9 1 M G K C Q E Y D K S L S V Q P 8 9 K S L S V Q P E K R T G L R D 8 14 Q P E K R T G L R D E N G E C 8 16 E K R T G L R D E N G E C G Q 8 19 T G L R D E N G E C G Q T F R 8 22 R D E N G E C G Q T F R L K E 8 23 D E N G E C G Q T F R L K E E 8 25 N G E C G Q T F R L K E E Q G 8 27 E C G Q T F R L K E E Q G R A 8 41 A F R G S S V H Q K L V N D P 8 59 Q E V F G G G V G D I V G R D 8 64 G G V G D I V G R D L S I S F 8 72 R D L S I S F R N S E T S A S 8 81 S E T S A S E E E K Y D M S G 8 82 E T S A S E E E K Y D M S G A 8 6 E Y D K S L S V Q P E K R T G 7 28 C G Q T F R L K E E Q G R A F 7 42 F R G S S V H Q K L V N D P R 7 74 L S I S F R N S E T S A S E E 7 35 K E E Q G R A F R G S S V H Q 6 45 S S V H Q K L V N D P R E T Q 6 70 V G R D L S I S F R N S E T S 5 5 Q E Y D K S L S V Q P E K R T 4 7 Y D K S L S V Q P E K R T G L 4 43 R G S S V H Q K L V N D P R E 4 50 K L V N D P R E T Q E V F G G 3 57 E T Q E V F G G G V G D I V G 3 4 C Q E Y D K S L S V Q P E K R 2 11 L S V Q P E K R T G L R D E E 2 13 V Q P E K R T G L R D E N G E 2 15 P E K R T G L R D E N G E C G 2 38 Q G R A F R G S S V H Q K L V 2 46 S V H Q K L V N D P R E T Q E 2 47 V H Q K L V N D P R E T Q E V 2 65 G V G D I V G R D L S I S F R 2 68 D I V G R D L S I S F R N S E 2 76 I S F R N S E T S A S E E E K 2 77 S F R N S E T S A S E E E K Y 2 83 T S A S E E E K Y D M S G A R 2 3 K C Q E Y D K S L S V Q P E K 1 12 S V Q P E K R T G L R D E N G 1 20 G L R D E N G E C G Q T F R L 1 24 E N G E C G Q T F R L K E E Q 1 36 E E Q G R A F R G S S V H Q K 1 37 E Q G R A F R G S S V H Q K L 1 53 N D P R E T Q E V F G G G V G 1 54 D P R E T Q E V F G G G V G D 1 55 P R E T Q E V F G G G V G D I 1 56 R E T Q E V F G G G V G D I v 1 60 E V F G G G V G D I V G R D L 1 61 V F G G G V G D I V G R D L S 1 69 I V G R D L S I S F R N S E T 1 84 S A S E E E K Y D M S G A R L 1 213P1F11 v.4: HLA-DRB1*0401 (DR4Dw4) 15-mers 10 S L S V Q P E K R T G L R D E 26 48 H Q K L V N D P R E T Q E V F 26 75 S I S F R N S E T S A S E E E 22 63 G G G V G D I V G R D L S I S 20 67 G D I V G R D L S I S F R N S 20 2 G K C Q E Y D K S L S V Q P E 18 30 Q T F R L K E E Q C R A F R G 18 35 K E E Q G R A F R G S S V H Q 18 41 A F R G S S V H Q K L V N D P 18 64 G G V G D I V G R D L S I S F 18 72 R D L S I S F R N S E T S A S 18 4 C Q E Y D K S L S V Q P E K R 16 39 G R A F R G S S V H Q K L V N 16 59 Q E V F G G G V G D I V G R D 16 73 D L S I S F R N S E T S A S E 15 8 D K S L S V Q P E K R T G L R 14 18 R T G L R D E N G E C G Q T F 14 31 T F R L K E E Q G R A F R G S 14 58 T Q E V F G G G V G D I V G R 14 71 G R D L S I S F R N S E T S A 14 6 E Y D K S L S V Q P E K R T G 12 14 Q P E K R T G L R D E N G E C 12 17 K R T G L R D E N G E C G Q T 12 23 D E N G E C G Q T F R L K E E 12 36 E E Q G R A F R G S S V H Q K 12 38 Q G R A F R G S S V H Q K L V 12 40 R A F R G S S V H Q K L V N D 12 45 S S V H Q K L V N D P R E T Q 12 51 L V N D P R E T Q E V F G G G 12 55 P R E T Q E V F G G G V G D I 12 69 I V G R D L S I S F R N S E T 12 70 V G R D L S I S F R N S E T S 12 76 I S F R N S E T S A S E E E K 12 79 R N S E T S A S E E E K Y D M 12 82 E T S A S E E E K Y D M S G A 12 83 T S A S E E E K Y D M S G A R 12 86 S E E E K Y D M S G A R L A L 12 66 V G D I V G R D L S I S F R N 9 49 Q K L V N D P R E T Q E V F G 8 50 K L V N D P R E T Q E V F G G 7 1 M G K C Q E Y D K S L S V Q P 6 5 Q E Y D K S L S V Q P E K R T 6 7 Y D K S L S V Q P E K R T G L 6 15 P E K R T G L R D E N G E C G 6 19 T G L R D E N G E C G Q T F R 6 20 G L R D E N G E C G Q T F R L 6 21 L R D E N G E C G Q T F R L K 6 22 R D E N G E C G Q T F R L K E 6 24 E N G E C G Q T F R L K E E Q 6 25 N G E C G Q T F R L K E E Q G 6 26 G E C G Q T F R L K E E Q G R 6 28 C G Q T F R L K E E Q G R A F 6 32 F R L K E E Q G R A F R G S S 6 42 F R G S S V H Q K L V N D P R 6 46 S V H Q K L V N D P R E T Q E 6 47 V H Q K L V N D P R E T Q E V 6 54 D P R E T Q E V F G G G V G D 6 56 R E T Q E V F G G G V G D I V 6 60 E V F G G G V G D I V G R D L 6 62 F G G G V G D I V G R D L S I 6 65 G V G D I V G R D L S T S F R 6 68 D I V G R D L S I S F R N S E 6 74 L S I S F R N S E T S A S E E 6 78 F R N S E T S A S E E E K Y D 6 81 S E T S A S E E E K Y D M S G 6 85 A S E E E K Y D M S G A R L A 6 29 G Q T F R L K E E Q G R A F R 5 44 G S S V H Q K L V N D P R E T 3 3 K C Q E Y D K S L S V Q P E K 1 11 L S V Q P E K R T G L R D E N 1 27 E C G Q T F R L K E E Q G R A 1 34 L K E E Q G R A F R G S S V H 1 37 E Q G R A F R G S S V H Q K L 1 12 S V Q P E K R T G L R D E N G -5 16 E K R T G L R D E N G E C G Q -5 84 S A S E E E K Y D M S G A R L -5 213P1F11 v.4: HLA-DRB1*1101 15-mers 64 G G V G D I V G R D L S 1S F 21 4 C Q E Y D K S L S V Q P E K R 16 75 S I S F R N S E T S A S E E E 16 1 M G K C Q E Y D K S L S V Q P 15 9 K S L S V Q P E K R T G L R D 15 63 G G G V G D I V G R D L S I S 15 10 S L S V Q P E K R T G L R D E 14 15 P E K R T G L R D E N G E C G 14 35 K E E Q G R A F R G S S V H Q 14 48 H Q K L V N D P R E T Q E V F 14 71 G R D L S I S F R N S E T S A 14 28 C G Q T F R L K E E Q G R A F 13 59 Q E V F G G G V G D I V G R D 13 60 E V F G G G V G D I V G R D L 13 66 V G D I V G R D L S I S F R N 13 70 V G R D L S I S F R N S E T S 13 18 R T G L R D E N G E C G Q T F 12 44 G S S V H Q K L V N D P R E T 12 73 D L S I S F R N S E T S A S E 12 40 R A F R G S S V H Q K L V N D 11 27 E C G Q T F R L K E E Q G R A 10 29 G Q T F R L K E E Q G R A F R 10 39 G R A F R G S S V H Q K L V N 10 42 F R G S S V H Q K L V N D P R 10 54 D P R E T Q E V F G G G V G D 10 8 D K S L S V Q P E K R T G L R 9 25 N G E C G Q T F R L K E E Q G 9 14 Q P E K R T G L R D E N G E C 8 30 Q T F R L K E E Q G R A F R G 8 32 F R L K E E Q G R A F R G S S 8 33 R L K E E Q G R A F R G S S V 8 45 S S V H Q K L V N D P R E T Q 8 46 S V H Q K L V N D P R E T Q E 8 56 R E T Q E V F G G G V G D I V 8 82 E T S A S E E E K Y D M S G A 8 7 Y D K S L S V Q P E K R T G L 7 31 T F R L K E E Q G R A F R G S 7 34 L K E E Q G R A F R G S S V H 7 41 A F R G S S V H Q K L V N D P 7 58 T Q E V F G G G V G D I V G R 7 74 L S I S F R N S E T S A S E E 7 5 Q E Y D K S L S V Q P E K R T 6 49 Q K L V N D P R E T Q E V F G 6 52 V N D P R E T Q E V F G G G V 6 53 N D P R E T Q E V F G G G V G 6 55 P R E T Q E V F G G G V G D I 6 67 G D I V G R D L S I S F R N S 6 68 D I V G R D L S I S F R N S E 6 83 T S A S E E E K Y D M S G A R 6 84 S A S E E E K Y D M S G A R L 6 85 A S E E E K Y D M S G A R L A 6 6 E Y D K S L S V Q P E K R T G 2 12 S V Q P E K R T G L R D E N G 2 23 D E N G E C G Q T F R L K E E 2 24 E N G E C G Q T F R L K E E Q 2 47 V H Q K L V N D P R E T Q E V 2 62 TABLE XIXC, part 5: MHC Class II 15-mer analysis of 2131F11 v.5 (aa 1-242) 213P1F11 v.5: HLA-DRB1*0101 15-mers 8 A R L A L I L R V T K A R E G 21 11 A L I L R V T K A R E G S E E 19 2 K Y D M S G A R L A L I L R V 16 3 Y D M S G A R L A L T L R V T 16 9 R L A L I L R V T K A R E G S 16 7 G A R L A L I L R V T K A R E 15 10 L A L I L R V T K A R E G S E 14 5 M S G A R L A L I L R V T K A 11 12 L I L R V T K A R E G S E E D 11 13 I L R V T K A R E C S E E D L 11 14 L R V T K A R E G S E E D L D 10 6 S G A R L A L I L R V T K A R 9 1 E K Y D M S G A R L A L I L R 8 4 D M S G A R L A L I L R V T K 8 15 R V T K A R E G S E E D L D A 7 213P1F11 v.5: HLA-DRB1*0301 (DR17) 15-mers 7 G A R L A L I L R V T K A R E 19 10 L A L I L R V T I C A R E G S E 19 1 E K Y D M S G A R L A L I L R 17 13 I L R V T K A R E G S E E D L 17 9 R L A L I L R V T K A R E G S 13 2 K Y D N S G A R L A L T L R V 12 11 A L I L R V T K A R E G S E E 12 3 Y D M S G A R L A L I L R V T 10 12 L I L R V T K A R E G S E E D 7 5 M S G A R L A L I L R V T K A 4 8 A R L A L I L R V T K A R E G 3 6 S G A R L A L T L R V T K A R 2 14 L R V T K A R E G S E E D L D 2 4 D M S G A R L A L I L R V T K 1 15 R V T K A R E G S E E D L D A 1 213P1F11 v.5: HLA-DRB1*0401 (DR4Dw4) 15-mers 7 G A R L A L I L R V T K A R E 26 2 K Y D M S G A R L A L I L R V 20 10 L A L I L R V T K A R E G S E 14 13 I L R V T K A R E G S E E D L 14 1 E K Y D M S G A R L A L I L R 12 5 M S G A R L A L I L R V T K A 12 6 S G A R L A L I L R V T K A R 12 8 A R L A L I L R V T K A R E G 12 15 R V T K A R E G S E E D L D A 12 9 R L A L I L R V T K A R E G S 9 11 A L I L R V T K A R E G S E E 8 12 L I L R V T K A R E G S E E D 7 4 D M S G A R L A L I L R V T K 6 3 Y D M S G A R L A L I L R V T 1 14 L R V T K A R E G S E E D L D −5 213P1F11 v.5: HLA-DRB1*1101 15-mers 10 L A L I L R V T K A R E G S E 22 7 G A R L A L I L R V T K A R E 20 11 A L I L R V T K A R E G S E E 14 12 L I L R V T K A R E G S E E D 14 8 A R L A L I L R V T K A R E G 13 1 E K Y D M S G A R L A L I L R 8 6 S G A R L A L I L R V T K A R 8 9 R L A L I L R V T K A R E G S 8 13 I L R V T K A R E G S E E D L 8 2 K Y D M S G A R L A L I L R V 7 4 D M S G A R L A L I L R V T K 7 TABLE XIXC, part 6: MHC Class II 15-mer analysis of 2131F11 v.6 (aa 1-242). 213P1F11 v.6: HLA-DRB1*0101 15-mers 2 D G E M V K L E N L F E A M N 20 3 G E M V K L E N L F E A M N N 18 9 E N L F E A M N N K N C Q A L 17 12 F E A M N N K N C Q A L R A K 17 14 A M N N K N C Q A L R A K P K 17 5 M V K L E N L F E A M N N K N 16 6 V K L E N L F E A M N N K N C 11 8 L E N L F E A M N N K N C Q A 9 1 E D G E M V K L E N L F E A M 8 4 E M V K L E N L F E A M N N K 8 11 L F E A M N N K N C Q A L R A 8 15 M N N K N C Q A L R A K P K V 8 13 E A M N N K N C Q A L R A K P 3 7 K L E N L F E A M N N K N C Q 1 10 N L F E A M N N K N C Q A L R 1 213P1F11 v.6: HLA-DRB1*0301 (DR17) 15-mers 12 F E A M N N K N C Q A L R A K 20 2 D G E H V K L E N L F E A M N 19 8 L E N L F E A M N N K N C Q A 19 1 E D G E M V K L E N L F E A M 17 5 M V K L E N L F E A M N N K N 17 9 E N L F E A M N N K N C Q A L 15 3 G E M V K L E N L F E A M N N 12 10 N L F E A M N N K N C Q A L R 8 13 E A M N N K N C Q A L R A K P 8 11 L F E A M N N K N C Q A L R A 7 4 E M V K L E N L F E A M N N K 5 7 K L E N L F E A M N N K N C Q 4 6 V K L E N L F E A M N N K N C 3 14 A M N N K N C Q A L R A K P K 2 213P1F11 v.6: HLA-DRB 1*0401 (DR4Dw4) 15-mers 8 L E N L F E A M N N K N C Q A 26 2 D G E M V K L E N L F E A M N 20 3 G E M V K L E N L F E A M N H 20 5 M V K L E N L F E A M N N K N 20 9 E N L F E A M N N K N C Q A L 16 6 V K L E N L F E A M N N K N C 12 7 K L E N L F E A M N N K N C Q 12 11 L F E A M N N K N C Q A L R A 12 12 F E A M N N K N C Q A L R A K 9 4 E M V K L E N L F E A M N N K 6 10 N L F E A M N H K N C Q A L R 6 13 E A M N N K N C Q A L R A K P 6 14 A M N N K N C Q A L R A K P K 6 15 M N N K N C Q A L R A K P K V 6 1 E D G E M V K L E N L F E A M -5 213P1F11 v.6: HLA-DRB1*1101 15-mers 9 E N L F E A M N N K N C Q A L 16 2 D G E M V K L E N L F E A M N 12 3 G E M V K L E N L F E A M N N 12 5 M V K L E N L F E A M N N K N 12 8 L E N L F E A M N N K N C Q A 8 10 N L F E A M N N K N C Q A L R 8 14 A M N N K N C Q A L R A K P K 8 11 L F E A M N N K N C Q A L R A 6 12 F E A M N N K N C Q A L R A K 6 1 E D G E M V K L E N L F E A M 2 13 E A M N N K N C Q A L R A K P 1
[0772] 25 TABLE XX Frequently Occurring Motifs avrg. % Name identity Description Potential Function zf-C2H2 34% Zinc finger, C2H2 Nucleic acid-binding type protein functions as transcription factor, nuclear location probable cytochrome_b 68% Cytochrome b(N- membrane bound N terminal)/b6/petB oxidase, generate superoxide ig 19% Immunoglobulin domains are one domain hundred amino acids long and include a conserved intradomain disulfide bond. WD40 18% WD domain, G-beta tandem repeats of repeat about 40 residues, each containing a Trp-Asp motif. Function in signal transduction and protein interaction PDZ 23% PDZ domain may function in targeting signaling molecules to sub- membranous sites LRR 28% Leucine Rich Repeat short sequence motifs involved in protein- protein interactions pkinase 23% Protein kinase domain conserved catalytic core common to both serine/threonine and tyrosine protein kinases contain- ing an ATP binding site and a catalytic site PH 16% PH domain pleckstrin homology involved in intracellular signaling or as constituents of the cyto skeleton EGF 34% EGF-like domain 30-40 amino- acid long found in the extracellular domain of membrane- bound proteins or in secreted proteins rvt 49% Reverse transcriptase (RNA-dependent DNA polymerase) ank 25% Ank repeat Cytoplasmic protein, associates integral membrane proteins to the cytoskeleton oxidored_ql 32% NADH- membrane associated. Ubiquinone/plastoquin Involved in proton one (complex I), translocation various chains membrane across the membrane efand 4% EF hand calcium-binding domain, consists of a12 residue loop flanked on both sides by a 12 residue alpha-helical domain rvp 79% Retroviral aspartyl Aspartyl or acid protease proteases, centered on a catalytic aspartyl residue Collagen 42% Collagen triple helix extracellular repeat (20 copies) structural proteins involved in formation of connective tissue. The sequence consists of the G-X-Y and the polypeptide chains forms a triple helix. fn3 20% Fibronectin type III Located in the extra- domain cellular ligand-binding region of receptors and is about 200 amino acid residues long with two pairs of cysteines involved in disulfide bonds 7tm_1 19% 7 transmembrane seven hydro- receptor (rhodopsin phobic transmembrane family) regions, with the N-terminus located extracellularly while the C-terminus is cytoplasmic. Signal through G proteins
[0773] 26 TABLE XXI Motifs and Post-translational Modifications of 213P1F11 A) Post-translational Modifications of 213P1F11-v.1 Tyrosine sulfation site. 5-19 rsleeekYdmsgarl Protein kinase C phosphorylation site 51-53 TmK 196-198 TkR 210-212 TrR. 234-236 TlR Casein kinase II phosphorylation site 6-9 SleE 32-35 SeeD 74-77 SreD 161-164 TytD 170-173 StvE 190-193 TlvD 227-230 TnpE Tyrosine kinase phosphorylation site 5-12 RsleEek.Y N-myristoylation site 16-21 GArlAL Bipartite nuclear targeting sequence 211-227 RRmaeaelvqegkarkt B) Post-translational Modifications of 213P1F11-v.2 Tyrosine sulfation site. 5-19 rsleeekYdmsgarl Protein kinase C phosphorylation site. 51-53 TmK 220-222 SrR Casein kinase II phosphorylation site. 6-9 SleE 32-35 SeeD 74-77 SreD 161-164 TytD 170-173 StvE 201-204 SptD Tyrosine kinase phosphorylation site 5-12 RsleEek.Y N-myristoylation site 16-21 GArlAL
[0774] 27 TABLE XXII Protein Properties of 213P1F11 Bioinformatic Program URL Outcome 213P1F11-v.1 ORF ORF finder 728 Protein length 242 Transmembrane TM Pred http://www.ch.embnet.orgl No TM region HMMTop http://www.enzim.hu/hmmtop/ No TM Sosui http://www.genome.ad.jp/SOSui No TM TMHMM http://www.cbs.dtu.dk/services/TMHMM TMP = 0 Signal Peptide Signal P http://www.cbs.dtu.dk/services/SignalP Non-Secretory Protein pI pI/MW tool http://www.expasy.ch/tools/ pI: 5.44 Molecular weight pI/MW tool http://www.expasy.ch/tools/ 28.0 kDa Localization PSORT http://psort.nibb.ac.jp/ endoplasmic reticulum 55.0% lysosome 19% PSORT II http://psort.nibb.ac.jp/ 56.5%: cytoplasmic 21.7%: nuclear Motifs Pfam http://www.sanger.ac.uk/Pfam/ Interleukin-1 converting enzyme (ICE) Prints http://www.biochem.ucl.ac.uk/ Interleukin-1B converting enzyme Blocks http://www.blocks.fhcrc.org/ ICE-like protease (caspase) p20 dom >213P1F11-v.2 ORF ORF finder 692 Protein length 230 Transmembrane TM Pred http://www.ch.embnet.org/ No TM region HMMTop http://www.enzim.hu/hmmtop/ No TM Sosui http://www.genome.ad.jp/SOSui/ Soluble Protein. 0 TMHMM http://www.cbs.dtu.dk/services/TMHMM No TM Signal Peptide Signal P http://www.cbs.dtu.dk/services/SignalP/ Non-Secretory Protein pI pI/MW tool http://www.expasy.ch/tools/ 4.94 Molecular weight pI/MW tool http://www.expasy.ch/tools/ 26.5 kDa Localization PSORT http://psort.nibb.ac.jp/ endoplasmic reticulum 55.0% lysosome 19% PSORT II http://psort.nibb.ac.jp/ 39.1%: nuclear 30.4%: mitochondrial Motifs Pfam http://www.sanger.ac.uk/Pfam/ Interleukin-1 converting enzyme (ICE) Prints http://www.biochem.ucl.ac.uk/ Interleukin-1 B converting enzyme Blocks http://www.blocks.fbcrc.org/ ICE-like protease (caspase) p20 dom 213P1F11-v.3 ORF ORF finder 440 Protein length 146 Transmembrane TM Pred http://www.ch.embnet.org/ No TM region HMMTop http://www.enzim.hu/hmmtop/ No TM Sosui http://www.genome.ad.jp/SOSui/ No TM TMHMM http://www.cbs.dtu.dk/dservices/TMHMM No TM Signal Peptide Signal P http://www.cbs.dtu.dk/services/SignalP/ Non-Secretory Protein pI pI/MW tool http://www.expasy.ch/tools/ Theoretical pI: 5.06 Molecular weight pI/MW tool http://www.expasy.ch/tools/ Theoretical MW: 16977.4 Localization PSORT http://psort.nibb.ac.jp/ endoplasmic reticulum 55.0% lysosome 19% PSORT II http://psort.nibb.ac.jp/ 60.9%: cytoplasmic 17.4%: nuclear Motifs Pfam http://www.sanger.ac.uk/Pfam/ Interleukin-1 converting enzyme (ICE) Prints http://www.biochem.ucl.ac.uk/ Interleukin-1B converting enzyme Blocks http://www.blocks.fhcrc.org/ ICE-like protease (caspase) p20 dom 213P1F11-v.4 Protein length 321 Transmembrane TM Pred http://www.ch.embnet.org/ TM = 0 region HMMTop http://www.enzim.hu/hmmtop/ TM = 0 Sosui http://sosui..ac.jp/ Soluble Protein. TM = 0 TMHMM http://www.cbs.dtu.dk/services/TMHMM TMP = Signal Peptide Signal P http://www.cbs.dtu.dk/services/SignalP/ pI pI/MW tool http://www.expasy.ch/tools/ Theoretical pI: 5.42 Molecular weight pI/MW tool http://www.expasy.ch/tools/ Theoretical MW: 36398.04 Localization PSORT http://psort.nibb.ac.jp/ endoplasmic reticulum (membrane) Certainty = 0.760 lysosome (lumen) Certainty = 0.100 microbody (peroxisome) Certainty = 0.300 mitochondrial matrix space Certainty = 0.100 PSORT II http://psort.nibb.ac.jp/ 47.8%: cytoplasmic 39.1%: nuclear 13.0%: vacuolar 8.7%: mitochondrial Motifs Pfam http://www.sanger.ac.uk/Pfam/ Interleukin-1 converting enzyme (ICE) p20 & p10 domain Prints http://www.biochem.ucl.ac.uk/ Interleukin-1B converting enzyme Blocks http://www.blocks.fhcrc.org/ ICE-like protease (caspase) p20 domain
[0775] 28 TABLE XXIIIA Exon compositions of 213P1F11 v.1 Exon Number Start End Exon 1 1 357 Exon 2 358 430 Exon 3 431 580 Exon 4 581 806 Exon 5 807 923 Exon 6 924 1027 Exon 7 1028 3336
[0776] 29 TABLE XXIIIB Exon compositions of 213P1F11 v.4 Exon Number Start End Exon 1 1 119 Exon 2 120 212 Exon 3 213 264 Exon 4 265 414 Exon 5 415 640 Exon 6 641 757 Exon 7 758 861 Exon 8 862 966
[0777] 30 TABLE XXIVA Nucleotide sequence of transcript variant 213P1F11 v.2 ctgactcatt tagactctct gcctaggcca cctttgccag agggagtccc ctcagccttg 60 cgatcactca tcccattggc gttggctcca tttccacacc acagctgtgt gccaagggtg 120 tgtcatgagg tttcttgagt gacagaaaac tcaccgacaa taaagggcca ggtgattgtg 180 ccacccgatt catagaccag gcttctcagg agaaaccccg ggagattcca cactgtcagc 240 cccttctcca agatcagtac gtgggcctga ctcctcctcg gtgcccagct cagtattggc 300 aactaggaga gtagtgagat tgaacttggc cttgaggaac agctgcctct agagttggat 360 cagacaaggg tgctgagagc cgggactcac aaccaaagga gaaatgagca atccgcggtc 420 tttggaagag gagaaatatg atatgtcagg tgcccgcctg gccctaatac tgtgtgtcac 480 caaagcccgg gaaggttccg aagaagacct ggatgctctg gaacacatgt ttcggcagct 540 gagattcgaa agcaccatga aaagagaccc cactgccgag caattccagg aagagctgga 600 aaaattccag caggccatcg attcccggga agatcccgtc agttgtgcct tcgtggtact 660 catggctcac gggagggaag gcttcctcaa gggagaagat ggggagatgg tcaagctgga 720 gaatctcttc gaggccctga acaacaagaa ctgccaggcc ctgcgagcta agcccaaggt 780 gtacatcata caggcctgtc gaggagaaca aagggacccc ggtgaaacag taggtggaga 840 tgagattgtg atggtcatca aagacagccc acaaaccatc ccaacataca cagatgcctt 900 gcacgtttat tccacggtag agggacccac gcccttccag gatcccctct acctaccctc 960 tgaagctccc ccgaacccac ctctctggaa ttcccaggat acatcgccta ccgacatgat 1020 cagaaaggct catgctttat ccagaccctg gtggatgtgt tcacgaagag gaaaggacat 1080 atcttggaac ttctgacaga ggtgacccgg cggatggcag aagcagagct ggttcaagaa 1140 ggaaaagcaa ggaaaacgaa ccctgaaatc caaagcaccc tccggaaacg gctgtatctg 1200 cagtagaagt agaaagacca ggaggagctt tccttccagc attctttctg tctcacagaa 1260 atttagaggc agctcttacc tctccccaag atcttctgtt cccaaggcca aatggcaccc 1320 agtttctttt ccatcacacc cttcatgcag gtcctcctgt ccttattaga gcaagccagc 1380 caaaacttag cacaaggcat ggtggcaaca ttaacatcac ctccctcagg ctggactttc 1440 tatctttatt aatgcaaccg aagagaccta agagtgcatt cacttatccc actttctgtt 1500 cctgtggtct tctttctccc atgaagcaga aactggataa agctcaagat tttccataga 1560 caaaccaaag cccactcatc ccctcctacc ccaatccaac ctctgctggc tcctgcatct 1620 cacttggagg tcaaacctcc tcctgaggcc aatgcattcc caacttccag ttctttcctt 1680 taccctggag agttagtaag gtaagaacca ttctttctct ccaaaaccac tcctccttgg 1740 ctggcaagtt ggtgtcctaa ctccgttctc ttcctagctc atggcctctc tagataataa 1800 agttgtctcc tcctttctgg atctcttcct cctaacaccc ctcccctgaa accctggact 1860 ctgccctctc tccaagaaaa tccatctatt caactattct tgcattcaat tactctaaat 1920 gagagcgtgt tggagctatg gcaaattccc tgttgtcacc ttgctatttt gcagacaaca 1980 taatatttaa cctctcataa ccagagaggt taaataattt gtcaaatgca atacagtaag 2040 acagaggcaa ggacaaggtt tgacttccag cccagcctct tttccacaac ctgctaaatc 2100 ctgatccatc tgaaaacttt tctaattagt gaagatgact aataaaaatt ttccctatct 2160 ccaaggtagg agctttctgg aagtttctag aaattttcaa taaccaccag ccaaggttac 2220 ctccaggtaa ccttgcagca ccaggctgga agtcagatcg gcttcactat cttccaactc 2280 tacagcctgt atctctccat ccccagcttt gacctttcct gctcaagtaa cctacgggca 2340 catccagcgt cactaaaaac tcagggcttt tcttcccggt tactcctcca agcgttccct 2400 ggtatcctca acctcagatc ccaggttcag atttctgcag tcaatctatg acccctctct 2460 tcttgcatcc ttcatatgcc accagacacc atgcccagtc cagcctgatt ttgaaacaac 2520 tttcatgccg gtcttctctt ccctgacatg ttactgtcca ggctcaagtc ctcagcttct 2580 catatctgca tctttgcaac caacttcctc ccttgcctct ctgcttttcc atcccacttt 2640 tcatgtgtcc tccataccat ctataacagt gatctccctg gaacactcaa gaagacacaa 2700 cataccatat tatttaaaga ccagggtact ggacagtggc tcacacctgt attcccgact 2760 ttgagagtct gaagcgggag gatcacttga ggccaggagt taagagacca gcctgggcaa 2820 cacagcaaga ccctgtctct aaaaaaaaaa attaattaac tgggtatggt ggcacatgcc 2880 tgtagtccca gctactcagg aggctgaggt gggaggatga cttgagccca ggagtttgag 2940 gctgcaagga gctatgatca tgccagtgca tcccagctct aggtgagaca gtgagatccg 3000 gtctccaaaa taaatcaatc aatcaaataa agaccaaagt caaaccgcac atcaggatct 3060 ctcacaccct tccaattttg ccatctacca gcacttagct aaacccatct cccatctctt 3120 ccaccatgaa ttcactcttt caaaaaggct aatgtcttct tactcaccct tgcctctaag 3180 cctttgctat caccatttcc cccaagctgg agggccctcc ctctcccttt acccctcttc 3240 cactacctcc cacccctact ttttccagaa agccatttcc tctctttttt ctgattgatc 3300 cttccctctc acccaggatt agatgctgga aatgaccact tctggagggc agggaacaag 3360 cccttaatct gcataatgag tgttcaataa acagttgtca aactttgaaa 3410
[0778] 31 TABLE XXIVB Nucleotide sequence of transcript variant 213P1F11 v.3 ctgactcatt tagactctct gcctaggcca cctttgccag agggagtccc ctcagccttg 60 cgatcactca tcccattggc gttggctcca tttccacacc acagctgtgt gccaagggtg 120 tgtcatgagg tttcttgagt gacagaaaac tcaccgacaa taaagggcca ggtgattgtg 180 ccacccgatt catagaccag gcttctcagg agaaaccccg ggagattcca cactgtcagc 240 cccttctcca agatcagtac gtgggcctga ctcctcctcg gtgcccagct cagtattggc 300 aactaggaga gtagtgagat tgaacttggc cttgaggaac agctgcctct agagttggat 360 cagacaaggg tgctgagagc cgggactcac aaccaaagga gaaatgagca atccgcggtc 420 tttggaagag gagaaatatg atatgtcagg tgcccgcctg gccctaatac tgtgtgtcac 480 caaagcccgg gaaggttccg aagaagacct ggatgctctg gaacacatgt ttcggcagct 540 gagattcgaa agcaccatga aaagagaccc cactgccgag caattccagg aagagctgga 600 aaaattccag caggccatcg attcccggga agatcccgtc agttgtgcct tcgtggtact 660 catggctcac gggagggaag gcttcctcaa gggagaagat ggggagatgg tcaagctgga 720 gaatctcttc gaggccctga acaacaagaa ctgccaggcc ctgcgagcta agcccaaggt 780 gtacatcata caggcctgtc gaggagccac cctgcccagc ccctttcctt acctttctct 840 ctgactttgc ctcctcctct tcttgttgtt tcagaacaaa gggaccccgg tgaaacagta 900 ggtggagatg agattgtgat ggtcatcaaa gacagcccac aaaccatccc aacatacaca 960 gatgccttgc acgtttattc cacggtagag ggatacatcg cctaccgaca tgatcagaaa 1020 ggctcatgct ttatccagac cctggtggat gtgttcacga agaggaaagg acatatcttg 1080 gaacttctga cagaggtgac ccggcggatg gcagaagcag agctggttca agaaggaaaa 1140 gcaaggaaaa cgaaccctga aatccaaagc accctccgga aacggctgta tctgcagtag 1200 aagtagaaag accaggagga gctttccttc cagcattctt tctgtctcac agaaatttag 1260 aggcagctct tacctctccc caagatcttc tgttcccaag gccaaatggc acccagtttc 1320 ttttccatca cacccttcat gcaggtcctc ctgtccttat tagagcaagc cagccaaaac 1380 ttagcacaag gcatggtggc aacattaaca tcacctccct caggctggac tttctatctt 1440 tattaatgca accgaagaga cctaagagtg cattcactta tcccactttc tgttcctgtg 1500 gtcttctttc tcccatgaag cagaaactgg ataaagctca agattttcca tagacaaacc 1560 aaagcccact catcccctcc taccccaatc caacctctgc tggctcctgc atctcacttg 1620 gaggtcaaac ctcctcctga ggccaatgca ttcccaactt ccagttcttt cctttaccct 1680 ggagagttag taaggtaaga accattcttt ctctccaaaa ccactcctcc ttggctggca 1740 agttggtgtc ctaactccgt tctcttccta gctcatggcc tctctagata ataaagttgt 1800 ctcctccttt ctggatctct tcctcctaac acccctcccc tgaaaccctg gactctgccc 1860 tctctccaag aaaatccatc tattcaacta ttcttgcatt caattactct aaatgagagc 1920 gtgttggagc tatggcaaat tccctgttgt caccttgcta ttttgcagac aacataatat 1980 ttaacctctc ataaccagag aggttaaata atttgtcaaa tgcaatacag taagacagag 2040 gcaaggacaa ggtttgactt ccagcccagc ctcttttcca caacctgcta aatcctgatc 2100 catctgaaaa cttttctaat tagtgaagat gactaataaa aattttccct atctccaagg 2160 taggagcttt ctggaagttt ctagaaattt tcaataacca ccagccaagg ttacctccag 2220 gtaaccttgc agcaccaggc tggaagtcag atcggcttca ctatcttcca actctacagc 2280 ctgtatctct ccatccccag ctttgacctt tcctgctcaa gtaacctacg ggcacatcca 2340 gcgtcactaa aaactcaggg cttttcttcc cggttactcc tccaagcgtt ccctggtatc 2400 ctcaacctca gatcccaggt tcagatttct gcagtcaatc tatgacccct ctcttcttgc 2460 atccttcata tgccaccaga caccatgccc agtccagcct gattttgaaa caactttcat 2520 gccggtcttc tcttccctga catgttactg tccaggctca agtcctcagc ttctcatatc 2580 tgcatctttg caaccaactt cctcccttgc ctctctgctt ttccatccca cttttcatgt 2640 gtcctccata ccatctataa cagtgatctc cctggaacac tcaagaagac acaacatacc 2700 atattattta aagaccaggg tactggacag tggctcacac ctgtattccc gactttgaga 2760 gtctgaagcg ggaggatcac ttgaggccag gagttaagag accagcctgg gcaacacagc 2820 aagaccctgt ctctaaaaaa aaaaattaat taactgggta tggtggcaca tgcctgtagt 2880 cccagctact caggaggctg aggtgggagg atgacttgag cccaggagtt tgaggctgca 2940 aggagctatg atcatgccag tgcatcccag ctctaggtga gacagtgaga tccggtctcc 3000 aaaataaatc aatcaatcaa ataaagacca aagtcaaacc gcacatcagg atctctcaca 3060 cccttccaat tttgccatct accagcactt agctaaaccc atctcccatc tcttccacca 3120 tgaattcact ctttcaaaaa ggctaatgtc ttcttactca cccttgcctc taagcctttg 3180 ctatcaccat ttcccccaag ctggagggcc ctccctctcc ctttacccct cttccactac 3240 ctcccacccc tactttttcc agaaagccat ttcctctctt ttttctgatt gatccttccc 3300 tctcacccag gattagatgc tggaaatgac cacttctgga gggcagggaa caagccctta 3360 atctgcataa tgagtgttca ataaacagtt gtcaaacttt gaaa 3404
[0779] 32 TABLE XXIVC Nucleotide sequence of transcript variant 213P1F11 v.4 atggggaaat gccaagagta tgacaaaagt ctgtctgtgc agccagagaa gagaacagga 60 ctcagagatg agaatggaga atgtggacag acattcagac tcaaggaaga gcaagggagg 120 gctttcaggg gaagttcagt ccaccagaag ctggtgaatg acccacggga gacacaggaa 180 gtttttgggg gcggagtggg ggacattgtg ggacgggatc tcagtattag cttcagaaac 240 tctgagacct ctgcaagtga ggaggagaaa tatgatatgt caggtgcccg cctggcccta 300 atactgtgtg tcaccaaagc ccgggaaggt tccgaagaag acctggatgc tctggaacac 360 atgtttcggc agctgagatt cgaaagcacc atgaaaagag accccactgc cgagcaattc 420 caggaagagc tggaaaaatt ccagcaggcc atcgattccc gggaagatcc cgtcagttgt 480 gccttcgtgg tactcatggc tcacgggagg gaaggcttcc tcaagggaga agatggggag 540 atggtcaagc tggagaatct cttcgaggcc ctgaacaaca agaactgcca ggccctgcga 600 gctaagccca aggtgtacat catacaggcc tgtcgaggag aacaaaggga ccccggtgaa 660 acagtaggtg gagatgagat tgtgatggtc atcaaagaca gcccacaaac catcccaaca 720 tacacagatg ccttgcacgt ttattccacg gtagagggat acatcgccta ccgacatgat 780 cagaaaggct catgctttat ccagaccctg gtggatgtgt tcacgaagag gaaaggacat 840 atcttggaac ttctgacaga ggtgacccgg cggatggcag aagcagagct ggttcaagaa 900 ggaaaagcaa ggaaaacgaa ccctgaaatc caaagcaccc tccggaaacg gctgtatctg 960 cagtag 966
[0780] 33 TABLE XXVA Nucleotide sequence alignment of 213P1F11 v.1 and 213P1F11 v.2 213P1F11v.1 CTGACTCATTTAGACTCTCTGCCTAGGCCACCTTTGCCAGAGGGAGTCCCCTCAGCCTTG 60 213P1F11v.2 CTGACTCATTTAGACTCTCTGCCTAGGCCACCTTTGCCAGAGGGAGTCCCCTCAGCCTTG 60 ************************************************************ 213P1F11v.1 CGATCACTCATCCCATTGGCGTTGGCTCCATTTCCACACCACAGCTGTGTGCCAAGGGTG 120 213P1F11v.2 CGATCACTCATCCCATTGCCGTTGGCTCCATTTCCACACCACAGCTGTGTGCCAAGGGTG 120 ************************************************************ 213P1F11v.1 TGTCATGAGGTTTCTTGAGTGACAGAAAACTCACCGACAATAAAGGGCCAGGTGATTGTG 180 213P1F11v.2 TGTCATGAGGTTTCTTGAGTGACAGAAAACTCACCGACAATAAAGGGCCAGGTGATTGTG 180 ************************************************************ 213P1F11v.1 CCACCCGATTCATAGACCAGGCTTCTCAGGAGAAACCCCGGGAGATTCCACACTGTCAGC 240 213P1F11v.2 CCACCCGATTCATAGACCAGGCTTCTCAGGAGAAACCCCGGGAGATTCCACACTGTCAGC 240 ************************************************************ 213P1F11v.1 CCCTTCTCCAAGATCAGTACGTGGGCCTGACTCCTCCTCGGTGCCCAGCTCAGTATTGGC 300 213P1F11v.2 CCCTTCTCCAAGATCAGTACGTGGGCCTGACTCCTCCTCGGTGCCCAGCTCAGTATTGGC 300 ************************************************************ 213P1F11v.1 AACTAGGAGAGTAGTGAGATTGAACTTGGCCTTGAGGAACAGCTGCCTCTAGAGTTGGAT 360 213P1F11v.2 AACTAGGAGAGTAGTGAGATTGAACTTGGCCTTGAGGAACAGCTGCCTCTAGAGTTGGAT 360 ************************************************************ 213P1F11v.1 CAGACAAGGGTGCTGAGAGCCGGGACTCACAACCAAAGGAGAAATGAGCAATCCGCGGTC 420 213P1F11v.2 CAGACAAGGGTGCTGAGAGCCGGGACTCACAACCAAAGGAGAAATGAGCAATCCGCGGTC 420 ************************************************************ 213P1F11v.1 TTTGGAAGAGGAGAAATATGATATGTCAGGTGCCCGCCTGGCCCTAATACTGTGTGTCAC 480 213P1F11v.2 TTTGGAAGAGGAGAAATATGATATGTCAGGTGCCCGCCTGGCCCTAATACTGTGTGTCAC 480 ************************************************************ 213P1F11v.1 CAAAGCCCGGGAAGGTTCCGAAGAAGACCTGGATGCTCTGGAACACATGTTTCGGCAGCT 540 213P1F11v.2 CAAAGCCCGGGAAGGTTCCGAAGAAGACCTGGATGCTCTGGAACACATGTTTCGGCAGCT 540 ************************************************************ 213P1F11v.1 GAGATTCGAAAGCACCATGAAAAGAGACCCCACTGCCGAGCAATTCCAGGAAGAGCTGGA 600 213P1F11v.2 GAGATTCGAAAGCACCATGAAAAGAGACCCCACTGCCGAGCAATTCCAGGAAGAGCTGGA 600 ************************************************************ 213P1F11v.1 AAAATTCCAGCAGGCCATCGATTCCCGGGAAGATCCCGTCAGTTGTGCCTTCGTGGTACT 660 213P1F11v.2 AAAATTCCAGCAGGCCATCGATTCCCGGGAAGATCCCGTCAGTTGTGCCTTCGTGGTACT 660 ************************************************************ 213P1F11v.1 CATGGCTCACGGGAGGGAAGGCTTCCTCAAGGGAGAAGATGGGGAGATGGTCAAGCTGGA 720 213P1F11v.2 CATGGCTCACGGGAGGGAAGGCTTCCTCAAGGGAGAAGATGGGGAGATGGTCAAGCTGGA 720 ************************************************************ 213P1F11v.1 GAATCTCTTCGAGGCCCTGAACAACAAGAACTGCCAGGCCCTGCGAGCTAAGCCCAAGGT 780 213P1F11v.2 GAATCTCTTCGAGGCCCTGAACAACAAGAACTGCCAGGCCCTGCGAGCTAAGCCCAAGGT 780 ************************************************************ 213P1F11v.1 GTACATCATACAGGCCTGTCGAGGAGAACAAAGGGACCCCGGTGAAACAGTAGGTGGAGA 840 213P1F11v.2 GTACATCATACAGGCCTGTCGAGGAGAACAAAGGGACCCCGGTGAAACAGTAGGTGGAGA 840 ************************************************************ 213P1F11v.1 TGAGATTGTGATGGTCATCAAAGACAGCCCACAAACCATCCCAACATACACAGATGCCTT 900 213P1F11v.2 TGAGATTGTGATGGTCATCAAAGACAGCCCACAAACCATCCCAACATACACAGATGCCTT 900 ************************************************************ 213P1F11v.1 GCACGTTTATTCCACGGTAGAGGGA----------------------------------- 925 213P1F11v.2 GCACGTTTATTCCACGGTAGAGGGACCCACGCCCTTCCAGGATCCCCTCTACCTACCCTC 960 ************************* 213P1F11v.1 ---------------------------------------TACATCGCCTACCGACATGAT 946 213P1F11v.2 TGAAGCTCCCCCGAACCCACCTCTCTGGAATTCCCAGGATACATCGCCTACCGACATGAT 1020 ********************* 213P1F11v.1 CAGAAAGGCTCATGCTTTATCCAGACCCTGGTGGATGTGTTCACGAAGAGGAAAGGACAT 1006 213P1F11v.2 CAGAAAGGCTCATGCTTTATCCAGACCCTGGTGGATGTGTTCACGAAGAGGAAAGGACAT 1080 ************************************************************ 213P1F11v.1 ATCTTGGAACTTCTGACAGAGGTGACCCGGCGGATGGCAGAAGCAGAGCTGGTTCAAGAA 1066 213P1F11v.2 ATCTTGGAACTTCTGACAGAGGTGACCCGGCGGATGGCAGAAGCAGAGCTGGTTCAAGAA 1140 ************************************************************ 213P1F11v.1 GGAAAAGCAAGGAAAACGAACCCTGAAATCCAAAGCACCCTCCGGAAACGGCTGTATCTG 1126 213P1F11v.2 GGAAAAGCAAGGAAAACGAACCCTGAAATCCAAAGCACCCTCCGGAAACGGCTGTATCTG 1200 ************************************************************ 213P1F11v.1 CAGTAGAAGTAGAAAGACCAGGAGGAGCTTTCCTTCCAGCATTCTTTCTGTCTCACAGAA 1186 213P1F11v.2 CAGTAGAAGTAGAAAGACCAGGAGGAGCTTTCCTTCCAGCATTCTTTCTGTCTCACAGAA 1260 ************************************************************ 213P1F11v.1 ATTTAGAGGCAGCTCTTACCTCTCCCCAAGATCTTCTGTTCCCAAGGCCAAATGGCACCC 1246 213P1F11v.2 ATTTAGAGGCAGCTCTTACCTCTCCCCAAGATCTTCTGTTCCCAAGGCCAAATGGCACCC 1320 ************************************************************ 213P1F11v.1 AGTTTCTTTTCCATCACACCCTTCATGCAGGTCCTCCTGTCCTTATTAGAGCAAGCCAGC 1366 213P1F11v.2 AGTTTCTTTTCCATCACACCCTTCATGCAGGTCCTCCTGTCCTTATTAGAGCAAGCCAGC 1380 ************************************************************ 213P1F11v.1 CAAAACTTAGCACAAGGCATGGTGGCAACATTAACATCACCTCCCTCAGGCTGGACTTTC 1366 213P1F11v.2 CAAAACTTAGCACAAGGCATGGTGGCAACATTAACATCACCTCCCTCAGGCTGGACTTTC 1440 ************************************************************ 213P1F11v.1 TATCTTTATTAATGCAACCGAAGAGACCTAAGAGTGCATTCACTTATCCCACTTTCTGTT 1426 213P1F11v.2 TATCTTTATTAATGCAACCGAAGAGACCTAAGAGTGCATTCACTTATCCCACTTTCTGTT 1500 ************************************************************ 213P1F11v.1 CCTGTGGTCTTCTTTCTCCCATGAAGCAGAAACTGGATAAAGCTCAAGATTTTCCATAGA 1486 213P1F11v.2 CCTGTGGTCTTCTTTCTCCCATGAAGCAGAAACTGGATAAAGCTCAAGATTTTCCATAGA 1560 ************************************************************ 213P1F11v.1 CAAACCAAAGCCCACTCATCCCCTCCTACCCCAATCCAACCTCTGCTGGCTCCTGCATCT 1546 213P1F11v.2 CAAACCAAAGCCCACTCATCCCCTCCTACCCCAATCCAACCTCTGCTGGCTCCTGCATCT 1620 ************************************************************ 213P1F11v.1 CACTTGGAGGTCAAACCTCCTCCTGAGGCCAATGCATTCCCAACTTCCAGTTCTTTCCTT 1606 213P1F11v.2 CACTTGGAGGTCAAACCTCCTCCTGAGGCCAATGCATTCCCAACTTCCAGTTCTTTCCTT 1680 ************************************************************ 213P1F11v.1 TACCCTGGAGAGTTAGTAAGGTAAGAACCATTCTTTCTCTCCAAAACCACTCCTCCTTGG 1666 213P1F11v.2 TACCCTGGAGAGTTAGTAAGGTAAGAACCATTCTTTCTCTCCAAAACCACTCCTCCTTGG 1740 ************************************************************ 213P1F11v.1 CTGGCAAGTTGGTGTCCTAACTCCGTTCTCTTCCTAGCTCATGGCCTCTCTAGATAATAA 1726 213P1F11v.2 CTGGCAAGTTGGTGTCCTAACTCCGTTCTCTTCCTAGCTCATGGCCTCTCTAGATAATAA 1800 ************************************************************ 213P1F11v.1 AGTTGTCTCCTCCTTTCTGGATCTCTTCCTCCTAACACCCCTCCCCTGAAACCCTGGACT 1786 213P1F11v.2 AGTTGTCTCCTCCTTTCTGGATCTCTTCCTCCTAACACCCCTCCCCTGAAACCCTGGACT 1860 ************************************************************ 213P1F11v.1 CTGCCCTCTCTCCAAGAAAATCCATCTATTCAACTATTCTTGCATTCAATTACTCTAAAT 1846 213P1F11v.2 CTGCCCTCTCTCCAAGAAAATCCATCTATTCAACTATTCTTGCATTCAATTACTCTAAAT 1920 ************************************************************ 213P1F11v.1 GAGAGCGTGTTGGAGCTATGGCAAATTCCCTGTTGTCACCTTGCTATTTTGCAGACAACA 1906 213P1F11v.2 GAGAGCGTGTTGGAGCTATGGCAAATTCCCTGTTGTCACCTTGCTATTTTGCAGACAACA 1980 ************************************************************ 213P1F11v.1 TAATATTTAACCTCTCATAACCAGAGAGGTTAAATAATTTGTCAAATGCAATACAGTAAG 1966 213p1F11v.2 TAATATTTAACCTCTCATAACCAGAGAGGTTAAATAATTTGTCAAATGCAATACAGTAAG 2040 ************************************************************ 213P1F11v.1 ACAGAGGCAAGGACAAGGTTTGACTTCCAGCCCAGCCTCTTTTCCACAACCTGCTAAATC 2026 213P1F11v.2 ACAGAGGCAAGGACAAGGTTTGACTTCCAGCCCAGCCTCTTTTCCACAACCTGCTAAATC 2100 ************************************************************ 213P1F11v.1 CTGATCCATCTGAAAACTTTTCTAATTAGTGAAGATGACTAATAAAAATTTTCCCTATCT 2086 213P1F11v.2 CTGATCCATCTGAAAACTTTTCTAATTAGTGAAGATGACTAATAAAAATTTTCCCTATCT 2160 ************************************************************ 213P1F11v.1 CCAAGGTAGGAGCTTTCTGGAAGTTTCTAGAAATTTTCAATAACCACCAGCCAAGGTTAC 2146 213P1F11v.2 CCAAGGTAGGAGCTTTCTGGAAGTTTCTAGAAATTTTCAATAACCACCAGCCAAGGTTAC 2220 ************************************************************ 213P1F11v.1 CTCCAGGTAACCTTGCAGCACCAGGCTGGAAGTCAGATCGGCTTCACTATCTTCCAACTC 2206 213P1F11v.2 CTCCAGGTAACCTTGCAGCACCAGGCTGGAAGTCAGATCGGCTTCACTATCTTCCAACTC 2280 ************************************************************ 213P1F11v.1 TACAGCCTGTATCTCTCCATCCCCAGCTTTGACCTTTCCTGCTCAAGTAACCTACGGGCA 2266 213P1F11v.2 TACAGCCTGTATCTCTCCATCCCCAGCTTTGACCTTTCCTGCTCAAGTAACCTACGGGCA 2340 ************************************************************ 213P1F11v.1 CATCCAGCGTCACTAAAAACTCAGGGCTTTTCTTCCCGGTTACTCCTCCAAGCGTTCCCT 2326 213P1F11v.2 CATCCAGCGTCACTAAAAACTCAGGGCTTTTCTTCCCGGTTACTCCTCCAAGCGTTCCCT 2400 ************************************************************ 213P1F11v.1 GGTATCCTCAACCTCAGATCCCAGGTTCAGATTTCTGCAGTCAATCTATGACCCCTCTCT 2386 213P1F11v.2 GGTATCCTCAACCTCAGATCCCAGGTTCACATTTCTGCAGTCAATCTATGACCCCTCTCT 2460 ************************************************************ 213P1F11v.1 TCTTGCATCCTTCATATGCCACCAGACACCATGCCCAGTCCAGCCTGATTTTGAAACAAC 2446 213P1F11v.2 TCTTGCATCCTTCATATGCCACCAGACACCATGCCCAGTCCAGCCTGATTTTGAAACAAC 2520 ************************************************************ 213P1F11v.1 TTTCATGCCGGTCTTCTCTTCCCTGACATGTTACTGTCCAGGCTCAAGTCCTCAGCTTCT 2506 213P1F11v.2 TTTCATGCCGGTCTTCTCTTCCCTGACATGTTACTGTCCAGGCTCAAGTCCTCAGCTTCT 2580 ************************************************************ 213P1F11v.1 CATATCTGCATCTTTGCAACCAACTTCCTCCCTTGCCTCTCTGCTTTTCCATCCCACTTT 2566 213P1F11v.2 CATATCTGCATCTTTGCAACCAACTTCCTCCCTTGCCTCTCTGCTTTTCCATCCCACTTT 2640 ************************************************************ 213P1F11v.1 TCATGTGTCCTCCATACCATCTATAACAGTGATCTCCCTGGAACACTCAAGAAGACACAA 2626 213P1F11v.2 TCATGTGTCCTCCATACCATCTATAACAGTGATCTCCCTGGAACACTCAAGAAGACACAA 2700 ************************************************************ ** 213P1F11v.1 CATACCATATTATTTAAAGACCAGGGTACTGGACAGTGGCTCACACCTGTATTCCCGACT 2686 213P1F11v.2 CATACCATATTATTTAAAGACCAGGGTACTGGACAGTGGCTCACACCTGTATTCCCGACT 2760 ************************************************************ 213P1F11v.1 TTGAGAGTCTGAAGCGGGAGGATCACTTGAGGCCAGGAGTTAAGAGACCAGCCTGGGCAA 2746 213P1F11v.2 TTGAGAGTCTGAAGCGGGAGGATCACTTGAGGCCAGGAGTTAAGAGACCAGCCTGGGCAA 2820 ************************************************************ 213P1F11v.1 CACAGCAAGACCCTGTCTCTAAAAAAAAAAATTAATTAACTGGGTATGGTGGCACATGCC 2806 213P1F11v.2 CACAGCAAGACCCTGTCTCTAAAAAAAAAAATTAATTAACTGGGTATGGTGGCACATGCC 2880 ************************************************************ 213P1F11v.1 TGTAGTCCCAGCTACTCAGGAGGCTGAGGTGGGAGGATGACTTGAGCCCAGGAGTTTGAG 2866 213P1F11v.2 TGTAGTCCCAGCTACTCAGGAGGCTGAGGTGGGAGGATGACTTGAGCCCAGGAGTTTGAG 2940 ************************************************************ 213P1F11v.1 GCTGCAAGGAGCTATGATCATGCCAGTGCATCCCAGCTCTAGGTGAGACAGTGAGATCCG 2926 213P1F11v.2 GCTGCAAGGAGCTATGATCATGCCAGTGCATCCCAGCTCTAGGTGAGACAGTGAGATCCG 3000 ************************************************************ 213P1F11v.1 GTCTCCAAAATAAATCAATCAATCAAATAAAGACCAAAGTCAAACCGCACATCAGGATCT 2986 213P1F11v.2 GTCTCCAAAATAAATCAATCAATCAAATAAAGACCAAAGTCAAACCGCACATCAGGATCT 3060 ************************************************************ 213P1F11v.1 CTCACACCCTTCCAATTTTGCCATCTACCAGCACTTAGCTAAACCCATCTCCCATCTCTT 3046 213P1F11v.2 CTCACACCCTTCCAATTTTGCCATCTACCAGCACTTAGCTAAACCCATCTCCCATCTCTT 3120 ************************************************************ 213P1F11v.1 CCACCATGAATTCACTCTTTCAAAAAGGCTAATGTCTTCTTACTCACCCTTGCCTCTAAG 3106 213P1F11v.2 CCACCATGAATTCACTCTTTCAAAAAGGCTAATGTCTTCTTACTCACCCTTGCCTCTAAG 3180 ************************************************************ 213P1F11v.1 CCTTTGCTATCACCATTTCCCCCAAGCTGGAGGGCCCTCCCTCTCCCTTTACCCCTCTTC 3166 213P1F11v.2 CCTTTGCTATCACCATTTCCCCCAAGCTGGAGGGCCCTCCCTCTCCCTTTACCCCTCTTC 3240 ************************************************************ 213P1F11v.1 CACTACCTCCCACCCCTACTTTTTCCAGAAAGCCATTTCCTCTCTTTTTTCTGATTGATC 3226 213P1F11v.2 CACTACCTCCCACCCCTACTTTTTCCAGAAAGCCATTTCCTCTCTTTTTTCTGATTGATC 3300 ************************************************************ 213P1F11v.1 CTTCCCTCTCACCCAGGATTAGATGCTGGAAATGACCACTTCTGGAGGGCAGGGAACAAG 3286 213P1F11v.2 CTTCCCTCTCACCCAGGATTAGATGCTGGAAATGACCACTTCTGGAGGGCAGGGAACAAG 3360 ************************************************************ 213P1F11v.1 CCCTTAATCTGCATAATGAGTGTTCAATAAACAGTTGTCAAACTTTGAAA 3336 213P1F11v.2 CCCTTAATCTGCATAATGAGTGTTCAATAAACAGTTGTCAAACTTTGAAA 3410 **************************************************
[0781] 34 TABLE XXVB Nucleotide sequence alignment of 213P1F11 v.1 and 213P1F11 v.3 213P1F11v.1 CTGACTCATTTAGACTCTCTGCCTAGGCCACCTTTGCCAGAGGGAGTCCCCTCAGCCTTG 60 213P1F11v.3 CTGACTCATTTAGACTCTCTGCCTAGGCCACCTTTGCCAGAGGGAGTCCCCTCAGCCTTG 60 ************************************************************ 213P1F11v.1 CGATCACTCATCCCATTGGCGTTGGCTCCATTTCCACACCACAGCTGTGTGCCAAGGGTG 120 213P1F11v.3 CGATCACTCATCCCATTGGCGTTGGCTCCATTTCCACACCACAGCTGTGTGCCAAGGGTG 120 ************************************************************ 213P1F11v.1 TGTCATGAGGTTTCTTGAGTGACAGAAAACTCACCGACAATAAAGGGCCAGGTGATTGTG 180 213P1F11v.3 TGTCATGAGGTTTCTTGAGTGACAGAAAACTCACCGACAATAAAGGGCCAGGTGATTGTG 180 ************************************************************ 213P1F11v.1 CCACCCGATTCATAGACCAGGCTTCTCAGGAGAAACCCCGGGAGATTCCACACTGTCAGC 240 213P1F11v.3 CCACCCGATTCATAGACCAGGCTTCTCAGGAGAAACCCCGGGAGATTCCACACTGTCAGC 240 ************************************************************ 213P1F11v.1 CCCTTCTCCAAGATCAGTACGTGGGCCTGACTCCTCCTCGGTGCCCAGCTCAGTATTGGC 300 213P1F11v.3 CCCTTCTCCAAGATCAGTACGTGGGCCTGACTCCTCCTCGGTGCCCAGCTCAGTATTGGC 300 ************************************************************ 213P1F11v.1 AACTAGGAGAGTAGTGAGATTGAACTTGGCCTTGAGGAACAGCTGCCTCTAGAGTTGGAT 360 213P1F11v.3 AACTAGGAGAGTAGTGAGATTGAACTTGGCCTTGAGGAACAGCTGCCTCTAGAGTTGGAT 360 ************************************************************ 213P1F11v.1 CAGACAAGGGTGCTGAGAGCCGGGACTCACAACCAAAGGAGAAATGAGCAATCCGCGGTC 420 213P1F11v.3 CAGACAAGGGTGCTGAGAGCCGGGACTCACAACCAAAGGAGAAATGAGCAATCCGCGGTC 420 ************************************************************ 213P1F11v.1 TTTGGAAGAGGAGAAATATGATATGTCAGGTGCCCGCCTGGCCCTAATACTGTGTGTCAC 480 213P1F11v.3 TTTGGAAGAGGAGAAATATGATATGTCAGGTGCCCGCCTGGCCCTAATACTGTGTGTCAC 480 ************************************************************ 213P1F11v.1 CAAAGCCCGGGAAGGTTCCGAAGAAGACCTGGATGCTCTGGAACACATGTTTCGGCAGCT 540 213P1F11v.3 CAAAGCCCGGGAAGGTTCCGAAGAAGACCTGGATGCTCTGGAACACATGTTTCGGCAGCT 540 ************************************************************ 213P1F11v.1 GAGATTCGAAAGCACCATGAAAAGAGACCCCACTGCCGAGCAATTCCAGGAAGAGCTGGA 600 213P1F11v.3 GAGATTCGAAAGCACCATGAAAAGAGACCCCACTGCCGAGCAATTCCAGGAAGAGCTGGA 600 ************************************************************ 213P1F11v.1 AAAATTCCAGCAGGCCATCGATTCCCGGGAAGATCCCGTCAGTTGTGCCTTCGTGGTACT 660 213P1F11v.3 AAAATTCCAGCAGGCCATCGATTCCCGGGAAGATCCCGTCAGTTGTGCCTTCGTGGTACT 660 ************************************************************ 213P1F11v.1 CATGGCTCACGGGAGGGAAGGCTTCCTCAAGGGAGAAGATGGGGAGATGGTCAAGCTGGA 720 213P1F11v.3 CATGGCTCACGGGAGGGAAGGCTTCCTCAAGGGAGAAGATGGGGAGATGGTCAAGCTGGA 720 ************************************************************ 213P1F11v.1 GAATCTCTTCGAGGCCCTGAACAACAAGAACTGCCAGGCCCTGCGAGCTAAGCCCAAGGT 780 213P1F11v.3 GAATCTCTTCGAGGCCCTGAACAACAAGAACTGCCAGGCCCTGCGAGCTAAGCCCAAGGT 780 ************************************************************ 213P1F11v.1 GTACATCATACAGGCCTGTCGAGGAG---------------------------------- 806 213P1F11v.3 GTACATCATACAGGCCTGTCGAGGAGCCACCCTGCCCAGCCCCTTTCCTTACCTTTCTCT 840 ************************** 213P1F11v.1 ----------------------------------AACAAAGGGACCCCGGTGAAACAGTA 832 213P1F11v.3 CTGACTTTGCCTCCTCCTCTTCTTGTTGTTTCAGAACAAAGGGACCCCGGTGAAACAGTA 900 ************************** 213P1F11v.1 GGTGGAGATGAGATTGTGATGGTCATCAAAGACAGCCCACAAACCATCCCAACATACACA 892 213P1F11v.3 GGTGGAGATGAGATTGTGATGGTCATCAAAGACAGCCCACAAACCATCCCAACATACACA 960 ************************************************************ 213P1F11v.1 GATGCCTTGCACGTTTATTCCACGGTAGAGGGATACATCGCCTACCGACATGATCAGAAA 952 213P1F11v.3 GATGCCTTGCACGTTTATTCCACGGTAGAGGGATACATCGCCTACCGACATGATCAGAAA 1020 ************************************************************ 213P1F11v.1 GGCTCATGCTTTATCCAGACCCTGGTGGATGTGTTCACGAAGAGGAAAGGACATATCTTG 1012 213P1F11v.3 GGCTCATGCTTTATCCAGACCCTGGTGGATGTGTTCACGAAGAGGAAAGGACATATCTTG 1080 ************************************************************ 213P1F11v.1 GAACTTCTGACAGAGGTGACCCGGCGGATGGCAGAAGCAGAGCTGGTTCAAGAAGGAAAA 1072 213P1F11v.3 GAACTTCTGACAGAGGTGACCCGGCGGATGGCAGAAGCAGAGCTGGTTCAAGAAGGAAAA 1140 ************************************************************ 213P1F11v.1 GCAAGGAAAACGAACCCTGAAATCCAAAGCACCCTCCGGAAACGGCTGTATCTGCAGTAG 1132 213P1F11v.3 GCAAGGAAAACGAACCCTGAAATCCAAAGCACCCTCCGGAAACGGCTGTATCTGCAGTAG 1200 ************************************************************ 213P1F11v.1 AAGTAGAAAGACCAGGAGGAGCTTTCCTTCCAGCATTCTTTCTGTCTCACAGAAATTTAG 1192 213P1F11v.3 AAGTAGAAAGACCAGGAGGAGCTTTCCTTCCAGCATTCTTTCTGTCTCACAGAAATTTAG 1260 ************************************************************ 213P1F11v.1 AGGCAGCTCTTACCTCTCCCCAAGATCTTCTGTTCCCAAGGCCAAATGGCACCCAGTTTC 1252 213P1F11v.3 AGGCAGCTCTTACCTCTCCCCAAGATCTTCTGTTCCCAAGGCCAAATGGCACCCAGTTTC 1320 ************************************************************ 213P1F11v.1 TTTTCCATCACACCCTTCATGCAGGTCCTCCTGTCCTTATTAGAGCAAGCCAGCCAAAAC 1312 213P1F11v.3 TTTTCCATCACACCCTTCATGCAGGTCCTCCTGTCCTTATTAGAGCAAGCCAGCCAAAAC 1380 ************************************************************ 213P1F11v.1 TTAGCACAAGGCATGGTGGCAACATTAACATCACCTCCCTCAGGCTGGACTTTCTATCTT 1372 213P1F11v.3 TTAGCACAAGGCATGGTGGCAACATTAACATCACCTCCCTCAGGCTGGACTTTCTATCTT 1440 ************************************************************ 213P1F11v.1 TATTAATGCAACCGAAGAGACCTAAGAGTGCATTCACTTATCCCACTTTCTGTTCCTGTG 1432 213P1F11v.3 TATTAATGCAACCGAAGAGACCTAAGAGTGCATTCACTTATCCCACTTTCTGTTCCTGTG 1500 ************************************************************ 213P1F11v.1 GTCTTCTTTCTCCCATGAAGCAGAAACTGGATAAAGCTCAAGATTTTCCATAGACAAACC 1492 213P1F11v.3 GTCTTCTTTCTCCCATGAAGCAGAAACTGGATAAAGCTCAAGATTTTCCATAGACAAACC 1560 ************************************************************ 213P1F11v.1 AAAGCCCACTCATCCCCTCCTACCCCAATCCAACCTCTGCTGGCTCCTGCATCTCACTTG 1552 213P1F11v.3 AAAGCCCACTCATCCCCTCCTACCCCAATCCAACCTCTGCTGGCTCCTGCATCTCACTTG 1620 ************************************************************ 213P1F11v.1 GAGGTCAAACCTCCTCCTGAGGCCAATGCATTCCCAACTTCCAGTTCTTTCCTTTACCCT 1612 213P1F11v.3 GAGGTCAAACCTCCTCCTGAGGCCAATGCATTCCCAACTTCCAGTTCTTTCCTTTACCCT 1680 ************************************************************ 213P1F11v.1 GGAGAGTTAGTAAGGTAAGAACCATTCTTTCTCTCCAAAACCACTCCTCCTTGGCTGGCA 1672 213P1F11v.3 GGAGAGTTAGTAAGGTAAGAACCATTCTTTCTCTCCAAAACCACTCCTCCTTGGCTGGCA 1740 ************************************************************ 213P1F11v.1 AGTTGGTGTCCTAACTCCGTTCTCTTCCTAGCTCATGGCCTCTCTAGATAATAAAGTTGT 1732 213P1F11v.3 AGTTGGTGTCCTAACTCCGTTCTCTTCCTAGCTCATGGCCTCTCTAGATAATAAAGTTGT 1800 ************************************************************ 213P1F11v.1 CTCCTCCTTTCTGGATCTCTTCCTCCTAACACCCCTCCCCTGAAACCCTGGACTCTGCCC 1792 213P1F11v.3 CTCCTCCTTTCTGGATCTCTTCCTCCTAACACCCCTCCCCTGAAACCCTGGACTCTGCCC 1860 ************************************************************ 213P1F11v.1 TCTCTCCAAGAAAATCCATCTATTCAACTATTCTTGCATTCAATTACTCTAAATGAGAGC 1852 213P1F11v.3 TCTCTCCAAGAAAATCCATCTATTCAACTATTCTTGCATTCAATTACTCTAAATGAGAGC 1920 ************************************************************ 213P1F11v.1 GTGTTGGAGCTATGGCAAATTCCCTGTTGTCACCTTGCTATTTTGCAGACAACATAATAT 1912 213P1F11v.3 GTGTTGGAGCTATGGCAAATTCCCTGTTGTCACCTTGCTATTTTGCAGACAACATAATAT 1980 ************************************************************ 213P1F11v.1 TTAACCTCTCATAACCAGAGAGGTTAAATAATTTGTCAAATGCAATACAGTAAGACAGAG 1972 213P1F11v.3 TTAACCTCTCATAACCAGAGAGGTTAAATAATTTGTCAAATGCAATACAGTAAGACAGAG 2040 ************************************************************ 213P1F11v.1 GCAAGGACAAGGTTTGACTTCCAGCCCAGCCTCTTTTCCACAACCTGCTAAATCCTGATC 2032 213P1F11v.3 GCAAGGACAAGGTTTGACTTCCAGCCCAGCCTCTTTTCCACAACCTGCTAAATCCTGATC 2100 ************************************************************ 213P1F11v.1 CATCTGAAAACTTTTCTAATTAGTGAAGATGACTAATAAAAATTTTCCCTATCTCCAAGG 2092 213P1F11v.3 CATCTGAAAACTTTTCTAATTAGTGAAGATGACTAATAAAAATTTTCCCTATCTCCAAGH 2160 ************************************************************ 213P1F11v.1 TAGGAGCTTTCTGGAAGTTTCTAGAAATTTTCAATAACCACCAGCCAAGGTTACCTCCAG 2152 213P1F11v.3 TAGGAGCTTTCTGGAAGTTTCTAGAAATTTTCAATAACCACCAGCCAAGGTTACCTCCAG 2220 ************************************************************ 213P1F11v.1 GTAACCTTGCAGGACCAGGCTGGAAGTCAGATCGGCTTCACTATCTTCCAACTCTACAGC 2212 213P1F11v.3 GTAACCTTGCAGGACCAGGCTGGAAGTCAGATCGGCTTCACTATCTTCCAACTCTACAGC 2280 ************************************************************ 213P1F11v.1 CTGTATCTCTCCATCCCCAGCTTTGACCTTTCCTGCTCAAGTAACCTACGGGCACATCCA 2272 213P1F11v.3 CTGTATCTCTCCATCCCCAGCTTTGACCTTTCCTGCTCAAGTAACCTACGGGCACATCCA 2340 ************************************************************ 213P1F11v.1 GCGTCACTAAAAACTCAGGGCTTTTCTTCCCGGTTACTCCTCCAAGCGTTCCCTGGTATC 2332 213P1F11v.3 GCGTCACTAAAAACTCAGGGCTTTTCTTCCCGGTTACTCCTCCAAGCGTTCCCTGGTATC 2400 ************************************************************ 213P1F11v.1 CTCAACCTCAGATCCCAGGTTCAGATTTCTGCAGTCAATCTATGACCCCTCTCTTCTTGC 2392 213P1F11v.3 CTCAACCTCAGATCCCAGGTTCAGATTTCTGCAGTCAATCTATGACCCCTCTCTTCTTGC 2460 ************************************************************ 213P1F11v.1 ATCCTTCATATGCCACCAGACACCATGCCCAGTCCAGCCTGATTTTGAAACAACTTTCAT 2452 213P1F11v.3 ATCCTTCATATGCCACCAGACACCATGCCCAGTCCAGCCTGATTTTGAAACAACTTTCAT 2520 ************************************************************ 213P1F11v.1 GCCGGTCTTCTCTTCCCTGACATGTTACTGTCCAGGCTCAAGTCCTCAGCTTCTCATATC 2512 213P1F11v.3 GCCGGTCTTCTCTTCCCTGACATGTTACTGTCCAGGCTCAAGTCCTCAGCTTCTCATATC 2580 ************************************************************ 213P1F11v.1 TGCATCTTTGCAACCAACTTCCTCCCTTGCCTCTCTGCTTTTCCATCCCACTTTTCATGT 2572 213P1F11v.3 TGCATCTTTGCAACCAACTTCCTCCCTTGCCTCTCTGCTTTTCCATCCCACTTTTCATGT 2640 ************************************************************ 213P1F11v.1 GTCCTCCATACCATCTATAACAGTGATCTCCCTGGAACACTCAAGAAGACACAACATACC 2632 213P1F11v.3 GTCCTCCATACCATCTATAACAGTGATCTCCCTGGAACACTCAAGAAGACACAACATACC 2700 ************************************************************ 213P1F11v.1 ATATTATTTAAAGACCAGGGTACTGGACAGTGGCTCACACCTGTATTCCCGACTTTGACA 2692 213P1F11v.3 ATATTATTTAAAGACCAGGGTACTGGACAGTGGCTCACACCTGTATTCCCGACTTTGACA 2760 ************************************************************ 213P1F11v.1 GTCTGAAGCGGGAGGATCACTTGAGGCCAGGAGTTAAGAGACCAGCCTGGGCAACACAGC 2752 213P1F11v.3 GTCTGAAGCGGGAGGATCACTTGAGGCCAGGAGTTAAGAGACCAGCCTGGGCAACACAGC 2820 ************************************************************ 213P1F11v.1 AAGACCCTGTCTCTAAAAAAAAAAATTAATTAACTGGGTATGGTGGCACATGCCTGTAGT 2812 213P1F11v.3 AAGACCCTGTCTCTAAAAAAAAAAATTAATTAACTGGGTATGGTGGCACATGCCTGTAGT 2880 ************************************************************ 213P1F11v.1 CCCAGCTACTCAGGAGGCTGAGGTGGGAGGATGACTTGAGCCCAGGAGTTTGAGGCTGCA 2872 213P1F11v.3 CCCAGCTACTCAGGAGGCTGAGGTGGGAGGATGACTTGAGCCCAGGAGTTTGAGGCTGCA 2940 ************************************************************ 213P1F11v.1 AGGAGCTATGATCATGCCAGTGCATCCCAGCTCTAGGTGAGACAGTGAGATCCGGTCTCC 2932 213P1F11v.3 AGGAGCTATCATCATGCCAGTGCATCCCAGCTCTAGGTGAGACAGTGAGATCCGGTCTCC 3000 ************************************************************ 213P1F11v.1 AAAATAAATCAATCAATCAAATAAAGACCAAAGTCAAACCGCACATCAGGATCTCTCACA 2992 213P1F11v.3 AAAATAAATCAATCAATCAAATAAAGACCAAAGTCAAACCGCACATCAGGATCTCTCACA 3060 ************************************************************ 213P1F11v.1 CCCTTCCAATTTTGCCATCTACCAGCACTTAGCTAAACCCATCTCCCATCTCTTCCACCA 3052 213P1F11v.3 CCCTTCCAATTTTGCCATCTACCAGCACTTAGCTAAACCCATCTCCCATCTCTTCCACCA 3120 ************************************************************ 213P1F11v.1 TGAATTCACTCTTTCAAAAAGGCTAATGTCTTCTTACTCACCCTTGCCTCTAAGCCTTTG 3112 213P1F11v.3 TGAATTCACTCTTTCAAAAAGGCTAATGTCTTCTTACTCACCCTTGCCTCTAAGCCTTTG 3180 ************************************************************ 213P1F11v.1 CTATCACCATTTCCCCCAAGCTGGAGGGCCCTCCCTCTCCCTTTACCCCTCTTCCACTAC 3172 213P1F11v.3 CTATCACCATTTCCCCCAAGCTGGAGGGCCCTCCCTCTCCCTTTACCCCTCTTCCACTAC 3240 ************************************************************ 213P1F11v.1 CTCCCACCCCTACTTTTTCCAGAAAGCCATTTCCTCTCTTTTTTCTGATTGATCCTTCCC 3232 213P1F11v.3 CTCCCACCCCTACTTTTTCCAGAAAGCCATTTCCTCTCTTTTTTCTGATTGATCCTTCCC 3300 ************************************************************ 213P1F11v.1 TCTCACCCAGGATTAGATGCTGGAAATGACCACTTCTGGAGGGCAGGGAACAAGCCCTTA 3292 213P1F11v.3 TCTCACCCAGGATTAGATGCTGGAAATGACCACTTCTGGAGGGCAGGGAACAAGCCCTTA 3360 ************************************************************ 213P1F11v.1 ATCTGCATAATGAGTGTTCAATAAACAGTTGTCAAACTTTGAAA 3336 213P1F11v.3 ATCTGCATAATGAGTGTTCAATAAACAGTTGTCAAACTTTGAAA 3404 ********************************************
[0782] 35 TABLE XXVC Nucleotide sequence alignment of 213P1F11 v.1 and 213P1F11 v.4 213P1F11v.1 CTGACTCATTTAGACTCTCTGCCTAGGCCACCTTTGCCAGAGGGAGTCCCCTCAGCCTTG 60 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 CGATCACTCATCCCATTGGCGTTGGCTCCATTTCCACACCACAGCTGTGTGCCAAGGGTG 120 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 TGTCATGAGGTTTCTTGAGTGACAGAAAACTCACCGACAATAAAGGGCCAGG-TGATTGT 179 213P1F11v.4 ---------------------ATGGGGAAAT-GCCAAGAGTATGACAAAAGTCTGTCTGT 38 * * ** * ** * * ** ** ** *** 213P1F11v.1 GCCACCCGATTCATAGACCAGGCTTCTCAGGAGAAACCCCGGGAGATTCCACACTGTCAG 239 213P1F11v.4 GCAGCCAGAGA-AGAGAACAGGACTCAGAGATGAGAAT---GGAGAATGTGGACAGACA- 93 ** ** ** * *** **** ** ** ** * ***** * ** * ** 213P1F11v.1 CCCCTTCTCCAAGATCAGTACGTGGGCCTGACTCCTCCTCGGTGCCCAGCTCAGTATTGG 299 213P1F11v.4 TTCAGACTCAAGGAAGAGCAAGGGAC---GGCTTTCAGGGGAAGTTCAGTCCACCAGAAG 150 * *** * ** ** * * * * * ** * * *** ** * * 213P1F11v.1 CAACTAGGAGAGTAGTGAGATTGAACTTGGCCTTGAGGAACAGCTGCCTCTAGAGTTGGA 359 213P1F11v.4 CTGGTGAATGACCCACGGGA---GACAC-------AGGAA-----GTTTTTGGGGGCGGA 195 * * ** * ** ** ***** * * * * * *** 213P1F11v.1 TCAGACAAGGGTGCTGAGAGCCGGGA-CTCACAACCAAAGG-AGAAATGAGCAATCCGCG 417 213P1F11v.4 GTGGG---GGACATTGTGGGACGGGATCTCAGTATTAGCTTCAGAAACTCTGAGACCTCT 252 * ** ** * * ***** **** * * ***** * ** * 213P1F11v.1 GTCTTTGGAAGAGGAGAAATATGATATGTCAGGTGCCCGCCTGGCCCTAATACTGTGTGT 477 213P1F11v.4 GCAAGTG-AGGAGGAGAAATATGATATGTCAGCTGCCCGCCTGGCCCTAATACTGTGTGT 311 * ** * ************************************************** 213P1F11v.1 CACCAAAGCCCGGGAAGGTTCCGAAGAAGACCTGGATGCTCTGGAACACATGTTTCGGCA 537 213P1F11v.4 CACCAAAGCCCGGGAAGGTTCCGAAGAAGACCTGGATGCTCTGGAACACATGTTTCGGCA 371 ************************************************************ 213P1F11v.1 GCTGAGATTCGAAAGCACCATGAAAAGAGACCCCACTGCCGAGCAATTCCAGGAAGAGCT 597 213P1F11v.4 GCTGAGATTCGAAAGCACCATGAAAAGAGACCCCACTGCCGAGCAATTCCAGGAAGAGCT 431 ************************************************************ 213P1F11v.1 GGAAAAATTCCAGCAGGCCATCGATTCCCGGGAAGATCCCGTCAGTTGTGCCTTCGTGGT 657 213P1F11v.4 GGAAAAATTCCAGCAGGCCATCGATTCCCGGGAAGATCCCGTCAGTTGTGCCTTCGTGGT 491 ************************************************************ 213P1F11v.1 ACTCATGGCTCACGGGAGGGAAGGCTTCCTCAAGGGAGAAGATGGGGAGATGGTCAAGCT 717 213P1F11v.4 ACTCATGGCTCACGGGAGGGAAGGCTTCCTCAAGGGAGAAGATGGGGAGATGGTCAAGCT 551 ************************************************************ 213P1F11v.1 GGAGAATCTCTTCGAGGCCCTGAACAACAAGAACTGCCAGGCCCTGCGAGCTAAGCCCAA 777 213P1F11v.4 GGAGAATCTCTTCGAGGCCCTGAACAACAAGAACTGCCAGGCCCTGCGAGCTAAGCCCAA 611 ************************************************************ 213P1Fl1v.1 GGTGTACATCATACAGGCCTGTCGAGGAGAACAAAGGGACCCCGGTGAAACAGTAGGTGG 837 213P1F11v.4 GGTGTACATCATACAGGCCTGTCGAGGAGAACAAAGGGACCCCGGTGAAACAGTAGGTGG 671 ************************************************************ 213P1F11v.1 AGATGAGATTGTGATGGTCATCAAAGACAGCCCACAAACCATCCCAACATACACAGATGC 897 213P1F11v.4 AGATGAGATTGTGATGGTCATCAAAGACAGCCCACAAACCATCCCAACATACACAGATGC 731 ************************************************************ 213P1F11v.1 CTTGCACGTTTATTCCACGGTAGAGGGATACATCGCCTACCGACATGATCAGAAAGGCTC 957 213P1F11v.4 CTTGCACGTTTATTCCACGGTAGAGGGATACATCGCCTACCGACATGATCAGAAAGGCTC 791 ************************************************************ 213P1F11v.1 ATGCTTTATCCAGACCCTGGTGGATGTGTTCACGAAGAGGAAAGGACATATCTTGGAACT 1017 213P1F11v.4 ATGCTTTATCCAGACCCTGGTGGATGTGTTCACGAAGAGGAAAGGACATATCTTGGAACT 851 ************************************************************ 213P1F11v.1 TCTGACAGAGGTGACCCGGCGGATGGCAGAAGCAGAGCTGGTTCAAGAAGGAAAAGCAAG 1077 213P1F11v.4 TCTGACAGAGGTGACCCGGCGGATGGCAGAAGCAGAGCTGGTTCAAGAAGGAAAAGCAAG 911 ************************************************************ 213P1F11v.1 GAAAACGAACCCTGAAATCCAAAGCACCCTCCGGAAACGGCTGTATCTGCAGTAGAAGTA 1137 213P1F11v.4 GAAAACGAACCCTGAAATCCAAAGCACCCTCCGGAAACGGCTGTATCTGCAGTAG----- 966 ******************************************************* 213P1F11v.1 GAAAGACCAGGAGGAGCTTTCCTTCCAGCATTCTTTCTGTCTCACAGAAATTTAGAGGCA 1197 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 GCTCTTACCTCTCCCCAAGATCTTCTGTTCCCAAGGCCAAATGGCACCCAGTTTCTTTTC 1257 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 CATCACACCCTTCATGCAGGTCCTCCTGTCCTTATTAGAGCAAGCCAGCCAAAACTTAGC 1317 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 ACAAGGCATGGTGGCAACATTAACATCACCTCCCTCAGGCTGGACTTTCTATCTTTATTA 1377 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 ATGCAACCGAAGAGACCTAAGAGTGCATTCACTTATCCCACTTTCTGTTCCTGTGGTCTT 1437 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 CTTTCTCCCATGAAGCAGAAACTGGATAAAGCTCAAGATTTTCCATAGACAAACCAAAGC 1497 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 CCACTCATCCCCTCCTACCCCAATCCAACCTCTGCTGGCTCCTGCATCTCACTTGGAGGT 1557 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 CAAACCTCCTCCTGAGGCCAATGCATTCCCAACTTCCAGTTCTTTCCTTTACCCTGGAGA 1617 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 GTTAGTAAGGTAAGAACCATTCTTTCTCTCCAAAACCACTCCTCCTTGGCTGGCAAGTTG 1677 213P1F11v.4 ------------------------------------------------------------ 213p1F11v.1 GTGTCCTAACTCCGTTCTCTTCCTAGCTCATGGCCTCTCTAGATAATAAAGTTGTCTCCT 1737 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 CCTTTCTGGATCTCTTCCTCCTAACACCCCTCCCCTGAAACCCTGGACTCTGCCCTCTCT 1797 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 CCAAGAAAATCCATCTATTCAACTATTCTTGCATTCAATTACTCTAAATGAGAGCGTGTT 1857 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 GGAGCTATGGCAAATTCCCTGTTGTCACCTTGCTATTTTGCAGACAACATAATATTTAAC 1917 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 CTCTCATAACCACAGAGGTTAAATAATTTGTCAAATGCAATACAGTAAGACAGAGGCAAG 1977 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 GACAAGGTTTGACTTCCAGCCCAGCCTCTTTTCCACAACCTGCTAAATCCTGATCCATCT 2037 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 GAAAACTTTTCTAATTAGTGAAGATGACTAATAAAAATTTTCCCTATCTCCAAGGTAGGA 2097 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 GCTTTCTGGAAGTTTCTAGAAATTTTCAATAACCACCAGCCAAGGTTACCTCCAGGTAAC 2157 213P1F11v4 ------------------------------------------------------------ 213P1F11v.1 CTTGCAGCACCAGGCTGGAAGTCAGATCGGCTTCACTATCTTCCAACTCTACAGCCTGTA 2217 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 TCTCTCCATCCCCAGCTTTGACCTTTCCTGCTCAAGTAACCTACGGGCACATCCAGCGTC 2277 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 ACTAAAAACTCAGGGCTTTTCTTCCCGGTTACTCCTCCAAGCGTTCCCTGGTATCCTCAA 2337 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 CCTCAGATCCCAGGTTCAGATTTCTGCAGTCAATCTATGACCCCTCTCTTCTTGCATCCT 2397 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 TCATATGCCACCAGACACCATGCCCAGTCCAGCCTGATTTTGAAACAACTTTCATGCCGG 2457 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 TCTTCTCTTCCCTGACATGTTACTGTCCAGGCTCAAGTCCTCAGCTTCTCATATCTGCAT 2517 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 CTTTGCAACCAACTTCCTCCCTTGCCTCTCTGCTTTTCCATCCCACTTTTCATGTGTCCT 2577 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 CCATACCATCTATAACAGTGATCTCCCTGGAACACTCAAGAAGACACAACATACCATATT 2637 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 ATTTAAAGACCAGGGTACTGGACAGTGGCTCACACCTGTATTCCCGACTTTGAGAGTCTG 2697 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 AAGCGGGAGGATCACTTGAGGCCAGGAGTTAAGAGACCAGCCTGGGCAACACAGCAAGAC 2757 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 CCTGTCTCTAAAAAAAAAAATTAATTAACTGGGTATGGTGGCACATGCCTGTAGTCCCAG 2817 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 CTACTCAGGAGGCTGAGGTGGGAGGATGACTTGAGCCCAGGAGTTTGAGGCTGCAAGGAG 2877 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 CTATGATCATGCCAGTGCATCCCAGCTCTAGGTGAGACAGTGAGATCCGGTCTCCAAAAT 2937 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 AAATCAATCAATCAAATAAAGACCAAAGTCAAACCGCACATCAGGATCTCTCACACCCTT 2997 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 CCAATTTTGCCATCTACCAGCACTTAGCTAAACCCATCTCCCATCTCTTCCACCATGAAT 3057 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 TCACTCTTTCAAAAAGGCTAATGTCTTCTTACTCACCCTTGCCTCTAAGCCTTTGCTATC 3117 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 ACCATTTCCCCCAAGCTGGAGGGCCCTCCCTCTCCCTTTACCCCTCTTCCACTACCTCCC 3177 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 ACCCCTACTTTTTCCAGAAAGCCATTTCCTCTCTTTTTTCTGATTGATCCTTCCCTCTCA 3237 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 CCCAGGATTAGATGCTGGAAATGACCACTTCTGGAGGGCAGGGAACAAGCCCTTAATCTG 3297 213P1F11v.4 ------------------------------------------------------------ 213P1F11v.1 CATAATGAGTGTTCAATAAACAGTTGTCAAACTTTGAAA 3336 213P1F11v.4 ---------------------------------------
[0783] 36 TABLE XXVIA Peptide sequences of protein coded by 213P1F11 v.2 MSNPRSLEEE KYDMSGARLA LILCVTKARE GSEEDLDALE HMFRQLRFES TMKRDPTAEQ 60 FQEELEKFQQ AIDSREDPVS CAFVVLMAHG REGFLKGEDG EMVKLENLFE ALNNKNCQAL 120 RAKPKVYIIQ ACRGEQRDPG ETVGGDEIVM VIKDSPQTIP TYTDALHVYS TVEGPTPFQD 180 PLYLPSEAPP NPPLWNSQDT SPTDMIRKAH ALSRPWWMCS RRGKDISWNF 230
[0784] 37 TABLE XXVIB Peptide sequences of protein coded by 213P1F11 v.3 MSNPRSLEEE KYDMSGARLA LILCVTKARE GSEEDLDALE HMFRQLRFES TMKRDPTAEQ 60 FQEELEKFQQ AIDSREDPVS CAFVVLMAHG REGFLKGEDG EMVKLENLFE ALNNKNCQAL 120 RAKPKVYIIQ ACRGATLPSP FPYLSL 146
[0785] 38 TABLE XXVIC Peptide sequences of protein coded by 213P1F11 v.4 MGKCQEYDKS LSVQPEKRTG LRDENGECGQ TFRLKEEQGR AFRGSSVHQK LVNDPRETQE 60 VFGGGVGDIV GRDLSISFRN SETSASEEEK YDMSGARLAL ILCVTKAREG SEEDLDALEH 120 MFRQLRFEST MKRDPTAEQF QEELEKFQQA IDSREDPVSC AFVVLMAHGR EGFLKGEDGE 180 MVKLENLFEA LNNKNCQALR AKPKVYIIQA CRGEQRDPGE TVGGDEIVMV IKDSPQTIPT 240 YTDALHVYST VEGYIAYRHD QKGSCFIQTL VDVFTKRKGH ILELLTEVTR RMAEAELVQE 300 GKARKTNPEI QSTLRKRLYL Q 321
[0786] 39 TABLE XXVIIA Amino acid sequence alignment of 213P1F11 v.1 and 213P1F11 v.2 213P1F11v.1 MSNPRSLEEEKYDMSGARLALILCVTKAREGSEEDLDALEHMFRQLRFESTMKRDPTAEQ 60 213P1F11V.2 MSNPRSLEEEKYDMSGARLALILCVTKAREGSEEDLDALEHMFRQLRFESTMKRDPTAEQ 60 ************************************************************ 213P1F11v.1 FQEELEKFQQAIDSREDPVSCAFVVLMAHGREGFLKGEDGEMVKLENFEALNNKNCQAL 120 213P1F11v.2 FQEELEKFQQAIDSREDPVSCAFVVLMAHGREGFLKGEDGEMVKLENFEALNNKNCQAL 120 *********************************************************** 213P1F11v.1 RAKPKVYIIQACRGEQRDPGETVGGDEIVMVIKDSPQTIPTYTDALHVYSTVEGYIAYRH 180 213P1F11v.2 RAKPKVYIIQACRGEQRDPGETVGGDEIVMVIKDSPQTIPTYTDALHVYSTVEGPTPFQD 180 ****************************************************** .::. 213P1F11v.1 DQKGSCFIQTLVDVFTKRKGHILELLTEVTRRMAEAELVQEGKARKTNPEIQSTLRKRLY 240 213P1F11v.2 PLYLPSEAPPNPPLWN-------------SQDTSPTDMIRKAHALSRPWWMCSRRGKDIS 227 .. . ::. :: : ::::::.:* . : * * : 213P1F11v.1 LQ- 242 213P1F11v.2 WNF 230
[0787] 40 TABLE XXVIIB Amino acid sequence alignment of 213P1F11 v.1 and 213P1F11 v.3 213P1F11v.1 MSNPRSLEEEKYDMSGARLALILCVTKAREGSEEDLDALEHMFRQLRFESTMKRDPTAEQ 60 213P1F11v.3 MSNPRSLEEEKYDMSGARLALILCVTKAREGSEEDLDALEHMFRQLRFESTMKRDPTAEQ 60 ************************************************************ 213P1F11v.1 FQEELEKFQQAIDSREDPVSCAFVVLMAHGREGFLKGEDGEMVKLENLFEALNNKNCQAL 120 213P1F11v.3 FQEELEKFQQATDSREDPVSCAFVVLMAHGREGFLKGEDGEMVKLENLFEALNNKNCQAL 120 ************************************************************ 213P1F11v.1 RAKPKVYIIQACRGEQRDPGETVGGDEIVMVIKDSPQTIPTYTDALHVYSTVEGYIAYRH 180 213P1F11v.3 RAKPKVYIIQACRG----------------------ATLPSPFPYLSL------------ 146 ************** *:*: * : 213P1F11v.1 DQKGSCFIQTLVDVFTKRKGHILELLTEVTRRMAEAELVQEGKARKTNPEIQSTLRKLY 240 213P1F11v.3 ----------------------------------------------------------- 213P1F11v.1 LQ 242 213P1F11v.3 --
[0788] 41 TABLE XXVIIC Amino acid sequence alignment of 213P1F11 v.1 and 213P1F11v.4 213P1F11v.1 ---------------------------------------------------MSNPR---- 5 213P1F11v.3 MGKCQEYDKSLSVQPEKRTGLRDENGECGQTFRLKEEQGRAFRGSSVHQKLVNDPRETQE 60 :.:** 213P1F11v.1 ------------------------SLEEEKYDMSCARLALILCVTKAREGSEEDLDALEH 41 213P1F11v.3 VFGGGVGDIVGRDLSISFRNSETSASEEEKYDMSGARLALILCVTKAREGSEEDLDALEH 120 : ********************************** 213P1F11V.1 MFRQLRFESTMKRDPTAEQFQEELEKFQQAIDSREDPVSCAFVVLMAHGREGFLKGEDGE 101 213P1F11v.3 MFRQLRFESTMKRDPTAEQFQEELEKFQQAIDSREDPVSCAFVVLMAHGREGFLKGEDGE 180 ************************************************************ 213P1F11v.1 MVKLENLFEALNNKNCQALRAKPKVYIIQACRGEQRDPGETVGGDEIVMVIKDSPQTIPT 161 213P1F11v.3 MVKLENLFEALNNKNCQALRAKPKVYIIQACRGEQRDPGETVGGDEIVMVIKDSPQTIPT 240 ************************************************************ 213P1F11v.1 YTDALHVYSTVEGYIAYRHDQKGSCFIQTLVDVFTKRKGHILELLTEVTRRMAEAELVQE 221 213P1F11v.3 YTDALHVYSTVEGYIAYRHDQKGSCFIQTLVDVFTKRKGHILELLTEVTRRMAEAELVQE 300 ************************************************************ 213P1F11v.1 GKARKTNPEIQSTLRKRLYLQ 242 213P1F11v.3 GKARKTNPEIQSTLRKRLYLQ 321 *********************
[0789] 42 TABLE XXVIII Clustal Alignment of 213P1F11 protein variants. v.1— ------------------------------------------------------------ v.2— ------------------------------------------------------------ v.3— ------------------------------------------------------------ v.4— MGKCQEYDKSLSVQPEKRTGLRDENGECGQTFRLKEEQGRAFRGSSVHQKLVNDPRETQE v.5— ------------------------------------------------------------ v.6— ------------------------------------------------------------ v.1— -------------------MSNPRSLEEEKYDMSGARLALILCVTKAREGSEEDLDALEH v.2— -------------------MSNPRSLEEEKYDMSGARLALILCVTKAREGSEEDLDALEH v.3— -------------------MSNPRSLEEEKYDMSGARLALILCVTKAREGSEEDLDALEH v.4— VFGGGVGDIVGRDLSISFRNSETSASEEEKYDMSGARLALILCVTKAREGSEEDLDALEH v.5— -------------------MSNPRSLEEEKYDMSGARLALILCVTKAREGSEEDLDALEH v.6— -------------------MSNPRSLEEEKYDMSGARLALILCVTKAREGSEEDLDALEH *:. : **************** ***************** v.1— MFRQLRFESTMKRDPTAEQFQEELEKFQQAIDSREDPVSCAFVVLMAHGREGFLKGEDGE v.2— MFRQLRFESTMKRDPTAEQFQEELEKFQQAIDSREDPVSCAFVVLMAHGREGFLKGEDGE v.3— MFRQLRFESTMKRDPTAEQFQEELEKFQQAIDSREDPVSCAFVVLMAHGREGFLKGEDHE v.4— MFRQLRFESTMKRDPTAEQFQEELEKFQQAIDSREDPVSCAFVVLMAHGREGFLKGEDGE v.5— MFRQLRFESTMKRDPTAEQFQEELEKFQQAIDSREDPVSCAFVVLMAHGREGFLKGEDGE v.6— MFRQLRFESTMKRDPTAEQFQEELEKFQQAIDSREDPVSCAFVVLMAHGREGFLKGEDHE ************************************************************ v.1— MVKLENLFEALNNKHCQALRAKPKVYIIQACRGEQRDPGETVGGDEIVMVIKDSPQTIPT v.2— MVKLENLFEALNNKNCQALRAKPKVYIIQACRGEQRDPGETVGGDEIVMVIKDSPQTIPT v.3— MVKLENLFEALNNKNCQALRAKPKVYIIQACRGATLPSPFPYLSL--------------- v.4— MVKLENLFEALNNKNCQALRAKPKVYIIQACRGEQRDPGETVGGDEIVMVIKDSPQTIPT v.5— MVKLENLFEALNNKNCQALRAKPKVYIIQACRGEQRDPGETVGGDEIVMVIKDSPQTIPT v.6— MVKLENLFEAMNNKNCQALRAKPKVYIIQACRGEQRDPGETVGGDEIVMVIKDSPQTIPT **********:********************** . . . v.1— YTDALHVYSTVEGYIAYRHDQKGSCFIQTLVDVFTKRKGHILELLTEVTRRMAEAELVQE v.2— YTDALHVYSTVEGPTPFQDPLYLPSEAPPNPPLWNSQDTSPTDMIRKAH-ALSRPWWMCS v.3— ------------------------------------------------------------ v.4— YTDALHVYSTVEGYIAYRHDQKGSCFIQTLVDVFTKRKGHILELLTEVTRRMAEAELVQE v.5— YTDALHVYSTVEGYIAYRHDQKGSCFIQTLVDVFTKRKGHILELLTEVTRRMAEAELVQE v.6— YTDALHVYSTVEGYIAYRHDQKGSCFIQTLVDVFTKRKGHILELLTEVTRRMAEAELVQE v.1— GKARKTNPEIQSTLRKRLYLQ v.2— RRGKDISWNF----------- v.3— --------------------- v.4— GKARKTNPEIQSTLRKRLYLQ v.5— GKARKTNPEIQSTLRKRLYLQ v.6— GKARKTNPEIQSTLRKRLYLQ
[0790] 43 TABLE XXIX Search Peptides 213P1F11 Variant 1: Nonamers, Decamers, 15-mers (aa. 1-242) 1 MSNPRSLEEE KYDMSGARLA LILCVTKARE GSEEDLDALE HMFRQLRFES TMKRDPTAEQ 61 FQEELEKFQQ AIDSREDPVS CAFVVLMAHG REGFLKGEDG EMVKLENLFE ALNNKNCQAL 121 RAKPKVYIIQ ACRGEQRDPG ETVGGDEIVM VIKDSPQTIP TYTDALHVYS TVEGYIAYRH 181 DQKGSCFIQT LVDVFTKRKG HILELLTEVT RRMAEAELVQ EGKARKTNPE IQSTLRKRLY 241 LQ 213P1F11 Variant 2: Nonamers (aa. 167-230) HVYS TVEGPTPFQD PLYLPSEAPP NPPLWNSQDT SPTDMIRKAH ALSRPWWMCS RRGKDISWNF Decamers (aa. 166-230) LHVYS TVEGPTPFQD PLYLPSEAPP NPPLWNSQDT SPTDMIRKAH ALSRPWWMCS RRGKDISWNF 15-mers (aa. 161-230) TYTDALHVYS TVEGPTPFQD PLYLPSEAPP NPPLWNSQDT SPTDMIRKAH ALSRPWWMCS RRGKDISWNF 213P1F11 Variant 3: Nonamers (aa. 127-146) YIIQ ACRGATLPSP FPYLSL Decamers (aa.126-146) VYIIQ ACRGATLPSP FPYLSL 15mers (aA. 121-146) RAKPKVYIIQ ACRGATLPSP FPYLSL 213P1F11 Variant 4: Nonamers (aa. 1-94) MGKCQEYDKS LSVQPEKRTG LRDENGECGQ TFRLKEEQGR AFRGSSVHQK LVNDPRETQE VFGGGVGDIV GRDLSISFRN SETSASEEEK YDMS Decamers (aa. 1-95) MGKCQEYDKS LSVQPEKRTG LRDENGECGQ TFRLKEEQGR AFRGSSVHQK LVNDPRETQE VFGGGVGDIV GRDLSISFRN SETSASEEEK YDMSG 15-mers (aa. 1-100) MGKCQEYDKS LSVQPEKRTG LRDENGECGQ TFRLKEEQGR AFRGSSVHQK LVNDPRETQE VFGGGVGDIV GRDLSISFRN SETSASEEEK YDMSGARLAL 213P1F11 Variant 5: Nonamers (aa. 16-32) GARLA LILRVTKARE GS Decamers (aa. 15-33) SGARLA LILRVTKARE GSE 15-mers (aa. 10-38) E KYDMSGARLA LILRVTKARE GSEEDLDA 213P1F11 Variant 6: Nonamers (aa. 104-120) KLENLFE AMNNKNQAL Decamers (aa. 103-121) VKLENLFE AMNNKNCQAL R 15-mers (aa. 98-126) EDG EMVKLENLFE AMNNKNCQAL RAKPKV
Claims
1. A composition comprising:
- a substance that a) modulates the status of 213P1F11, or b) a molecule that is modulated by 213P1F11 whereby the status of a cell that expresses 213P1F11 is modulated.
2. A composition of claim 1, further comprising a physiologically acceptable carrier.
3. A pharmaceutical composition that comprises the composition of claim 1 in a human unit dose form.
4. A composition of claim 1 wherein the substance comprises an antibody or fragment thereof that specifically binds to a 213P1F11-related protein.
5. An antibody or fragment thereof of claim 4, which is monoclonal.
6. An antibody of claim 4, which is a human antibody, a humanized antibody or a chimeric antibody.
7. A non-human transgenic animal that produces an antibody of claim 4.
8. A hybridoma that produces an antibody of claim 5.
9. A method of delivering a cytotoxic agent or a diagnostic agent to a cell that expresses 213P1F11, said method comprising:
- providing the cytotoxic agent or the diagnostic agent conjugated to an antibody or fragment thereof of claim 4; and,
- exposing the cell to the antibody-agent or fragment-agent conjugate.
10. A composition of claim 1 wherein the substance comprises a polynucleotide that encodes an antibody or fragment thereof either of which immunospecifically bind to a 213P1F11-related protein.
11. A composition of claim 1 wherein the substance comprises a 213P1F11-related protein.
12. A protein of claim 11 that is at least 90% homologous to an entire amino acid sequence shown in FIG. 2 (SEQ ID NOS:______).
13. A composition of claim 1 wherein the substance comprises a peptide of eight, nine, ten, or eleven contiguous amino acids of FIG. 2 or Tables V to XIX, or an analog thereof (SEQ ID NOS:______).
14. A composition of claim 1 wherein the substance comprises a CTL polypeptide of the amino acid sequence of FIG. 2 (SEQ ID NOS:______).
15. A composition of claim 14 further limited by a proviso that the epitope is not an entire amino acid sequence of FIG. 2 (SEQ ID NOS:).
16. A composition of claim 14 wherein the substance comprises a CTL polypeptide set forth in Tables V to XIX (SEQ ID NOS:______).
17. A composition of claim 16 further limited by a proviso that the polypeptide is not an entire amino acid sequence of FIG. 2 (SEQ ID NOS:______).
18. A composition of claim 1 wherein the substance comprises an antibody polypeptide epitope of the amino acid sequence of FIG. 2 (SEQ ID NOS:______).
19. A composition of claim 18 further limited by a proviso that the epitope is not an entire amino acid sequence of FIG. 2 (SEQ ID NOS:______).
20. A composition of claim 18 wherein the antibody epitope comprises a peptide region of at least 5 amino acids of FIG. 2 (SEQ ID NOS:______) in any whole number increment up to 242 that includes an amino acid position selected from: an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5, an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 6; an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7; an amino acid position having a value greater than 0.5 in the Average Flexibility profile on FIG. 8; or an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIG. 9.
21. A composition of claim 20 further limited by a proviso that the epitope is not an entire amino acid sequence of FIG. 2 (SEQ ID NOS:______).
22. A polynucleotide that encodes a protein of claim 11.
23. A polynucleotide of claim 22 that comprises a nucleic acid molecule set forth in FIG. 2.
24. A polynucleotide of claim 22 further limited by a proviso that the encoded protein is not an entire amino acid sequence of FIG. 2 (SEQ ID NOS:______).
25. A polynucleotide of claim 22 wherein T is substituted with U.
26. A composition of claim 1 wherein the substance comprises a polynucleotide comprising a coding sequence of a nucleic acid sequence of FIG. 2 (SEQ ID NOS:______).
27. A polynucleotide of claim 24 further comprising a polynucleotide that encodes a 213P1F11-related protein that is at least 90% homologous to an entire amino acid sequence shown in FIG. 2 (SEQ ID NOS:______).
28. A composition comprising a polynucleotide that is fully complementary to a polynucleotide of claim 22.
29. A composition comprising a polynucleotide that is fully complementary to a polynucleotide of claim 25.
30. A composition comprising a polynucleotide that is fully complementary to a polynucleotide of claim 27.
31. A composition of claim 1 wherein the substance comprises a) a ribozyme that cleaves a polynucleotide having 213P1F11 coding sequence, or b) a nucleic acid molecule that encodes the ribozyme; and, a physiologically acceptable carrier.
32. A composition comprising the composition of claim 1 wherein the substance comprises human T cells, wherein said T cells specifically recognize a 213P1F11 peptide sequence in the context of a particular HLA molecule.
33. A method of inhibiting growth of cancer cells that expresses 213P1F11, the method comprising:
- administering to the cells the composition of claim 1.
34. A method of claim 33 of inhibiting growth of cancer cells that express 213P1F11, the, method comprising steps of:
- administering to said cells an antibody or fragment thereof, either of which specifically bind to a 213P1F11-related protein.
35. A method of claim 33 of inhibiting growth of cancer cells that express 213P1F11, the method comprising steps of:
- administering to said cells a 213P1F11-related protein.
36. A method of claim 33 of inhibiting growth of cancer cells that express 213P1F11, the method comprising steps of:
- administering to said cells a polynucleotide comprising a 213P1F11-related protein coding sequence or a polynucleotide complementary to a polynucleotide having a 213P1F11 coding sequence.
37. A method of claim 33 of inhibiting growth of cancer cells that express 213P1F11, the method comprising steps of:
- administering to said cells a ribozyme that cleaves a polynucleotide having 213P1F11 coding sequence.
38. A method of claim 33 of inhibiting growth of cancer cells that express 213P1F11 and a particular HLA molecule, the method comprising steps of:
- administering to said cells human T cells, wherein said T cells specifically recognize a 213P1F11 peptide subsequence in the context of the particular HLA molecule.
39. A method of claim 33, the method comprising steps of:
- administering a vector that delivers a single chain monoclonal antibody coding sequence, whereby the encoded single chain antibody is expressed intracellularly within cancer cells that express 213P1F11.
40. A method of generating a mammalian immune response directed to 213P1F11, the method comprising:
- exposing cells of the mammal's immune system to a portion of
- a) a 213P1F11-related protein and/or
- b) a nucleotide sequence that encodes said protein,
- whereby an immune response is generated to 213P1F1.
41. A method of generating an immune response of claim 40, said method comprising:
- providing a 213P1F11-related protein that comprises at least one T cell or at least one B cell epitope; and,
- contacting the epitope with a mammalian immune system T cell or B cell respectively, whereby the T cell or B cell is induced.
42. A method of claim 41 wherein the immune system cell is a B cell, whereby the induced B cell generates antibodies that specifically bind to the 213P1F11-related protein.
43. A method of claim 41 wherein the immune system cell is a T cell that is a cytotoxic T cell (CTL), whereby the activated CTL kills an autologous cell that expresses the 213P1F11-related protein.
44. A method of claim 41 wherein the immune system cell is a T cell that is a helper T cell (HTL), whereby the activated HTL secretes cytokines that facilitate the cytotoxic activity of a cytotoxic T cell (CTL) or the antibody-producing activity of a B cell.
45. A method for detecting the presence of a 213P1F11-related protein or polynucleotide in a sample comprising steps of:
- contacting the sample with a substance of claim 1 that specifically binds to the 213P1F11-related protein or polynucleotide, respectively; and,
- determining that there is a complex of the substance and 213P1F11-related protein or the substance and 213P1F11-related polynucleotide, respectively.
46. A method of claim 45 for detecting the presence of a 213P1F11-related protein in a sample comprising steps of:
- contacting the sample with an antibody or fragment thereof either of which specifically bind to the 213P1F11-related protein; and,
- determining that there is a complex of the antibody or fragment thereof and 213P1F11-related protein.
47. A method of claim 45 further comprising a step of: taking the sample from a patient who has or who is suspected of having cancer.
48. A method of claim 45 for detecting the presence of 213P1F11 mRNA in a sample comprising:
- producing cDNA from the sample by reverse transcription using at least one primer;
- amplifying the cDNA so produced using 213P1F11 polynucleotides as sense and antisense primers, wherein the 213P1F11 polynucleotides used as the sense and antisense primers serve to amplify 213P1F11 cDNA; and,
- detecting the presence of the amplified 213P1F11 cDNA.
49. A method of claim 45 for monitoring 213P1F11 gene products in a biological sample from a patient who has or who is suspected of having cancer, the method comprising:
- determining the status of 213P1F11 gene products expressed by cells in a tissue sample from an individual;
- comparing the status so determined to the status of 213P1F11 gene products in a corresponding normal sample; and,
- identifying the presence of aberrant 213P1F11 gene products in the sample relative to the normal sample.
50. A method of monitoring the presence of cancer in an individual comprising: performing the method of claim 49 whereby the presence of elevated gene products 213P1F11 mRNA or 213P1F11 protein in the test sample relative to the normal tissue sample indicates the presence or status of a cancer.
51. A method of claim 50 wherein the cancer occurs in a tissue set forth in Table I.
Type: Application
Filed: Apr 1, 2002
Publication Date: Jan 29, 2004
Inventors: Pia M. Challita-Eid (Encino, CA), Arthur B. Raitano (Los Angeles, CA), Mary Faris (Los Angeles, CA), Rene S. Hubert (Los Angeles, CA), Robert Kendall Morrison (Santa Monica, CA), Wangmao GE (Culver City, CA), Aya Jakobovits (Beverly Hills, CA)
Application Number: 10114432
International Classification: A01K067/027; A61K039/395; C12N005/06; C07K016/40;
