Nucleic acids and corresponding proteins entitled 251P5G2 useful in treatment and detection of cancer
A novel gene 251P5G2 and its encoded protein, and variants thereof, are described wherein 251P5G2 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, 251P5G2 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 251P5G2 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 251P5G2 can be used in active or passive immunization.
[0001] This application is a non-provisional utility patent application that claims priority from U.S. provisional patent application USSN 60/404,306, filed Aug. 16, 2002 and this application claims priority from U.S. provisional patent application USSN 60/423,290, fled Nov. 01, 2002. The contents of the applications listed in this paragraph are fully incorporated by reference herein.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH[0002] Not applicable.
FIELD OF THE INVENTION[0003] The invention described herein relates to genes and their encoded proteins, termed 251P5G2, expressed in certain cancers, and to diagnostic and therapeutic methods and compositions useful in the management of cancers that express 251P5G2.
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 Sep. 2, 1996 (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. Nati. 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 targets 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 eight 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 significanuy 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 251P5G2, that has now been found to be over-expressed in the cancer(s) listed in Table I. Northern blot expression analysis of 251P5G2 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 251P5G2 are provided. The tissue-related profile of 251P5G2 in normal adult tissues, combined with the over-expression observed in the tissues listed in Table I, shows that 251P5G2 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 251P5G2 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 251P5G2-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 251P5G2-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 251P5G2 genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 251P5G2 genes, mRNAs, or to 251P5G2-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding 251P5G2. Recombinant DNA molecules containing 251P5G2 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 251P5G2 gene products are also provided. The invention further provides antibodies that bind to 251P5G2 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 251P5G2 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 251P5G2. A typical embodiment of this invention provides methods for monitoring 251P5G2 gene products in a tissue or hematology sample having or suspected of having some form of growth dysregulabon such as cancer.
[0030] The invention further provides various immunogenic or therapeutic compositions and strategies for treating cancers that express 251P5G2 such as cancers of tissues listed in Table I, including. therapies aimed at inhibiting the transcription, translation, processing or function of 251P5G2 as well as cancer vaccines. In one aspect, the invention provides compositions, and methods comprising them, for treating a cancer that expresses 251P5G2 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 251P5G2. Preferably, the carrier is a uniquely human carrier. In another aspect of the invention, the agent is a moiety that is immunoroactive with 251P5G2 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 251P5G2 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 251P5G2 as described above. The one or more than one nucleic acid molecule may also be, or encodes, a molecule that inhibits production of 251P5G2. Non-limiting examples of such molecules include, but are not limited to, those complementary to a nucleotide sequence essential for production of 251P5G2 (e.g. antisense sequences or molecules that form a triple helix with a nucleotide double helix essential for 251P5G2 production) or a ribozyme effective to lyse 251P5G2 mRNA.
[0032] Note that to determine the starting position of any peptide set forth in Tables VIIl-XXI and XXII to XLIX (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 VII. 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 VII. Accordingly, if a Search Peptide begins at position “X”, one must add the value “X−1” to each position in Tables VIII-XXI and XXII to XLIX 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 its 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.
[0033] One embodiment of the invention comprises an HLA peptide, that occurs at least twice in Tables VIII-XXI and XXII to XLIX 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 VIII-XXI and at least once in tables XXI! to XLIX, or an oligonucleotide that encodes the HLA peptide.
[0034] 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:
[0035] 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;
[0036] 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;
[0037] 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;
[0038] 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
[0039] 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.
BRIEF DESCRIPTION OF THE FIGURES[0040] FIG. 1. The 251P5G2 SSH sequence of 162 nucleotides.
[0041] FIG. 2. A) The cDNA and amino acid sequence of 251P5G2 variant 1 (also called “251P5G2v.1” or “251P5G2 variant 1”) is shown in FIG. 2A. The start methionine is underlined. The open reading frame extends from nucleic acid 722-1489 including the stop codon.
[0042] B) The cDNA and amino acid sequence of 251P5G2 variant 2 (also called “251P5G2 v.2”) is shown in FIG. 2B. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 722-1489 including the stop codon.
[0043] C) The cDNA and amino acid sequence of 251P5G2 variant 3 (also called “251P5G2 v.3”) is shown in FIG. 2C. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 722-1489 including the stop codon.
[0044] D) The cDNA and amino acid sequence of 251P5G2 variant 4 (also called “251P5G2 v.4”) is shown in FIG. 2D. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 722-1489 including the stop codon.
[0045] E) The cDNA and amino acid sequence of 251P5G2 variant 5 (also called “251P5G2 v.5”) is shown in FIG. 2E. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 722-1489 including the stop codon.
[0046] F) The cDNA and amino acid sequence of 251P5G2 variant 6 (also called “251P5G2 v.6”) is shown in FIG. 2F. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 722-1489 including the stop codon.
[0047] G) The cDNA and amino acid sequence of 251P5G2 variant 7 (also called “251P5G2 v.7”) is shown in FIG. 2G. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 722-1489 including the stop codon.
[0048] H) The cDNA and amino acid sequence of 251P5G2 variant 8 (also called “251P5G2 v.8”) is shown in FIG. 2H. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 722-1489 including the stop codon.
[0049] I) The cDNA and amino acid sequence of 251P5G2 variant 9 (also called “251P5G2 v.9”) is shown in FIG. 2I. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 722-1489 including the stop codon.
[0050] J) The cDNA and amino acid sequence of 251P5G2 variant 10 (also called “251P5G2 v.10”) is shown in FIG. 2J. The codon for the start methionine is underlined: The open reading frame extends from nucleic acid 722-1489 including the stop codon.
[0051] K) The cDNA and amino acid sequence of 251P5G2 variant 11 (also called “251P5G2 v.11”) is shown in FIG. 2K. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 722-1489 including the stop codon.
[0052] L) The cDNA and amino acid sequence of 251P5G2 variant 12 (also called “251P5G2 v.12”) is shown in FIG. 2L. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 722-4522 including the stop codon.
[0053] M) The cDNA and amino acid sequence of 251P5G2 variant 13 (also called 251P5G2 v.13) is shown in FIG. 2M. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 1-3801 including the stop codon.
[0054] FIG. 3.
[0055] A) The amino acid sequence of 251P5G2 v.1 is shown in FIG. 3A; it has 255 amino acids.
[0056] B) The amino acid sequence of 251P5G2 v.2 is shown in FIG. 3B; it has 255 amino acids.
[0057] C) The amino acid sequence of 251P5G2 v.3 is shown in FIG. 3C; it has 255 amino acids.
[0058] D) The amino acid sequence of 251P5G2 v.4 is shown in FIG. 3D; it has 255 amino acids.
[0059] E) The amino acid sequence of 251P5G2 v.12 is shown in FIG. 3E; it has 1266 amino acids.
[0060] As used herein, a reference to 251P5G2 includes all variants thereof, including those shown in FIGS. 2, 3, 10, and 11, unless the context clearly indicates otherwise.
[0061] FIG. 4. FIG. 4A. Alignment of 251P5G2 v.1 with the mouse vomeronasal 1 receptor C3. FIG. 4B. Amino acid alignment of 251P5G2 v.12 with the protein XM—063686 predicted from GenomeScan.
[0062] FIG. 5. Hydrophilicity amino acid profile of 251P5G2 v.1 and v.12 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 located on the World Wide Web at (expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
[0063] FIG. 6. Hydropathicity amino acid profile of 251P5G2 v.1 and v.12 determined by computer algorithm sequence analysis using the method of Kyte and Doolittle (Kyte J., Dooliltle R. F., 1982. J. Mol. Biol. 157:105-132) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
[0064] FIG. 7. Percentaccessible residues amino acid profile of 251P5G2 v.1 and v.12 determined by computer algorithm sequence analysis using the method of Janin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
[0065] FIG. 8. Average flexibility amino acid profile of 251P5G2 v.1 and v.12 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 located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
[0066] FIG. 9. Beta-turn amino acid profile of 251P5G2 v.1 and v.12 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 located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
[0067] FIG. 10. Schematic alignment of SNP variants of 251P5G2. Variants 251P5G2 v.2 through v.11 are variants with single nucleotide differences. Though these SNP variants are shown separately, they could also occur in any combinations and in any transcript variants that contains the base pairs. Numbers correspond to those of 251P5G2 v.1. Black box shows the same sequence as 251P5G2 v.1. SNPs are indicated above the box.
[0068] FIG. 11. Schematic alignment of protein variants of 251P5G2. Protein variants correspond to nucleotide variants. Nucleotide variants 251P5G2 v.5 through v.11 in FIG. 10 code for the same protein as 251P5G2 v.1. Nucleotide variants 251P5G2 v.12 and v.13 as shown in FIG. 12 code the same protein. Single amino acid differences were indicated above the boxes. Black boxes represent the same sequence as 251P5G2 v.1. Numbers underneath the box correspond to 251P5G2 v.1.
[0069] FIG. 12. Exon compositions of transcript variants of 251P5G2. Variant 251P5G2 v.12 and v.13 are transcript variants each with 19 exons. The first two exons of variants 251P5G2 v.12 and v.13 matches part of variant 251P5G2 v.1. Compared with 251P5G2 v.12, 251P5G2 v.13 has a shorter first exon (starting at base 722) but the other 18 exons are the same. Numbers in “( )” underneath the boxes correspond to those of 251P5G2 v.1. Lengths of introns and exons are not proportional.
[0070] FIG. 13. FIGS. 13(A) (SEQ ID NO: 82) and 13(B) (SEQ ID NO: 83) Secondary structure and transmembrane domains prediction for 251P5G2 protein variants.
[0071] The secondary structure of 251P5G2 protein variants 1 and 12 (FIGS. 31A and 13B, respectively) were predicted using the HNN—Hierarchical Neural Network method (Guermeur, 1997, hftp://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html), accessed from the ExPasy molecular biology server located on the World Wide Web at (.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.
[0072] FIGS. 13(C) and 13(E): Schematic representations of the probability of existence of transmembrane regions and orientation of 251P5G2 variants 1 and 12, respectively, based on the TMpred algorithm of Hofmann and Stoffel which utilizes TMBASE (K. Hofmann, W. Stoffel. TMBASE—A database of membrane spanning protein segments Biol. Chem. Hoppe-Seyler 374:166, 1993). FIGS. 13(D) and (F): Schematic representations of the probability of the existence of transmembrane regions and the extracellular and intracellular orientation of 251P5G2 variants 1 and 12, respectively, based on the TMHMM-algorith™ of Sonnhammer, von Heijne, and Krogh (Erik L. L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein sequences. In Proc. of Sixth Int. Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAI Press, 1998). The TMpred and TMHMM algorithms are accessed
[0073] FIG. 14. Expression of 251P5G2 by RT-PCR. First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), prostate cancer metastasis to lymph node, prostate cancer pool, bladder cancer pool, and cancer metastasis pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 251P5G2, was performed at 26 and 30 cycles of amplification. Results show strong expression of 251P5G2 in prostate cancer metastasis, prostate cancer pool, and cancer metastasis pool. Expression of 251P5G2 was also detected in bladder cancer pool, but not in vital pool 1 and vital pool 2.
[0074] FIG. 15. Expression of 251P5G2 in normal tissues. Two multiple tissue northern blots (Clontech) both with 2 ug of mRNA/lane were probed with the 251P5G2 sequence. Size standards in kilobases (kb) are indicated on the side. Results show weak expression of 251P5G2 in prostate and testis, but not in any other normal tissue tested.
[0075] FIG. 16. Expression of 251P5G2 in Patient Cancer Specimens and Normal Tissues. RNA was extracted from two prostate cancer metastasis to lymph node isolated from two different patients (Met1 and Met2), as well as from normal bladder (NB), normal kidney (NK), normal lung (NL) and normal breast (NBr), normal ovary (NO), and normal pancreas (NPa). Northern blot with 10 Gig of total RNA/lane was probed with 251P5G2 SSH sequence. Size standards in kilobases (kb) are indicated on the side. The 251P5G2 transcript was detected in the prostate cancer metastasis specimens, but not in the normal tissues tested.
[0076] FIG. 17. Expression of 251P5G2 in Prostate Cancer Patient Specimens. RNA was extracted from prostate cancer xenografts (LAPCAAD, LAPC4AI, LAPC-9AD, LAPC-9AI), prostate cancer cell lines (LNCaP and PC3), normal prostate (N), and prostate cancer patient tumors (T). Northern blots with 10 ug of total RNA were probed with the 251P5G2 SSH fragment. Size standards in kilobases are on the side. Results show expression of 251P5G2 in the LAPC-9AD xenograft and in prostate tumor tissues. Lower level expression was detected in the other xenograft tissues and LNCaP cell line but not in PC3. The lower panel represents ethidium-bromide staining of the gel confirming the quality of the RNA.
[0077] FIG. 18. Expression of 251P5G2 in Human Normal and Cancer Tissues. First strand cDNA was prepared from a panel of 13 normal tissues, prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, pancreas cancer pool, 2 different prostate cancer metastasis specimens to lymph node, and a pool of prostate cancer LAPC xenografts (LAPC4AD, LAPC-4AI, LAPC-9AD, and LAPC-9AI). Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 251P5G2, was performed at 26 and 30 cycles of amplification. A standard curve was generated using plasmid DNA containing 251P5G2 of known copy number. The experiment was performed in duplicate. Results show strong expression of 251P5G2 in prostate cancer metastasis, prostate cancer pool, and cancer metastasis pool. Expression of 251P5G2 was also detected in bladder cancer pool. Amongst normal tissues, very weak expression was detected in hear, prostate, skeletal muscle and testis but not in any other normal tissue tested.
[0078] FIG. 19. Expression of 251P5G2 in Prostate Cancer Patient Specimens. First strand cDNA was prepared from normal prostate, prostate cancer cell lines (PC3, DU145, LNCaP, 293T), and a panel of prostate cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 251P5G2, was performed at 26 and 30 cycles of amplification. Results show expression of 251P5G2 in 10 out of 19 patient specimens. Very strong expression was detected in 5 out of the 10 expressing tumors. Expression was also detected in LNCaP but not in the other cell lines tested nor in normal prostate.
[0079] FIG. 20. Expression of 251P5G2 in Bladder Cancer Patient Specimens. First strand cDNA was prepared from normal bladder, bladder cancer cell lines (UM-UC-3, TCCSUP, J82), and a panel of bladder cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 251P5G2, was performed at 26 and 30 cycles of amplification. Results show expression of 251P5G2 in 5 out of 9 patient specimens, but not in the cell lines tested nor in normal bladder.
DETAILED DESCRIPTION OF THE INVENTION Outline of Sections[0080] I.) Definitions
[0081] II.) 251P5G2 Polynucleotides
[0082] II.A.) Uses of 251P5G2 Polynucleotides
[0083] II.A.1.) Monitoring of Genetic Abnormalities
[0084] II.A.2.) Antisense Embodiments
[0085] II.A.3.) Primers and Primer Pairs
[0086] II.A.4.) Isolation of 251P5G2-Encoding Nucleic Acid Molecules
[0087] II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
[0088] III.) 251P5G2-related Proteins
[0089] III.A.) Motif-bearing Protein Embodiments
[0090] III.B.) Expression of 251P5G2-related Proteins
[0091] III.C.) Modifications of 251P5G2-related Proteins
[0092] III.D.) Uses of 251P5G2-related Proteins
[0093] IV.) 251P5G2 Antibodies
[0094] V.) 251P5G2 Cellular Immune Responses
[0095] VI.) 251P5G2 Transgenic Animals
[0096] VII.) Methods for the Detection of 251P5G2
[0097] VIII.) Methods for Monitoring the Status of 251P5G2-related Genes and Their Products
[0098] IX.) Identification of Molecules That Interact With 251P5G2
[0099] X.) Therapeutic Methods and Compositions
[0100] X.A.) Anti-Cancer Vaccines
[0101] X.B.) 251P5G2 as a Target for Antibody-Based Therapy
[0102] X.C.) 251P5G2 as a Target for Cellular Immune Responses
[0103] X.C.1. Minigene Vaccines
[0104] X.C.2. Combinations of CTL Peptides with Helper Peptides
[0105] X.C.3. Combinations of CTL Peptides with T Cell Priming Agents
[0106] X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides
[0107] X.D.) Adoptive Immunotherapy
[0108] X.E.) Administration of Vaccines for Therapeutic or Prophylactic Purposes
[0109] XI.) Diagnostic and Prognostic Embodiments of 251P5G2.
[0110] XII.) Inhibition of 251P5G2 Protein Function
[0111] XII.A.) Inhibition of 251P5G2 With Intracellular Antibodies
[0112] XII.B.) Inhibition of 251P5G2 with Recombinant Proteins
[0113] XII.C.) Inhibition of 251P5G2 Transcription or Translation
[0114] XII.D.) General Considerations for Therapeutic Strategies
[0115] XIII.) Identification, Characterization and Use of Modulators of 251P5G2
[0116] XIV.) KITS/Articles of Manufacture
[0117] I.) Definitions:
[0118] 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.
[0119] 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.
[0120] “Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence 251P5G2 (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 251P5G2. 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.
[0121] The term “analog” refers to a molecule which is structurally similar or shares similar or corresponding attributes with another molecule (e.g. a 251P5G2-related protein). For example, an analog of a 251P5G2 protein can be specifically bound by an antibody or T cell that specifically binds to 251P5G2.
[0122] 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-251P5G2 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.
[0123] 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-251P5G2 antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-251P5G2 antibody compositions with polyepitopic specificity.
[0124] 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.”
[0125] A “combinatorial library” is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical “building blocks” such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide (e.g., mutein) library, is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Numerous chemical compounds are synthesized through such combinatorial mixing of chemical building blocks (Gallop et al., J. Med. Chem. 37(9): 1233-1251 (1994)).
[0126] Preparation and screening of combinatorial libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Pept. Prot. Res. 37:487-493 (1991), Houghton et al., Nature, 354:84-88 (1991)), peptoids (PCT Publication No WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random bio-oligomers (PCT Publication WO 92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho, et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)). See, generally, Gordon et al., J. Med. Chem. 37:1385 (1994), nucleic acid libraries (see, e.g., Stratagene, Corp.), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology 14(3): 309-314 (1996), and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science 274:1520-1522 (1996), and U.S. Pat. No. 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Baum, C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506, 337; benzodiazepines, U.S. Pat. No. 5,288,514; and the like).
[0127] Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 NIPS, 390 NIPS, Advanced Chem Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A, Applied Biosystems, Foster City, Calif.; 9050, Plus, Millipore, Bedford, Mass.). A number of well-known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations such as the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate H, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.), which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J.; Asinex, Moscow, RU; Tdpos, Inc., St. Louis, Mo.; ChemStar, Ltd, Moscow, RU; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md.; etc.).
[0128] 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 auristatins, auromycins, maytansinoids, yttrium, bismuth, ricin, ricin A-chain, combrestatin, duocarmycins, dolostatins, doxorubicin, daunorubicin, taxol, cisplatin, ccl065, 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 or 213, P32 and radioactive isotopes of Lu including Lu177. Antibodies may also be conjugated to an anti-cancer pro-drug activating enzyme capable of converting the pro-drug to its active form.
[0129] The “gene product” is sometimes referred to herein as a protein or mRNA. For example, a “gene product of the invention” is sometimes referred to herein as a “cancer amino acid sequence”, “cancer protein”, “protein of a cancer listed in Table I”, a “cancer mRNA”, “mRNA of a cancer listed in Table I”, etc. In one embodiment, the cancer protein is encoded by a nucleic acid of FIG. 2. The cancer protein can be a fragment, or alternatively, be the full-length protein to the fragment encoded by the nucleic acids of FIG. 2. In one embodiment, a cancer amino acid sequence is used to determine sequence identity or similarity. In another embodiment, the sequences are naturally occurring allelic variants of a protein encoded by a nucleic acid of FIG. 2. In another embodiment, the sequences are sequence variants as further described herein.
[0130] “High throughput screening” assays for the presence, absence, quantification, or other properties of particular nucleic acids or protein products are well known to those of skill in the art. Similarly, binding assays and reporter gene assays are similarly well known. Thus, e.g., U.S. Pat. No. 5,559,410 discloses high throughput screening methods for proteins; U.S. Pat. No. 5,585,639 discloses high throughput screening methods for nucleic acid binding (i.e., in arrays); while U.S. Pat. Nos. 5,576,220 and 5,541,061 disclose high throughput methods of screening for ligand/antibody binding.
[0131] In addition, high throughput screening systems are commercially available (see, e.g., Amersham Biosdences, Piscataway, N.J.; Zymark Corp., Hopkinton, Mass.; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick, Mass.; etc.). These systems typically automate entire procedures, including all sample and reagent pipetling, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for various high throughput systems. Thus, e.g., Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.
[0132] 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.
[0133] “Human Leukocyte Antigen” or “HLA” is a human class I or class II Major Histocompatibility Complex (MHC) protein (see, e.g., Stites, et at., IMMUNOLOGY, 8TH ED., Lange Publishing, Los Altos, Calif. (1994).
[0134] 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.
[0135] 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 251P5G2 genes or that encode polypeptides other than 251P5G2 gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 251P5G2 polynucleotide. A protein is said to be “isolated,” for example, when physical, mechanical or chemical methods are employed to remove the 251P5G2 proteins from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated 251P5G2 protein. Alternatively, an isolated protein can be prepared by chemical means.
[0136] 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.
[0137] 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.
[0138] The term “modulator” or “test compound” or “drug candidate” or grammatical equivalents as used herein describe any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for the capacity to directly or indirectly alter the cancer phenotype or the expression of a cancer sequence, e.g., a nucleic acid or protein sequences, or effects of cancer sequences (e.g., signaling, gene expression, protein interaction, etc.) In one aspect, a modulator will neutralize the effect of a cancer protein of the invention. By “neutralize” is meant that an activity of a protein is inhibited or blocked, along with the consequent effect on the cell. In another aspect, a modulator will neutralize the effect of a gene, and its corresponding protein, of the invention by normalizing levels of said protein. In preferred embodiments, modulators alter expression profiles, or expression profile nucleic acids or proteins provided herein, or downstream effector pathways. In one embodiment, the modulator suppresses a cancer phenotype, e.g. to a normal tissue fingerprint. In another embodiment, a modulator induced a cancer phenotype. Generally, a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.
[0139] Modulators, drug candidates or test compounds encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 Daltons. Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less than 500 D. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Modulators also comprise biomolecules such as peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides. One class of modulators are peptides, for example of from about five to about 35 amino acids, with from about five to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. Preferably, the cancer modulatory protein is soluble, includes a non-transmembrane region, and/or, has an N-terminal Cys to aid in solubility. In one embodiment, the C-terminus of the fragment is kept as a free acid and the N-terminus is a free amine to aid in coupling, i.e., to cysteine. In one embodiment, a cancer protein of the invention is conjugated to an immunogenic agent as discussed herein. In one embodiment, the cancer protein is conjugated to BSA. The peptides of the invention, e.g., of preferred lengths, can be linked to each other or to other amino acids to create a longer peptide/protein. The modulatory peptides can be digests of naturally occurring proteins as is outlined above, random peptides, or “biased” random peptides. In a preferred embodiment, peptide/protein-based modulators are antibodies, and fragments thereof, as defined herein.
[0140] Modulators of cancer can also be nucleic acids. Nucleic acid modulating agents can be naturally occurring nucleic acids, random nucleic acids, or “biased” random nucleic acids. For example, digests of prokaryotic or eukaryotic genomes can be used in an approach analogous to that outlined above for proteins.
[0141] 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.
[0142] A “motif”, as in biological motif of a 251P5G2-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.
[0143] 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.
[0144] “Pharmaceutically acceptable” refers to a non-toxic, inert, and/or composition that is physiologically compatible with humans or other mammals.
[0145] 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 “oligonuleotide”. 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).
[0146] 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”.
[0147] 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. Alternatively, in another embodiment, the primary anchor residues of a peptide binds 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.
[0148] “Radioisotopes” include, but are not limited to the following (non-limiting exemplary uses are also set forth):
[0149] Examples of Medical Isotopes: 1 Isotope Description of use Actinium-225 See Thorium-229 (Th-229) (AC-225) Actinium-227 Parent of Radium-223 (Ra-223) which is an alpha emitter used to treat metastases in the (AC-227) skeleton resulting from cancer (i.e., breast and prostate cancers), and cancer radioimmunotherapy Bismuth-212 See Thorium-228 (Th-228) (Bi-212) Bismuth-213 See Thorium-229 (Th-229) (Bi-213) Cadmium-109 Cancer detection (Cd-109) Cobalt-60 Radiation source for radiotherapy of cancer, for food irradiators, and for sterilization of (Co-60) medical supplies Copper-64 A positron emitter used for cancer therapy and SPECT imaging (Cu-64) Copper-67 Beta/gamma emitter used in cancer radioimmunotherapy and diagnostic studies (i.e., breast (Cu-67) and colon cancers, and lymphoma) Dysprosium-166 Cancer radioimmunotherapy (Dy-166) Erbium-169 Rheumatoid arthritis treatment, particularly for the small joints associated with fingers and (Er-169) toes Europium-152 Radiation source for food irradiation and for sterilization of medical supplies (Eu-152) Europium-154 Radiation source for food irradiation and for sterilization of medical supplies (Eu-154) Gadolinium-153 Osteoporosis detection and nuclear medical quality assurance devices (Gd-153) Gold-198 Implant and intracavity therapy of ovarian, prostate, and brain cancers (Au-198) Holmium-166 Multiple myeloma treatment in targeted skeletal therapy, cancer radioimmunotherapy, bone (Ho-166) marrow ablation, and rheumatoid arthritis treatment Iodine-125 Osteoporosis detection, diagnostic imaging, tracer drugs, brain cancer treatment, (I-125) radiolabeling, tumor imaging, mapping of receptors in the brain, interstitial radiation therapy, brachytherapy for treatment of prostate cancer, determination of glomerular filtration rate (GFR), determination of plasma volume, detection of deep vein thrombosis of the legs Iodine-131 Thyroid function evaluation, thyroid disease detection, treatment of thyroid cancer as well as (I-131) other non-malignant thyroid diseases (i.e., Graves disease, goiters, and hyperthyroidism), treatment of leukemia, lymphoma, and other forms of cancer (e.g., breast cancer) using radioimmunotherapy Iridium-192 Brachytherapy, brain and spinal cord tumor treatment, treatment of blocked arteries (i.e., (Ir-192) arteriosclerosis and restenosis), and implants for breast and prostate tumors Lutetium-177 Cancer radioimmunotherapy and treatment of blocked arteries (i.e., arteriosclerosis and (Lu-177) restenosis) Molybdenum-99 Parent of Technetium-99 m (Tc-99 m) which is used for imaging the brain, liver, lungs, heart, (Mo-99) and other organs. Currently, Tc-99 m is the most widely used radioisotope used for diagnostic imaging of various cancers and diseases involving the brain, heart, liver, lungs; also used in detection of deep vein thrombosis of the legs Osmium-194 Cancer radioimmunotherapy (Os-194) Palladium-103 Prostate cancer treatment (Pd-103) Platinum-195 m Studies on biodistribution and metabolism of cisplatin, a chemotherapeutic drug (Pt-195 m) Phosphorus-32 Polycythemia rubra vera (blood cell disease) and leukemia treatment, bone cancer (P-32) diagnosis/treatment; colon, pancreatic, and liver cancer treatment; radiolabeling nucleic acids for in vitro research, diagnosis of superficial tumors, treatment of blocked arteries (i.e., arteriosclerosis and restenosis), and intracavity therapy Phosphorus-33 Leukemia treatment, bone disease diagnosis/treatment, radiolabeling, and treatment of (P-33) blocked arteries (i.e., arteriosclerosis and restenosis) Radium-223 See Actinium-227 (Ac-227) (Ra-223) Rhenium-186 Bone cancer pain relief, rheumatoid arthritis treatment, and diagnosis and treatment of (Re-186) lymphoma and bone, breast, colon, and liver cancers using radioimmunotherapy Rhenium-188 Cancer diagnosis and treatment using radioimmunotherapy, bone cancer pain relief, (Re-188) treatment of rheumatoid arthritis, and treatment of prostate cancer Rhodium-105 Cancer radioimmunotherapy (Rh-105) Samarium-145 Ocular cancer treatment (Sm-145) Samarium-153 Cancer radioimmunotherapy and bone cancer pain relief (Sm-153) Scandium-47 Cancer radioimmunotherapy and bone cancer pain relief (Sc-47) Selenium-75 Radiotracer used in brain studies, imaging of adrenal cortex by gamma-scintigraphy, lateral (Se-75) locations of steroid secreting tumors, pancreatic scanning, detection of hyperactive parathyroid glands, measure rate of bile acid loss from the endogenous pool Strontium-85 Bone cancer detection and brain scans (Sr-85) Strontium-89 Bone cancer pain relief, multiple myeloma treatment, and osteoblastic therapy (Sr-89) Technetium-99 m See Molybdenum-99 (Mo-99) (Tc-99 m) Thorium-228 Parent of Bismuth-212 (Bi-212) which is an alpha emitter used in cancer radioimmunotherapy (Th-228) Thorium-229 Parent of Actinium-225 (Ac-225) and grandparent of Bismuth-213 (Bi-213) which are alpha (Th-229) emitters used in cancer radioimmunotherapy Thulium-170 Gamma source for blood irradiators, energy source for implanted medical devices (Tm-170) Tin-117 m Cancer immunotherapy and bone cancer pain relief (Sn-117 m) Tungsten-188 Parent for Rhenium-188 (Re-188) which is used for cancer diagnostics/treatment, bone (W-188) cancer pain relief, rheumatoid arthritis treatment, and treatment of blocked arteries (i.e., arteriosclerosis and restenosis) Xenon-127 Neuroimaging of brain disorders, high resolution SPECT studies, pulmonary function tests, (Xe-127) and cerebral blood flow studies Ytterbium-175 Cancer radioimmunotherapy (Yb-175) Yttrium-90 Microseeds obtained from irradiating Yttrium-89 (Y-89) for liver cancer treatment (Y-90) Yttrium-91 A gamma-emitting label for Yttrium-90 (Y-90) which is used for cancer radioimmunotherapy (Y-91) (i.e., lymphoma, breast, colon, kidney, lung, ovarian, prostate, pancreatic, and inoperable liver cancers)
[0150] By “randomized” or grammatical equivalents as herein applied to nucleic acids and proteins is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. These random peptides (or nucleic acids, discussed herein) can incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.
[0151] In one embodiment, a library is “fully randomized,” with no sequence preferences or constants at any position. In another embodiment, the library is a “biased random” library. That is, some positions within the sequence either are held constant, or are selected from a limited number of possibilities. For example, the nucleotides or amino acid residues are randomized within a defined class, e.g., of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.
[0152] A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular manipulation in vitro.
[0153] Non-limiting examples of small molecules include compounds that bind or interact with 251P5G2, ligands including hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit 251P5G2 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, 251P5G2 protein; are not found in naturally occurring metabolic pathways; and/or are more soluble in aqueous than non-aqueous solutions
[0154] “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 lnterscience Publishers, (1995).
[0155] “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.
[0156] An HLA “supermotif” is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles. Overall phenotypic frequencies of HLA-supertypes in different ethnic populations are set forth in Table IV (F). The non-limiting constituents of various supetypes are as follows:
[0157] A2: A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*6802, A*6901, A*0207
[0158] A3: A3, A11, A31, A*3301, A*6801, A*0301, A*1101, A*3101
[0159] B7: B7, B*3501-03, B*51, B*5301, B*5401, B*5501, B*5502, B*5601, B*6701, B*7801, B*0702, B*5101, B*5602
[0160] B44: B*3701, B*4402, B*4403, B*60 (B*4001), B61 (B*4006)
[0161] A1: A*0102, A*2604, A*3601, A*4301, A*8001
[0162] A24: A*24, A*30, A*2403, A*2404, A*3002, A*3003
[0163] B27: B*1401-02, B*1503, B*1509, B*1510, B*1518, B*3801-02, B*3901, B*3902, B*3903-04, B*4801-02, B*7301, B*2701-08
[0164] B58: B*1516, B*1517, B*5701, B*5702, B58
[0165] B62: B*4601, B52, B*1501 (B62), B*1502 (B75), B*1513 (B77)
[0166] Calculated population coverage afforded by different HLA-supertype combinations are set forth in Table IV (G).
[0167] 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.
[0168] 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.
[0169] 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 anywhole unitintegerfrom 1-150 ormore, 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, or 150 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.
[0170] 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 251P5G2 protein shown in FIG. 2 or FIG. 3. An analog is an example of a variant protein. Splice isoforms and single nucleotides polymorphisms (SNPs) are further examples of variants.
[0171] The “251P5G2-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 251P5G2 proteins or fragments thereof, as well as fusion proteins of a 251P5G2 protein and a heterologous polypeptide are also included. Such 251P5G2 proteins are collectively referred to as the 251P5G2-related proteins, the proteins of the invention, or 251P5G2. The term “251P5G2-related protein” refers to a polypeptide fragment or a 251P5G2 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, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 576 or more amino acids.
[0172] II.) 251P5G2 Polynucleotides
[0173] One aspect of the invention provides polynucleotides corresponding or complementary to all or part of a 251P5G2 gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding a 251P5G2-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to a 251P5G2 gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to a 251P5G2 gene, mRNA, or to a 251P5G2 encoding polynucleotide (collectively, “251P5G2 polynucleotides”). In all instances when referred to in this section, T can also be U in FIG. 2.
[0174] Embodiments of a 251P5G2 polynucleotide include: a 251P5G2 polynucleotide having the sequence shown in FIG. 2, the nucleotide sequence of 251P5G2 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 251P5G2 nucleotides comprise, without limitation:
[0175] (I) a polynucleotide comprising, consisting essentially of, or consisting of a sequence as shown in FIG. 2, wherein T can also be U;
[0176] (II) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2A, from nucleotide residue number 722 through nucleotide residue number 1489, including the stop codon, wherein T can also be U;
[0177] (III) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2B, from nucleotide residue number 722 through nucleotide residue number 1489, including the stop codon, wherein T can also be U;
[0178] (IV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2C, from nucleotide residue number 722 through nucleotide residue number 1489, including the a stop codon, wherein T can also be U;
[0179] (V) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2D, from nucleotide residue number 722 through nucleotide residue number 1489, including the stop codon, wherein T can also be U;
[0180] (VI) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2E, from nucleotide residue number 722 through nucleotide residue number 1489, including the stop codon, wherein T can also be U;
[0181] (VII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2F, from nucleotide residue number 722 through nucleotide residue number 1489, including the stop codon, wherein T can also be U;
[0182] (VIII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2G, from nucleotide residue number 722 through nucleotide residue number 1489, including the stop codon, wherein T can also be U;
[0183] (IX) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2H, from nucleotide residue number 722 through nucleotide residue number 1489, including the stop codon, wherein T can also be U;
[0184] (X) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2I, from nucleotide residue number 722 through nucleotide residue number 1489, including the stop codon, wherein T can also be U;
[0185] (XI) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2J, from nucleotide residue number 722 through nucleotide residue number 1489, including the stop codon, wherein T can also be U;
[0186] (XII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2K, from nucleotide residue number 722 through nucleotide residue number 1489, including the stop codon, wherein T can also be U;
[0187] (XIII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2L, from nucleotide residue number 722 through nucleotide residue number 4522, including the stop codon, wherein T can also be U;
[0188] (XIV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2M, from nucleotide residue number 1 through nucleotide residue number 3801, including the stop codon, wherein T can also be U;
[0189] (XV) a polynucleotide that encodes a 251P5G2-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homologous to an entire amino acid sequence shown in FIGS. 2A-M;
[0190] (XVI) a polynucleotide that encodes a 251P5G2-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an entire amino acid sequence shown in FIGS. 2A-M;
[0191] (XVII) a polynucleotide that encodes at least one peptide set forth in Tables VIII-XXI and XXII-XLIX;
[0192] (XVIII) a polynucleotide that encodes a peptide region of at least 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 amino acids of a peptide of FIG. 3A-D in any whole number increment up to 255 that includes at least 1, 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 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
[0193] (XIX) a polynucleotide that encodes a peptide region of at least 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 amino acids of a peptide of FIG. 3A-D in any whole number increment up to 255 that includes 1, 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 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
[0194] (XX) a polynucleotide that encodes a peptide region of at least 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 amino acids of a peptide of FIG. 3A-D in any whole number increment up to 255 that includes 1, 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 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
[0195] (XXI) a polynucleotide that encodes a peptide region of at least 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 amino acids of a peptide of FIG. 3A-D in any whole number increment up to 255 that includes 1, 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 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
[0196] (XXII) a polynucleotide that encodes a peptide region of at least 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 amino acids of a peptide of FIGS. 3A-D in any whole number increment up to 255 that includes 1, 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 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;
[0197] (XXIII) a polynucleotide that encodes a peptide region of at least 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 amino acids of a peptide of FIG. 3E in any whole number increment up to 1266 that includes 1, 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 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
[0198] (XXIV) a polynucleotide that encodes a peptide region of at least 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 amino acids of a peptide of FIG. 3E in any whole number increment up to 1266 that includes 1, 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 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
[0199] (XXV) a polynucleotide that encodes a peptide region of at least 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 amino acids of a peptide of FIG. 3E in any whole number increment up to 1266 that includes 1, 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 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
[0200] (XXVI) a polynucleotide that encodes a peptide region of at least 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 amino acids of a peptide of FIG. 3E in any whole number increment up to 1266 that includes 1, 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 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
[0201] (XXVII) a polynucleotide that encodes a peptide region of at least 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 amino acids of a peptide of FIG. 3E in any whole number increment up to 1266 that indudes 1, 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 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9
[0202] (XXVIII) a polynucleotide that is fully complementary to a polynucleotide of any one of (I)-(XXVII).
[0203] (XXIX) a peptide that is encoded by any of (I) to (XXVIII); and
[0204] (XXX) a composition comprising a polynucleotide of any of (I)-(XXVIII) or peptide of (XXIX) together with a pharmaceutical excipient and/or in a human unit dose form.
[0205] (XXXI) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a method to modulate a cell expressing 251P5G2,
[0206] (XXXII) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 251P5G2
[0207] (XXXIII) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 251P5G2, said cell from a cancer of a tissue listed in Table I;
[0208] (XXIV) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a method to diagnose, prophylax, prognose, or treat a a cancer;
[0209] (XXXV) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XXIX) or a composition of (XXX) in a method to diagnose, prophylax, prognose, or treat a a cancer of a tissue listed in Table I; and,
[0210] (XXXVI) a method of using a polynucleotide of any (I)-(XXVIII) or peptide of (XXIX) or a composiion of (XXX) in a method to identify or characterize a modulator of a cell expressing 251P5G2.
[0211] As used herein, a range is understood to disclose specifically all whole unit positions thereof.
[0212] Typical embodiments of the invention disclosed herein include 251P5G2 polynucleotides that encode specific portions of 251P5G2 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:
[0213] (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, 250, or 255 or more contiguous amino acids of 251P5G2 variant 1; the maximal lengths relevant for other variants are: variant 2, 255 amino acids; variant 3, 255 amino acids, variant 4, 255 amino acids, and variant 12, 1266 amino acids.
[0214] 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 251P5G2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 251P5G2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 251P5G2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 251P5G2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the 251P5G2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 50 to about amino acid 60 of the 251P5G2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the 251P5G2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 70 to about amino acid 80 of the 251P5G2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 80 to about amino acid 90 of the 251P5G2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90 to about amino acid 100 of the 251P5G2 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 251P5G2 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.
[0215] Polynucleotides encoding relatively long portions of a 251P5G2 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 251P5G2 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 251P5G2 sequence as shown in FIG. 2.
[0216] Additional illustrative embodiments of the invention disclosed herein include 251P5G2 polynucleotide fragments encoding one or more of the biological motifs contained within a “251P5G2 protein” or variant sequence, including one or more of the motif-bearing subsequences of a 251P5G2 protein or varianr set forth in Tables VIII-XXI and XXII-XLIX. In another embodiment, typical polynucleotide fragments of the invention encode one or more of the regions of 251P5G2 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 251P5G2 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.
[0217] Note that to determine the starting position of any peptide set forth in Tables VIII-XXI and Tables XXII to XLIX (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 listed in Table VII. Generally, a unique Search Peptide is used to obtain HLA peptides for a particular variant. The position of each Search Peptide relative to its respective parent molecule is listed in Table VII. Accordingly, if a Search Peptide begins at position “X”, one must add the value “X minus 1” to each position in Tables VIII-XXI and Tables XXII-IL 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 its 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.
[0218] II.A.) Uses of 251P5G2 Polynucleotides
[0219] II.A.1.) Monitoring of Genetic Abnormalities
[0220] The polynucleotides of the preceding paragraphs have a number of different specific uses. The human 251P5G2 gene maps to the chromosomal location set forth in the Example entitled “Chromosomal Mapping of 251P5G2.” For example, because the 251P5G2 gene maps to this chromosome, polynucleotides that encode different regions of the 251P5G2 proteins are used to characterize cytogenefic 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 251P5G2 proteins provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes 251P5G2 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)).
[0221] Furthermore, as 251P5G2 was shown to be highly expressed in prostate and other cancers, 251P5G2 polynucleotides are used in methods assessing the status of 251P5G2 gene products in normal versus cancerous tissues. Typically, polynucleotides that encode specific regions of the 251P5G2 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 251P5G2 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.
[0222] II.A.2.) Antisense Embodiments
[0223] Other specifically contemplated nucleic acid related embodiments of the invention disclosed herein are genomic DNA, cDNAs, dibozymes, 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 indude molecules capable of inhibiting the RNA or protein expression of 251P5G2. 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 251P5G2 polynucleotides and polynucleotide sequences disclosed herein.
[0224] 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., 251P5G2. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The 251P5G2 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 phosphor6thioates) 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 a., J. Am. Chem. Soc. 112:1253-1254 (1990). Additional 251P5G2 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).
[0225] The 251P5G2 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 251P5G2 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 251P5G2 mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 251P5G2 antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 251P5G2 mRNA. Optionally, 251P5G2 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 251P5G2. Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of 251P5G2 expression, see, e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).
[0226] II.A.3.) Primers and Primer Pairs
[0227] Further specific embodiments of these 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 251P5G2 polynucleotide in a sample and as a means for detecting a cell expressing a 251P5G2 protein.
[0228] Examples of such probes indude polypeptides comprising all or part of the human 251P5G2 cDNA sequence shown in FIG. 2. Examples of primer pairs capable of specifically amplifying 251P5G2 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 251P5G2 mRNA.
[0229] The 251P5G2 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 251P5G2 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 251P5G2 polypeptides; as tools for modulating or inhibiting the expression of the 251P5G2 gene(s) and/or translation of the 251P5G2 transcript(s); and as therapeutic agents.
[0230] The present invention includes the use of any probe as described herein to identify and isolate a 251P5G2 or 251P5G2 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.
[0231] II.A.4.) Isolation of 251P5G2-Encoding Nucleic Acid Molecules
[0232] The 251P5G2 cDNA sequences described herein enable the isolation of other polynucleotides encoding 251P5G2 gene product(s), as well as the isolation of polynucleotides encoding 251P5G2 gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of a 251P5G2 gene product as well as polynucleotides that encode analogs of 251P5G2-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding a 251P5G2 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 doning methodologies can be conveniently employed, using commercially available cloning systems (e.g., Lambda ZAP Express, Stratagene). Phage clones containing 251P5G2 gene cDNAs can be identified by probing with a labeled 251P5G2 cDNA or a fragment thereof. For example, in one embodiment, a 251P5G2 cDNA (e.g., FIG. 2) or a portion thereof can be synthesized and used as a probe to retrieve overlapping and full-length cDNAs corresponding to a 251P5G2 gene. A 251P5G2 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 251P5G2 DNA probes or primers.
[0233] II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
[0234] The invention also provides recombinant DNA or RNA molecules containing a 251P5G2 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).
[0235] The invention further provides a host-vector system comprising a recombinant DNA molecule containing a 251P5G2 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 251P5G2 or a fragment, analog or homolog thereof can be used to generate 251P5G2 proteins or fragments thereof using any number of host-vector systems routinely used and widely known in the art.
[0236] A wide range of host-vector systems suitable for the expression of 251P5G2 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, 251P5G2 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 251P5G2 protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 251P5G2 and 251P5G2 mutations or analogs.
[0237] Recombinant human 251P5G2 protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 251P5G2-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding 251P5G2 or fragment, analog or homolog thereof, a 251P5G2-related protein is expressed in the 293T cells, and the recombinant 251P5G2 protein is isolated using standard purification methods (e.g., affinity purification using anti-251P5G2 antibodies). In another embodiment, a 251P5G2 coding sequence is subcloned into the retroviral vector pSR&agr;MSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPrl, 293 and rat-1 in order to establish 251P5G2 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 251P5G2 coding sequence can be used for the generation of a secreted form of recombinant 251P5G2 protein.
[0238] As discussed herein, redundancy in the genetic code permits variation in 251PSG2 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 dna.affrc.go.jp/-nakamura/codon.html.
[0239] Additional sequence modifications are known to enhance protein expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exonlintron splice site signals, transposon-like repeats, and/or other such well-haracterized 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)).
[0240] III.) 251P5G2-Related Proteins
[0241] Another aspect of the present invention provides 251P5G2-related proteins. Specific embodiments of 251P5G2 proteins comprise a polypeptide having all or part of the amino acid sequence of human 251P5G2 as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of 251P5G2 proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 251P5G2 shown in FIG. 2 or FIG. 3.
[0242] Embodiments of a 251P5G2 polypeptide include: a 251P5G2 polypeptide having a sequence shown in FIG. 2, a peptide sequence of a 251P5G2 as shown in FIG. 2 wherein T is U; at least 10 contiguous nucleotides of a polypeptide having the sequence as shown in FIG. 2; or, at least 10 contiguous peptides of a polypeptide having the sequence as shown in FIG. 2 where T is U. For example, embodiments of 251P5G2 peptides comprise, without limitation:
[0243] (I) a protein comprising, consisting essentially of, or consisting of an amino acid sequence as shown in FIGS. 2A-M or FIGS. 3A-E;
[0244] (II) a 251P5G2-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homologous to an entire amino acid sequence shown in FIGS. 2A-M or 3A-E;
[0245] (III) a 251P5G2-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an entire amino acid sequence shown in FIGS. 2A-M or 3A-E;
[0246] (IV) a protein that comprises at least one peptide set forth in Tables VIII to XLIX, optionally with a proviso that it is not an entire protein of FIG. 2;
[0247] (V) a protein that comprises at least one peptide set forth in Tables VIII-XXI, collectively, which peptide is also set forth in Tables XXII to XLIX, collectively, optionally with a proviso that it is not an entire protein of FIG. 2;
[0248] (VI) a protein that comprises at least two peptides selected from the peptides set forth in Tables VIII-XLIX, optionally with a proviso that it is not an entire protein of FIG. 2;
[0249] (VII) a protein that comprises at least two peptides selected from the peptides set forth in Tables VII to XLIX collectively, with a proviso that the protein is not a contiguous sequence from an amino acid sequence of FIG. 2;
[0250] (VIII) a protein that comprises at least one peptide selected from the peptides set forth in Tables VII-XXI; and at least one peptide selected from the peptides set forth in Tables XXII to XLIX, with a proviso that the protein is not a contiguous sequence from an amino acid sequence of FIG. 2;
[0251] (IX) a polypeptide comprising at least 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 amino acids of a protein of FIGS. 3A-D or 3E, in any whole number increment up to 255 or 1266 respectively that includes at least 1, 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 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
[0252] (X) a polypeptide comprising at least 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 amino acids of a protein of FIGS. 3A-D or 3E, in any whole number increment up to 255 or 1266 respectively that includes at least at least 1, 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 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
[0253] (XI) a polypeptide comprising at least 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 amino acids of a protein of FIGS. 3A-D, or 3E, in any whole number increment up to 255 or 1266 respectively that includes at least at least 1, 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 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
[0254] (XII) a polypeptide comprising at least 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 amino acids of a protein of FIGS. 3A-D or 3E, in any whole number increment up to 255 or 1266 respectively that includes at least at least 1, 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 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
[0255] (XIII) a polypeptide comprising at least 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, amino acids of a protein of FIGS. 3A-D or 3E in any whole number increment up to 255 or 1266 respectively that includes at least at least 1, 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 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;
[0256] (XIV) a peptide that occurs at least twice in Tables VIII-XXI and XXII to XLIX, collectively;
[0257] (XV) a peptide that occurs at least three times in Tables VIII-XXI and XXII to XLIX, collectively;
[0258] (XVI) a peptide that occurs at least four times in Tables VIII-XXI and XXII to XLIX, collectively;
[0259] (XVII) a peptide that occurs at least five times in Tables VIII-XXI and XXII to XLIX, collectively;
[0260] (XVIII) a peptide that occurs at least once in Tables VIII-XXI, and at least once in, tables XXII to XLIX;
[0261] (XIX) a peptide that occurs at least once in Tables VIII-XXI, and at least twice in tables XXII to XLIX;
[0262] (XX) a peptide that occurs at least twice in Tables VIII-XXI, and at least once in tables XXII to XLIX;
[0263] (XXI) a peptide that occurs at least twice in Tables VIII-XXI, and at least twice in tables XXII to XLIX;
[0264] (XXII) a peptide which comprises one two, three, four, or five of the following characteristics, or an oligonucleotide encoding such peptide:
[0265] i) a 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;
[0266] ii) a 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;
[0267] iii) a 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 indudes 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;
[0268] iv) a 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,
[0269] v) a 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;
[0270] (XXIII) a composition comprising a peptide of (I)-(XXII) or an antibody or binding region thereof together with a pharmaceutical excipient and/or in a human unit dose form.
[0271] (XXIV) a method of using a peptide of (I)-(XXII), or an antibody or binding region thereof or a composition of (XXIII) in a method to modulate a cell expressing 251P5G2,
[0272] (XXV) a method of using a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition of (XXIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 251P5G2
[0273] (XXVI) a method of using a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition (XXIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 251P5G2, said cell from a cancer of a tissue listed in Table I;
[0274] (XXVII) a method of using a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition of (XXIII) in a method to diagnose, prophylax, prognose, or treat a a cancer;
[0275] (XXVIII) a method of using a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition of (XXIII) in a method to diagnose, prophylax, prognose, or treat a a cancer of a tissue listed in Table I; and,
[0276] (XXIX) a method of using a a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition (XXIII) in a method to identify or characterize a modulator of a cell expressing 251P5G2.
[0277] As used herein, a range is understood to specifically disclose all whole unit positions thereof.
[0278] Typical embodiments of the invention disclosed herein include 251P5G2 polynucleotides that encode specific portions of 251P5G2 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:
[0279] (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, 250, or 255 or more contiguous amino acids of 251P5G2 variant 1; the maximal lengths relevant for other variants are: variant 2, 255 amino acids; variant 3, 255 amino acids, variant 4, 255 amino acids, and variant 12, 1266 amino acids.
[0280] In general, naturally occurring allelic variants of human 251P5G2 share a high degree of structural identity and homology (e.g., 90% or more homology). Typically, allelic variants of a 251P5G2 protein contain conservative amino acid substitutions within the 251P5G2 sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 251P5G2. One dass of 251P5G2 allelic variants are proteins that share a high degreeof homology with at least a small region of a particular 251P5G2 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.
[0281] 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 (Q) 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).
[0282] Embodiments of the invention disclosed herein include a wide variety of art-accepted variants or analogs of 251P5G2 proteins such as polypeptides having amino acid insertions, deletions and substitutions. 251P5G2 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 251P5G2 variant DNA.
[0283] 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.
[0284] As defined herein, 251P5G2 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that is “cross reactive” with a 251P5G2 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 251P5G2 variant also specifically binds to a 251P5G2 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 251P5G2 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.
[0285] Other classes of 251P5G2-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 251P5G2 protein variants or analogs comprises one or more of the 251P5G2 biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of 251P5G2 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.
[0286] As discussed herein, embodiments of the claimed invention include polypeptides containing less than the full amino acid sequence of a 251P5G2 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 251P5G2 protein shown in FIG. 2 or FIG. 3.
[0287] Moreover, representative embodiments of the invention disclosed herein include polypeptides consisting of about amino acid 1 to about amino acid 10 of a 251P5G2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of a 251P5G2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 20 to about amino acid 30 of a 251P5G2 protein shown in FIG. 2 or FIG. 3, polypepbdes consisting of about amino acid 30 to about amino acid 40 of a 251P5G2 protein shown in FIG. 2 or FIG. 3, polypepbdes consisting of about amino acid 40 to about amino acid 50 of a 251P5G2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of a 251P5G2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of a 251P5G2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of a 251P5G2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of a 251P5G2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 to about amino acid 100 of a 251P5G2 protein shown in FIG. 2 or FIG. 3, etc. throughout the entirety of a 251P5G2 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 251P5G2 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.
[0288] 251P5G2-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 251P5G2-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of a 251P5G2 protein (or variants, homologs or analogs thereof).
[0289] III.A.) Motif-bearing Protein Embodiments
[0290] Additional illustrative embodiments of the invention disclosed herein include 251P5G2 polypeptides comprising the amino acid residues of one or more of the biological motifs contained within a 251P5G2 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/; searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html; psort.ims.u-tokyo.ac.jp/; cbs.dtu.dk/; ebi.ac.uk/interpro/scan.html; expasy.ch/tools/scnpsit1.html; Epimatrix™ and Epimer™, Brown University, brown.edu/ResearchrTB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, bimas.dcrt.nih.gov/.).
[0291] Motif bearing subsequences of all 251P5G2 variant proteins are set forth and identified in Tables VIII-XXI and XXII-XLIX.
[0292] Table V sets forth several frequently occurring motifs based on pfam searches (see URL address pfam.wustl.edu/). The columns of Table V 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.
[0293] Polypeptides comprising one or more of the 251P5G2 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 251P5G2 motifs discussed above are associated with growth dysregulation and because 251P5G2 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 a., 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 my ristoylafion 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)).
[0294] 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 VIII-XXI and XXII-XLIX. CTL epitopes can be determined using specific algorithms to identify peptdes within a 251P5G2 protein that are capable of optimally binding to specified HLA alleles (e.g., Table IV; Epimatrix™ and Epimer™, Brown University, URL 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.
[0295] 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, on the basis of residues defined in Table IV, one can substitute out a deleterious residue in favor of any other residue, such as a preferred residue; substitute a less-preferred residue with a preferred residue; or substitute an originally-occurring preferred residue with another preferred residue. Substitutions can occur at primary anchor positions or at other positions in a peptide; see, e.g., Table IV.
[0296] 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 97/33602 to Chesnut et al.; Selte, Immunogenetics 1999 50(34): 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 a!, 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 etaL, J. Immunol. 149:3580-7 (1992); Parker et al., 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.
[0297] Related embodiments of the invention include polypeptdes comprising combinations of the different motifs set forth in Table VI, and/or, one or more of the predicted CTL epitopes of Tables VIII-XXI and XXII-XLIX, and/or, one or more of the predicted HTL epitopes of Tables XLVI-XLIX, 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 within 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.
[0298] 251P5G2-related proteins are embodied in many forms, preferably in isolated form. A purified 251P5G2 protein molecule will be substantially free of other proteins or molecules that impair the binding of 251P5G2 to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 251P5G2-related proteins include purified 251P5G2-related proteins and functional, soluble 251P5G2-related proteins. In one embodiment, a functional, soluble 251P5G2 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.
[0299] The invention also provides 251P5G2 proteins comprising biologically active fragments of a 251P5G2 amino acid sequence shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the starting 251P5G2 protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the starting 251P5G2 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.
[0300] 251P5G2-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 based on immunogenicity. Fragments that contain such structures are particularly useful in generating subunit-specific anti-251P5G2 antibodies or T cells or in identifying cellular factors that bind to 251P5G2. 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 idenfified, 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.
[0301] CTL epitopes can be determined using specific algorithms to identify peptides within a 251P5G2 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 (brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and BIMAS, URL bimas.dcrt.nih.gov/). Illustrating this, peptide epitopes from 251P5G2 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 (see, e.g., Tables VIII-XXI, XXII-XLIX). Specifically, the complete amino acid sequence of the 251P5G2 protein and relevant portions of other variants, i.e., for HLA Class I predictions 9 flanking residues on either side of a point mutation or exon juction, and for HLA Class II predictions 14 flanking residues on either side of a point mutation or exon junction corresponding to that variant, 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/.
[0302] 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, and 10-mer 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 251P5G2 predicted binding peptides are shown in Tables VIII-XXI and XXII-XLIX herein. In Tables VIII-XXI and XXII-XLVII, selected candidates, 9-mers and 10-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. In Tables XLVI-XLIX, selected candidates, 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.
[0303] 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.
[0304] It is to be appreciated that every epitope predicted by the BIMAS site, Epimer™ and Epimatdx™ 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.dcrtnih.gov/) are to be “applied” to a 251P5G2 protein in accordance with the invention. As used in this context “applied” means that a 251P5G2 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 251P5G2 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.
[0305] III.B.) Expression of 251P5G2-related Proteins
[0306] In an embodiment described in the examples that follow, 251P5G2 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 251P5G2 with a C-terminal 6XHis 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 251P5G2 protein in transfected cells. The secreted HIS-tagged 251P5G2 in the culture media can be purified, e.g., using a nickel column using standard techniques.
[0307] III.C.) Modifications of 251P5G2-related Proteins
[0308] Modifications of 251P5G2-related proteins such as covalent modifications are included within the scope of this invention. One type of covalent modification indudes reacting targeted amino acid residues of a 251P5G2 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of a 251P5G2 protein. Another type of covalent modification of a 251P5G2 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 251P5G2 comprises linking a 251P5G2 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.
[0309] The 251P5G2-related proteins of the present invention can also be modified to form a chimeric molecule comprising 251P5G2 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 251P5G2 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 251P5G2. A chimeric molecule can comprise a fusion of a 251P5G2-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 251P5G2 protein. In an alternative embodiment, the chimeric molecule can comprise a fusion of a 251P5G2-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 “immunoadhesin”), 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 251P5G2 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, CHI, CH2 and CH3 regions of an IgGI molecule. For the production of immunoglobulin fusions see, e.g., U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
[0310] III.D.) Uses of 251P5G2-related Proteins
[0311] The proteins of the invention have a number of different specific uses. As 251P5G2 is highly expressed in prostate and other cancers, 251P5G2-related proteins are used in methods that assess the status of 251P5G2 gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of a 251P5G2 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 251P5G2-related proteins comprising the amino acid residues of one or more of the biological motifs contained within a 251P5G2 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, 251P5G2-related proteins that contain the amino acid residues of one or more of the biological motifs in a 251P5G2 protein are used to screen for factors that interact with that region of 251P5G2.
[0312] 251P5G2 protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of a 251P5G2 protein), for identifying agents or cellular factors that bind to 251P5G2 or a particular structural domain thereof, and in various therapeutic and diagnostic contexts, induding but not limited to diagnostic assays, cancer vaccines and methods of preparing such vaccines.
[0313] Proteins encoded by the 251P5G2 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 251P5G2 gene product Antibodies raised against a 251P5G2 protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 251P5G2 protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. 251P5G2-related nucleic acids or proteins are also used in generating HTL or CTL responses.
[0314] Various immunological assays useful for the detection of 251P5G2 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. Antbodies can be labeled and used as immunological imaging reagents capable of detecting 251P5G2-expressing cells (e.g., in radioscintigraphic imaging methods). 251P5G2 proteins are also paricularly useful in generating cancer vaccines, as further described herein.
[0315] IV.) 251P5G2Antibodies
[0316] Another aspect of the invention provides antibodies that bind to 251P5G2-related proteins. Preferred antibodies specifically bind to a 251P5G2-related protein and do not bind (or bind weakly) to peptdes or proteins that are not 251P5G2-related proteins under physiological conditions. In this context, examples of physiological conditions include: 1) phosphate buffered saline; 2) Tris-buffered saline containing 25 mM Tris and 150 mM NaCl; or normal saline (0.9% NaCl); 4) animal serum such as human serum; or, 5) a combination of any of 1) through 4); these reactions preferably taking place at pH 7.5, alternatively in a range of pH 7.0 to 8.0, or alternatively in a range of pH 6.5 to 8.5; also, these reactions taking place at a temperature between 4° C. to 37° C. For example, antibodies that bind 251P5G2 can bind 251P5G2-related proteins such as the homologs or analogs thereof.
[0317] 251P5G2 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 251P5G2 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 251P5G2 is involved, such as advanced or metastatic prostate cancers.
[0318] The invention also provides various immunological assays useful for the detection and quantification of 251P5G2 and mutant 251P5G2-related proteins. Such assays can comprise one or more 251P5G2 antibodies capable of recognizing and binding a 251P5G2-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, enzymelinked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.
[0319] Immunological non-antibody assays of the invention also comprise T cell immunogenicity assays (inhibitory or stimulatory) as well as major histocompatbility complex (MHC) binding assays.
[0320] In addition, immunological imaging methods capable of detecting prostate cancer and other cancers expressing 251P5G2 are also provided by the invention, including but not limited to radioscinigraphic imaging methods using labeled 251P5G2 antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of 251P5G2 expressing cancers such as prostate cancer.
[0321] 251P5G2 antibodies are also used in methods for purifying a 251P5G2-related protein and for isolating 251P5G2 homologues and related molecules. For example, a method of purifying a 251P5G2-related protein comprises incubating a 251P5G2 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 251P5G2-related protein under conditions that permit the 251P5G2 antibody to bind to the 251P5G2-related protein; washing the solid matrix to eliminate impurities; and eluting the 251P5G2-related protein from the coupled antibody. Other uses of 251P5G2 antibodies in accordance with the invention include generating anti-idiotypic antibodies that mimic a 251P5G2 protein.
[0322] 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 251P5G2-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 251P5G2 can also be used, such as a 251P5G2 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 251P5G2-related protein is synthesized and used as an immunogen.
[0323] In addition, naked DNA immunization techniques known in the art are used (with or without purified 251P5G2-related protein or 251P5G2 expressing cells) to generate an immune response to the encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).
[0324] The amino acid sequence of a 251P5G2 protein as shown in FIG. 2 or FIG. 3 can be analyzed to select specific regions of the 251P5G2 protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of a 251P5G2 amino acid sequence are used to identify hydrophilic regions in the 251P5G2 structure. Regions of a 251P5G2 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-Doolittie, 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 251P5G2 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 251P5G2 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.
[0325] 251P5G2 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 251P5G2-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.
[0326] The antibodies or fragments of the invention can also be produced, by recombinant means. Regions that bind specifically to the desired regions of a 251P5G2 protein can also be produced in the context of chimeric or complementarity-determining region (CDR) grafted antibodies of multiple species origin. Humanized or human 251P5G2 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 Sims et al., 1993, J. Immunol. 151: 2296.
[0327] 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 251P5G2 monoclonal antibodies can be generated using doning 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 Applicatons 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 251P5G2 monodonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application WO98/24893, Kucherlapat and Jakobovits et al., published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607414; U.S. Pat. Nos. 6,162,963 issued Dec. 19, 2000; 6,150,584 issued Nov. 12, 2000; and, 6,114598 issued Sep. 5, 2000). This method avoids the in vitro manipulation required with phage display technology and efficienty produces high affinity authentic human antibodies.
[0328] Reactivity of 251P5G2 antibodies with a 251P5G2-related protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, 251P5G2-related proteins, 251P5G2-expressing cells or extracts thereof. A 251P5G2 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 251P5G2 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).
[0329] V.) 251P5G2 Cellular Immune Responses
[0330] 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.
[0331] 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 Bodmer, 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 a., SYFPEITHI, access via World Wide Web at URL (134.2.96.221/scripts.hlaserver.dll/home.htm); Sette, A. and Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994; Selle, 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 1999 November; 50(3-4):201-12, Review).
[0332] 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; Stern 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.)
[0333] 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).
[0334] 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.
[0335] Various strategies can be utilized to evaluate cellular immunogenicity, including:
[0336] 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.
[0337] 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 vftro 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.
[0338] 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.
[0339] VI.) 251P5G2 Transgenic Animals
[0340] Nucleic acids that encode a 251P5G2-related protein can also be used to generate either transgenic animals or “knock out” animals that, in tum, are useful in the development and screening of therapeutically useful reagents. In accordance with established techniques, cDNA encoding 251P5G2 can be used to clone genomic DNA that encodes 251P5G2. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 251P5G2. 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. Nos. 4,736,866 issued Apr. 12, 1988, and 4,870,009 issued Sep. 26, 1989. Typically, particular cells would be targeted for 251P5G2 transgene incorporation with tissue-specific enhancers.
[0341] Transgenic animals that include a copy of a transgene encoding 251P5G2 can be used to examine the effect of increased expression of DNA that encodes 251P5G2. 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.
[0342] Alternatively, non-human homologues of 251P5G2 can be used to construct a “251P5G2 ” knock outs animal that has a defective or altered gene encoding 251P5G2 as a result of homologous recombination between the endogenous gene encoding 251P5G2 and altered genomic DNA encoding 251P5G2 introduced into an embryonic cell of the animal. For example, cDNA that encodes 251P5G2 can be used to clone genomic DNA encoding 251P5G2 in accordance with established techniques. A portion of the genomic DNA encoding 251P5G2 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 251P5G2 polypeptide.
[0343] VII.) Methods for the Detection of 251P5G2
[0344] Another aspect of the present invention relates to methods for detecting 251P5G2 polynucleotides and 251P5G2-related proteins, as well as methods for identfying a cell that expresses 251P5G2. The expression profile of 251P5G2 makes it a diagnostic marker for metastasized disease. Accordingly, the status of 251P5G2 gene products provides information useful for predicting a variety of factors including suscepubility to advanced stage disease, rate of progression, and/or tumor aggressiveness. As discussed in detail herein, the status of 251P5G2 gene products in patient samples can be analyzed by a variety protocols that are well known in the art induding immunohistochemical analysis, the variety of Northern blotting techniques including in situ hybridizabon, RT-PCR analysis (for example on laser capture micro-dissected samples), Western blot analysis and tissue array analysis.
[0345] More particularly, the invention provides assays for the detection of 251P5G2 polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like. Detectable 251P5G2 polynucleotides include, for example, a 251P5G2 gene or fragment thereof, 251P5G2 mRNA, alternative splice variant 251P5G2 mRNAs, and recombinant DNA or RNA molecules that contain a 251P5G2 polynucleotide. A number of methods for amplifying and/or detecting the presence of 251P5G2 polynucleotides are well known in the art and can be employed in the practice of this aspect of the invention.
[0346] In one embodiment, a method for detecting a 251P5G2 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 251P5G2 polynucleotides as sense and antisense primers to amplify 251P5G2 cDNAs therein; and detecting the presence of the amplified 251P5G2 cDNA. Optionally, the sequence of the amplified 251P5G2 cDNA can be determined.
[0347] In another embodiment, a method of detecting a 251P5G2 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 251P5G2 polynucleotides as sense and antisense primers; and detecting the presence of the amplified 251P5G2 gene. Any number of appropriate sense and antisense probe combinations can be designed from a 251P5G2 nucleotide sequence (see, e.g., FIG. 2) and used for this purpose.
[0348] The invention also provides assays for detecting the presence of a 251P5G2 protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like. Methods for detecting a 251P5G2-related protein are also well known and indude, for example, immunoprecipitaton, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELISA, ELIFA and the like. For example, a method of detecting the presence of a 251P5G2-related protein in a biological sample comprises first contacting the sample with a 251P5G2 antibody, a 251P5G2-reactive fragment thereof, or a recombinant protein containing an antigen-binding region of a 251P5G2 antibody; and then detecting the binding of 251P5G2-related protein in the sample.
[0349] Methods for identifying a cell that expresses 251P5G2 are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a 251P5G2 gene comprises detecting the presence of 251P5G2 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 sdu hybridization using labeled 251P5G2 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 251P5G2, 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 251P5G2 gene comprises detecting the presence of 251P5G2-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 251P5G2-related proteins and cells that express 251P5G2-related proteins.
[0350] 251P5G2 expression analysis is also useful as a tool for identifying and evaluating agents that modulate 251P5G2 gene expression. For example, 251P5G2 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 251P5G2 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 251P5G2 expression by RT-PCR, nucleic acid hybridization or antibody binding.
[0351] VIII.) Methods for Monitoring the Status of 251P5G2-related Genes and Their Products
[0352] 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 251P5G2 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 251P5G2 in a biological sample of interest can be compared, for example, to the status of 251P5G2 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 251P5G2 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 251P5G2 status in a sample.
[0353] 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 251P5G2 expressing cells) as well as the level, and biological activity of expressed gene products (such as 251P5G2 mRNA, polynucleotides and polypeptides). Typically, an alteration in the status of 251P5G2 comprises a change in the location of 251P5G2 and/or 251P5G2 expressing cells and/or an increase in 251P5G2 mRNA and/or protein expression.
[0354] 251P5G2 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 251P5G2 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 251P5G2 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 251P5G2 gene), Northern analysis and/or PCR analysis of 251P5G2 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 251P5G2 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 251P5G2 proteins and/or associations of 251P5G2 proteins with polypeptide binding partners). Detectable 251P5G2 polynucleotides include, for example, a 251P5G2 gene or fragment thereof, 251P5G2 mRNA, alternative splice variants, 251P5G2 mRNAs, and recombinant DNA or RNA molecules containing a 251P5G2 polynucleotide.
[0355] The expression profile of 251P5G2 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 251P5G2 provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining 251P5G2 status and diagnosing cancers that express 251P5G2, such as cancers of the tissues listed in Table I. For example, because 251PSG2 mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue, assays that evaluate the levels of 251P5G2 mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 251P5G2 dysregulaton, and can provide prognostic information useful in defining appropriate therapeutic options.
[0356] The expression status of 251P5G2 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 251P5G2 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.
[0357] As described above, the status of 251P5G2 in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of 251P5G2 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 251P5G2 expressing cells (e.g. those that express 251P5G2 mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when 251P5G2-expressing cells arefound in a biological sample that does not normally contain such cells (such as a lymph node), because such alterations in the status of 251P5G2 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 1995 August 154(2 Pt 1):474-8).
[0358] In one aspect, the invention provides methods for monitoring 251P5G2 gene products by determining the status of 251P5G2 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 251P5G2 gene products in a corresponding normal sample. The presence of aberrant 251P5G2 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.
[0359] In another aspect, the invention provides assays useful in determining the presence of cancer in an individual, comprising detecting a significant increase in 251P5G2 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 251P5G2 mRNA can, for example, be evaluated in tissues including but not limited to those listed in Table I. The presence of significant 251P5G2 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 251P5G2 mRNA or express it at lower levels.
[0360] In a related embodiment, 251P5G2 status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of 251P5G2 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 251P5G2 expressed in a corresponding normal sample. In one embodiment, the presence of 251P5G2 protein is evaluated, for example, using immunohistochemical methods. 251P5G2 antibodies or binding partners capable of detecting 251P5G2 protein expression are used in a variety of assay formats well known in the art for this purpose.
[0361] In a further embodiment, one can evaluate the status of 251P5G2 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules. These perturbations can indude 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 251P5G2 may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in 251P5G2 indicates a potential loss of function or increase in tumor growth.
[0362] 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 251P5G2 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. Nos. 5,382,510 issued Sep. 7, 1999, and 5,952,170 issued Jan. 17, 1995).
[0363] Additionally, one can examine the methylaton status of a 251P5G2 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 hypermethylaton 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-azacytdine 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 methylaton 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 deave sequences that contain methylated CpG sites to assess the methylation status of CpG islands. In addition, MSP (methylation specific PCR) can rapidly profile the methylaton 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 unmethylated 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.
[0364] Gene amplification is an additional method for assessing the status of 251P5G2. 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.
[0365] 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 251P5G2 expression. The presence of RT-PCR amplifiable 251P5G2 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 indude 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).
[0366] 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 251P5G2 mRNA or 251P5G2 protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 251P5G2 mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of 251P5G2 in prostate or other tissue is examined, with the presence of 251P5G2 in the sample providing an indication of prostate cancer susceptibility (or the emergence or existence of a prostate tumor). Similarly, onecan evaluate the integrity 251P5G2 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 251P5G2 gene products in the sample is an indication of cancer susceptibility (or the emergence or existence of a tumor).
[0367] 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 251P5G2 mRNA or 251P5G2 protein expressed by tumor cells, comparing the level so determined to the level of 251P5G2 mRNA or 251P5G2 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 251P5G2 mRNA or 251P5G2 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 251P5G2 is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of 251P5G2 nucleotde 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.
[0368] 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 251P5G2 mRNA or 251P5G2 protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 251P5G2 mRNA or 251P5G2 protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 251P5G2 mRNA or 251P5G2 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 251P5G2 expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 251P5G2 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.
[0369] 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 251P5G2 gene and 251P5G2 gene products (or perturbations in 251P5G2 gene and 251P5G2 gene products) and a factor that is assodated 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 251P5G2 gene and 251P5G2 gene products (or perturbations in 251P5G2 gene and 251P5G2 gene products) and another factor that is associated with malignancy are useful, for example, because the presence of a set of specic factors that coincide with disease provides information crucial for diagnosing and prognosticating the status of a tissue sample.
[0370] In one embodiment, methods for observing a coincidence between the expression of 251P5G2 gene and 251P5G2 gene products (or perturbations in 251P5G2 gene and 251P5G2 gene products) and another factor associated with malignancy entails detecting the overexpression of 251P5G2 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 251P5G2 mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment, the expression of 251P5G2 and PSA mRNA in prostate issue is examined, where the coincidence of 251P5G2 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.
[0371] Methods for detecting and quantifying the expression of 251P5G2 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 quantificaton of 251P5G2 mRNA include in situ hybridization using labeled 251P5G2 riboprobes, Northern blot and related techniques using 251P5G2 polynucdeotde probes, RT-PCR analysis using primers specific for 251P5G2, 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 251P5G2 mRNA expression. Any number of primers capable of amplifying 251P5G2 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 251P5G2 protein can be used in an immunohistochemical assay of biopsied tissue.
[0372] IX.) Identification of Molecules That Interact With 251P5G2
[0373] The 251P5G2 protein and nucleic add sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 251P5G2, as well as pathways activated by 251P5G2 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. Nos. 5,955,280 issued Sep. 21, 1999, 5,925,523 issued Jul. 20, 1999, 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).
[0374] Alternatively one can screen peptide libraries to identify molecules that interact with 251P5G2 protein sequences. In such methods, peptides that bind to 251P5G2 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 251P5G2 protein(s).
[0375] 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 251P5G2 protein sequences are disclosed for example in U.S. Pat. Nos. 5,723,286 issued Mar. 3, 1998 and 5,733,731 issued Mar. 31, 1998.
[0376] Alternatively, cell lines that express 251P5G2 are used to identify protein-protein interactions mediated by 251P5G2. Such interactions can be examined using immunopredipitation techniques (see, e.g., Hamilton B. J., et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). 251P5G2 protein can be immunoprecipitated from 251P5G2-expressing cell lines using anti-251P5G2 antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express fusions of 251P5G2 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.
[0377] Small molecules and ligands that interact with 251P5G2 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 251P5G2'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 251P5G2-related ion channel, protein pump, or cell communication functions are identified and used to treat patients that have a cancer that expresses 251P5G2 (see, e.g., Hille, B., Ionic Channels of Excitable Membranes 2nd Ed., Sinauer Assoc., Sunderland, Mass., 1992). Moreover, ligands that regulate 251P5G2 function can be identified based on their ability to bind 251P5G2 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 251P5G2 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 251P5G2.
[0378] An embodiment of this invention comprises a method of screening for a molecule that interacts with a 251P5G2 amino acid sequence shown in FIG. 2 or FIG. 3, comprising the steps of contacting a population of molecules with a 251P5G2 amino acid sequence, allowing the population of molecules and the 251P5G2 amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 251P5G2 amino acid sequence, and then separating molecules that do not interact with the 251P5G2 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 251P5G2 amino acid sequence. The identified molecule can be used to modulate a function performed by 251P5G2. In a preferred embodiment, the 251P5G2 amino acid sequence is contacted with a library of peptides.
[0379] X.) Therapeutic Methods and Compositions
[0380] The identification of 251P5G2 as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in cancers such as those listed in Table I, opens a number of therapeutic approaches to the treatment of such cancers.
[0381] Of note, targeted antitumor therapies have been useful even when the targeted protein is expressed on normal tissues, even vital normal organ tissues. A vital organ is one that is necessary to sustain life, such as the heart or colon. A non-vital organ is one that can be removed whereupon the individual is still able to survive. Examples of non-vital organs are ovary, breast, and prostate.
[0382] For example, Herceptin® is an FDA approved pharmaceutical that has as its active ingredient an antibody which is immunoreactive with the protein variously known as HER2, HER2/neu, and erb-b-2. It is marketed by Genentech and has been a commercially successful antitumor agent. Herceptin sales reached almost $400 million in 2002. Herceptin is a treatment for HER2 positive metastatic breast cancer. However, the expression of HER2 is not limited to such tumors. The same protein is expressed in a number of normal tissues. In particular, it is known that HER2/neu is present in normal kidney and heart, thus these tissues are present in all human recipients of Herceptin. The presence of HER2/neu in normal kidney is also confirmed by Latif, Z., et al., B.J.U. International (2002) 89:5-9. As shown in this artide (which evaluated whether renal cell carcinoma should be a preferred indication for anti-HER2 antibodies such as Herceptin) both protein and mRNA are produced in benign renal tissues. Notably, HER2/neu protein was strongly overexpressed in benign renal tissue. Despite the fact that HER2/neu is expressed in such vital tissues as heart and kidney, Herceptin is a very useful, FDA approved, and commercially successful drug. The effect of Herceptin on cardiac tissue, i.e., “cardiotoxicity,” has merely been a side effect to treatment. When patients were treated with Herceptin alone, significant cardiotoxicity occurred in a very low percentage of patients.
[0383] Of particular note, although kidney tissue is indicated to exhibit normal expression, possibly even higher expression than cardiac tissue, kidney has no appreciable Herceptin side effect whatsoever. Moreover, of the diverse array of normal tissues in which HER2 is expressed, there is very little occurrence of any side effect. Only cardiac tissue has manifested any appreciable side effect at all. A tissue such as kidney, where HER2/neu expression is especially notable, has not been the basis for any side effect.
[0384] Furthermore, favorable therapeutic effects have been found for antitumor therapies that target epidermal growth factor receptor (EGFR). EGFR is also expressed in numerous normal tissues. There have been very limited side effects in normal tissues following use of anti-EGFR therapeutics.
[0385] Thus, expression of a target protein in normal tissue, even vital normal tissue, does not defeat the utility of a targeting agent for the protein as a therapeutic for certain tumors in which the protein is also overexpressed.
[0386] Accordingly, therapeutic approaches that inhibit the activity of a 251P5G2 protein are useful for patients suffering from a cancer that expresses 251P5G2. These therapeutic approaches generally fall into two classes. One class comprises various methods for inhibiting the binding or association of a 251P5G2 protein with its binding partner or with other proteins. Another class comprises a variety of methods for inhibiting the transcription of a 251P5G2 gene or translation of 251P5G2 mRNA.
[0387] X.A.) Anti-Cancer Vaccines
[0388] The invention provides cancer vaccines comprising a 251P5G2-related protein or 251P5G2-related nucleic acid. In view of the expression of 251P5G2, cancer vaccines prevent and/or treat 251P5G2-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 cellrnediated 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).
[0389] Such methods can be readily practiced by employing a 251P5G2-related protein, or a 251P5G2-encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the 251P5G2 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 1999 February 31(1):66-78; Maruyama et a., Cancer Immunol Immunother 2000 Jun 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 251P5G2 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 251P5G2 immunogen contains a biological motif, see e.g., Tables VIII-XXI and XXII-XLIX, or a peptide of a size range from 251P5G2 indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.
[0390] The entire 251P5G2 protein, immunogenic regions or epitopes thereof can be combined and delivered by various means. Such vaccine compositions can indude, 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 at., 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., Tam, 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 peptdes, viral delivery vectors (Perkus, M. E. et al., In: Concepts in vaccine development, Kaufmann, 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 orsyntheticorigin (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.
[0391] In patients with 251P5G2-assodated 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.
[0392] Cellular Vaccines:
[0393] CTL epitopes can be determined using specific algorithms to identify peptides within 251P5G2 protein that bind corresponding HLA alleles (see e.g., Table IV; Epimer™ and Epimatrix™, Brown University (URL brown.edulResearch/TB-HIV_Lab/epimatrix/epimatrix.html); and, BIMAS, (URL bimas.dcrtnih.gov/; SYFPEITHI at URL syfpeithi.bmi-heidelberg.com/). In a preferred embodiment, a 251P5G2 immunogen contains one or more amino adid sequences identified using techniques well known in the art, such as the sequences shown in Tables VIII-XXI and XXII-XLIX 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 allached 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.
[0394] Antibody-based Vaccines
[0395] 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 251P5G2 protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to 251P5G2 in a host, by contacting the host with a sufficient amount of at least one 251P5G2 B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the 251P5G2 B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a 251P5G2-related protein or a man-made multiepitopic peptide comprising: administering 251P5G2 immunogen (e.g. a 251P5G2 protein or a peptide fragment thereof, a 251P5G2 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 251P5G2 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 251P5G2 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 251P5G2, in order to generate a response to the target antigen.
[0396] Nucleic Acid Vaccines:
[0397] 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 251P5G2. Constructs comprising DNA encoding a 251P5G2-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 251P5G2 proteinlimmunogen. Alternatively, a vaccine comprises a 251P5G2-related protein. Expression of the 251P5G2-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear a 251P5G2 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 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 particlemediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).
[0398] 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 indude, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-associated virus, lenfivirus, 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 251P5G2-related protein into the patient (e.g., intramuscularly or intradenmally) to induce an anfi-tumor response.
[0399] 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 Calmelle 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.
[0400] Thus, gene delivery systems are used to deliver a 251P5G2-related nucleic acid molecule. In one embodiment, the full-length human 251P5G2 cDNA is employed. In another embodiment, 251P5G2 nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopes are employed.
[0401] Ex Vivo Vaccines
[0402] 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 dendritc cells (DC) to present 251P5G2 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 251P5G2 peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with 251P5G2 peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete 251P5G2 protein. Yet another embodiment involves engineering the overexpression of a 251P5G2 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 at., 1997, J. Exp. Med. 186:1177-1182). Cells that express 251P5G2 can also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents.
[0403] X.B.) 251P5G2 as a Target for Antibody-based Therapy
[0404] 251P5G2 is an allractive 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 251P5G2 is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of 251P5G2-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 251P5G2 are useful to treat 251P5G2-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.
[0405] 251P5G2 antibodies can be introduced into a patient such that the antibody binds to 251P5G2 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 251P5G2, inhibition of ligand binding or signal transduction pathways, modulation of tumor cell differentiation, alteration of tumor angiogenesis factor profiles, and/or apoptosis.
[0406] 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 251P5G2 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. 251P5G2), the cytotoxic agent will exert its known biological effect (i.e. cytotoxicity) on those cells.
[0407] 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-251P5G2 antibody) that binds to a marker (e.g. 251P5G2) 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 251P5G2, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 251P5G2 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.
[0408] Cancer immunotherapy using anti-251P5G2 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, 251P5G2 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., Mylotarglm, 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).
[0409] Although 251P5G2 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.
[0410] Although 251P5G2 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.
[0411] Cancer patients can be evaluated for the presence and level of 251P5G2 expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 251P5G2 imaging, or other techniques that reliably indicate the presence and degree of 251P5G2 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.
[0412] Anti-251P5G2 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-251P5G2 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-251P5G2 mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 251P5G2. 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-251P5G2 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.
[0413] 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 251P5G2 antigen with high affinity but exhibit low or no antigenicity in the patient.
[0414] Therapeutic methods of the invention contemplate the administration of single anti-251P5G2 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-251P5G2 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-251P5G2 mAbs are administered in their “naked” or unconjugated form, or can have a therapeutic agent(s) conjugated to them.
[0415] Anti-251P5G2 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-251P5G2 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.
[0416] Based on clinical experience with the Herceptn™ 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-251P5G2 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 251P5G2 expression in the patient, the extent of circulating shed 251P5G2 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.
[0417] Optionally, patients should be evaluated for the levels of 251P5G2 in a given sample (e.g. the levels of circulating 251P5G2 antigen and/or 251P5G2 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 andlor ImmunoCyt levels in bladder cancer therapy, or by analogy, serum PSA levels in prostate cancer therapy).
[0418] Anti-idiotypic anti-251P5G2 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 251P5G2-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-251P5G2 antibodies that mimic an epitope on a 251P5G2-related protein (see, for example, Wagner et al., 1997, Hybridoma 16: 3340; 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.
[0419] X.C.) 251P5G2 as a Target for Cellular Immune Responses
[0420] 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., recombinanuy or by chemical synthesis.
[0421] 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))
[0422] 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 251P5G2 antigen, or derives at least some therapeutic benefit when the antigen was tumor-associated.
[0423] 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).
[0424] 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.
[0425] 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.
[0426] 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 frequenuy-expressed TAAs.
[0427] 2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenidity: 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.
[0428] 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.
[0429] 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.
[0430] 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.
[0431] 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.
[0432] 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.
[0433] X.C.1. Minigene Vaccines
[0434] 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.
[0435] 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 251P5G2, the PADRE® universal helper T cell epitope or multiple HTL epitopes from 251P5G2 (see e.g., Tables VIII-XXI and XXII to XLIX), and an endoplasmic reticulum-translocating signal sequence can be engineered. A vaccine may also comprise epitopes that are derived from other TAAs.
[0436] 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.
[0437] 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.
[0438] 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.
[0439] 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.
[0440] 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.
[0441] 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.
[0442] 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.
[0443] 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.
[0444] 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.
[0445] 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 Feigner, 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.
[0446] 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 cellslare 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.
[0447] 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.
[0448] 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.
[0449] 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.
[0450] X.C.2. Combinations of CTL Peptides with Helper Peptides
[0451] 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.
[0452] 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 peptde 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.
[0453] 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: 37), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 38), and Streptococcus 18kD protein at positions 116-131 (GAVDSILGGVATYGM; SEQ ID NO: 39). Other examples include peptides bearing a DR 1-4-7 supermotif, or either of the DR3 motifs.
[0454] 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, most preferably, to bind most HLA-DR (human HLA class II) molecules. For instance, a pan-DR-binding epitope peptide having the formula: XKXVAAWTLKMX (SEQ ID NO: 40), 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.
[0455] 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.
[0456] X.C.3. Combinations of CTL Peptides with T Cell Priming Agents
[0457] 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 allached 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.
[0458] 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 prime specifically 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.
[0459] X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL andlor HTL Peptides
[0460] 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.
[0461] The DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL responses to 251P5G2. 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 251P5G2.
[0462] X.D. Adoptive Immunotherapy
[0463] Antigenic 251P5G2-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 patients, 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.
[0464] X.E. Administration of Vaccines for Therapeutic or Prophylactic Purposes
[0465] Pharmaceutical and vaccine compositions of the invention are typically used to treat and/or prevent a cancer that expresses or overexpresses 251P5G2. In therapeutic applications, peptide and/or nucleic acid compositions are administered to a patient in 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.
[0466] 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 251P5G2. 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.
[0467] For therapeutic use, administration should generally begin at the first diagnosis of 251P5G2-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 251P5G2, a vaccine comprising 251P5G2-specific CTL may be more efficacious in killing tumor cells in patient with advanced disease than alternative embodiments.
[0468] It is generally important to provide an amount of the peptide epitope delivered by a mode of administration sufficient to stimulate effectively a cytptoxic T cell response; compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the invention.
[0469] 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.
[0470] 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.
[0471] 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 &mgr;g 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.
[0472] 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.
[0473] 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.
[0474] The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, welting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
[0475] 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.
[0476] 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 add (0.1 to 1000 &mgr;g) can also be administered using a gene gun. Following an incubation period of 34 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.
[0477] For antibodies, a treatment generally involves repeated administration of the anti-251P5G2 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-251P5G2 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 251P5G2 expression in the patient, the extent of circulating shed 251P5G2 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; 0.5mg, 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.
[0478] 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.
[0479] 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.
[0480] 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.
[0481] 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.
[0482] 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%.
[0483] 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.
[0484] XI.) Diagnostic and Prognostic Embodiments of 251P5G2.
[0485] As disclosed herein, 251P5G2 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 251P5G2 in normal tissues, and patient specimens”).
[0486] 251P5G2 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. Aug; 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 1999 July 4(1):99-102 and Minimoto et al., Cancer Detect Prev 2000; 24(1):1-12). Therefore, this disclosure of 251P5G2 polynucleotides and polypeptides (as well as 251P5G2 polynucleotide probes and anti-251P5G2 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.
[0487] Typical embodiments of diagnostic methods which utilize the 251P5G2 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 251P5G2 polynucleotides described herein can be utilized in the same way to detect 251P5G2 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 251P5G2 polypeptides described herein can be utilized to generate antibodies for use in detecting 251P5G2 overexpression or the metastasis of prostate cells and cells of other cancers expressing this gene.
[0488] 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 251P5G2 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 251P5G2-expressing cells (lymph node) is found to contain 251P5G2-expressing cells such as the 251P5G2 expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.
[0489] Alternatively 251P5G2 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 251P5G2 or express 251P5G2 at a different level are found to express 251P5G2 or have an increased expression of 251P5G2 (see, e.g., the 251P5G2 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 251P5G2) such as PSA, PSCA etc. (see, e.g., Alanen et al, Pathol. Res. Pract. 192(3): 233-237 (1996)).
[0490] Just as PSA polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA, 251P5G2 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 251P5G2 in normal tissues, and patient specimens,” where a 251P5G2 polynucleotide fragment is used as a probe to show the expression of 251P5G2 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 November-December 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 251P5G2 polynucleotide shown in FIG. 2 or variant thereof) under conditions of high stringency.
[0491] 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. 251P5G2 polypeptide fragments and polypeptide analogs or variants can also be used in an analogous manner. This practice of using polypeptide fragments or polypeptde 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 251P5G2 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 251P5G2 polypeptide shown in FIG. 3).
[0492] As shown herein, the 251P5G2 polynucleotides and polypeptides (as well as the 251P5G2 polynucleotide probes and anti-251P5G2 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 251P5G2 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 prostatc 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 251P5G2 polynucleotides and polypeptides (as well as the 251P5G2 polynucleotide probes and anti-251P5G2 antibodies used to identify the presence of these molecules) need to be employed to confirm a metastases of prostatic origin.
[0493] Finally, in addition to their use in diagnostic assays, the 251P5G2 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 251P5G2 gene maps (see the Example entitled “Chromosomal Mapping of 251P5G2” below). Moreover, in addition to their use in diagnostic assays, the 251P5G2-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).
[0494] Additionally, 251P5G2-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of 251P5G2. 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 251P5G2 antigen. Antibodies or other molecules that react with 251P5G2 can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.
[0495] XII.) Inhibition of 251P5G2 Protein Function
[0496] The invention includes various methods and compositions for inhibiting the binding of 251P5G2 to its binding partner or its association with other protein(s) as well as methods for inhibiting 251P5G2 function.
[0497] XII.A.) Inhibition of 251P5G2 With Intracellular Antibodies
[0498] In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 251P5G2 are introduced into 251P5G2 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti-251P5G2 antibody is expressed intracellularly, binds to 251P5G2 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 etaaL, 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beedi et al., 1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).
[0499] 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 target precisely 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.
[0500] In one embodiment, intrabodies are used to capture 251P5G2 in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engineered into such 251P5G2 intrabodies in order to achieve the desired targeting. Such 251P5G2 intrabodies are designed to bind specifically to a particular 251P5G2 domain. In another embodiment, cytosolic intrabodies that specifically bind to a 251P5G2 protein are used to prevent 251P5G2 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g., preventing 251P5G2 from forming transcription complexes with other factors).
[0501] 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 promoterlenhancer can be utilized (See, for example, U.S. Pat. No. 5,919,652 issued Jul. 6, 1999).
[0502] XII.B.) Inhibition of 251P5G2 with Recombinant Proteins
[0503] In another approach, recombinant molecules bind to 251P5G2 and thereby inhibit 251P5G2 function. For example, these recombinant molecules prevent or inhibit 251P5G2 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 251P5G2 specific antibody molecule. In a particular embodiment, the 251P5G2 binding domain of a 251P5G2 binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 251P5G2 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 assodated with the expression of 251P5G2, whereby the dimeric fusion protein specifically binds to 251P5G2 and blocks 251P5G2 interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.
[0504] XII.C.) Inhibition of 251P5G2 Transcription or Translation
[0505] The present invention also comprises various methods and compositions for inhibiting the transcription of the 251P5G2 gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 251P5G2 mRNA into protein.
[0506] In one approach, a method of inhibiting the transcription of the 251P5G2 gene comprises contacting the 251P5G2 gene with a 251P5G2 antisense polynucleotide. In another approach, a method of inhibiting 251P5G2 mRNA translation comprises contacting a 251P5G2 mRNA with an antisense polynucleotide. In another approach, a 251P5G2 specific ribozyme is used to cleave a 251P5G2 message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 251P5G2 gene, such as 251P5G2 promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a 251P5G2 gene transcription factor are used to inhibit 251P5G2 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.
[0507] Other factors that inhibit the transcription of 251P5G2 by interfering with 251P5G2 transcriptional activation are also useful to treat cancers expressing 251P5G2. Similarly, factors that interfere with 251P5G2 processing are useful to treat cancers that express 251P5G2. Cancer treatment methods utilizing such factors are also within the scope of the invention.
[0508] XIII.D.) General Considerations for Therapeutic Strategies
[0509] Gene transfer and gene therapy technologies can be used to deliver therapeutic polynucleotide molecules to tumor cells synthesizing 251P5G2 (i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other 251P5G2 inhibitory molecules). A number of gene therapy approaches are known in the art. Recombinant vectors encoding 251P5G2 antisense polynucleotides, fibozymes, factors capable of interfering with 251P5G2 transcription, and so forth, can be delivered to target tumor cells using such gene therapy approaches.
[0510] 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.
[0511] 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 251P5G2 to a binding partner, etc.
[0512] In vivo, the effect of a 251P5G2 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.
[0513] 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.
[0514] 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 antitumor 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).
[0515] 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.
[0516] 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.
[0517] XIII.) Identification, Characterization and Use of Modulators of 251P5G2
[0518] Methods to Identify and Use Modulators
[0519] In one embodiment, screening is performed to identify modulators that induce or suppress a particular expression profile, suppress or induce specific pathways, preferably generating the associated phenotype thereby. In another embodiment, having identified differentially expressed genes important in a particular state; screens are performed to identify modulators that alter expression of individual genes, either increase or decrease. In another embodiment, screening is performed to identify modulators that alter a biological function of the expression product of a differentially expressed gene. Again, having identified the importance of a gene in a particular state, screens are performed to identify agents that bind and/or modulate the biological activity of the gene product.
[0520] In addition, screens are done for genes that are induced in response to a candidate agent. After identifying a modulator (one that suppresses a cancer expression pattern leading to a normal expression pattern, or a modulator of a cancer gene that leads to expression of the gene as in normal tissue) a screen is performed to identify genes that are specifically modulated in response to the agent. Comparing expression profiles between normal tissue and agent-treated cancer tissue reveals genes that are not expressed in normal tissue or cancer tissue, but are expressed in agent treated tissue, and vice versa. These agent-specific sequences are identified and used by methods described herein for cancer genes or proteins. In particular these sequences and the proteins they encode are used in marking or identifying agent-treated cells. In addition, antibodies are raised against the agent-induced proteins and used to target novel therapeutics to the treated cancer tissue sample.
[0521] Modulator-related Identification and Screening Assays:
[0522] Gene Expression-related Assays
[0523] Proteins, nucleic acids, and antibodies of the invention are used in screening assays. The cancer-associated proteins, antibodies, nucleic acids, modified proteins and cells containing these sequences are used in screening assays, such as evaluating the effect of drug candidates on a “gene expression profile,” expression profile of polypeptides or alteration of biological function. In one embodiment, the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent (e.g., Davis, G F, et al, J Biol Screen 7:69 (2002); Zlokarnik, et al., Science 279:84-8 (1998); Heid, Genome Res 6:986-94, 1996).
[0524] The cancer proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified cancer proteins or genes are used in screening assays. That is, the present invention comprises methods for screening for compositions which modulate the cancer phenotype or a physiological function of a cancer protein of the invention. This is done on a gene itself or by evaluating the effect of drug candidates on a “gene expression profile” or biological function. In one embodiment, expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring after treatment with a candidate agent, see Zlokamik, supra.
[0525] A variety of assays are executed directed to the genes and proteins of the invention. Assays are run on an individual nucleic acid or protein level. That is, having identified a particular gene as up regulated in cancer, test compounds are screened for the ability to modulate gene expression or for binding to the cancer protein of the invention. “Modulation” in this context includes an increase or a decrease in gene expression. The preferred amount of modulation will depend on the original change of the gene expression in normal versus tissue undergoing cancer, with changes of at least 10%, preferably 50%, more preferably 100-300%, and in some embodiments 300-1000% or greater. Thus, if a gene exhibits a 4-fold increase in cancer tissue compared to normal tissue, a decrease of about four-fold is often desired; similarly, a 10-fold decrease in cancer tissue compared to normal tissue a target value of a 10-fold increase in expression by the test compound is often desired. Modulators that exacerbate the type of gene expression seen in cancer are also useful, e.g., as an upregulated target in further analyses.
[0526] The amount of gene expression is monitored using nucleic acid probes and the quantification of gene expression levels, or, alternatively, a gene product itself is monitored, e.g., through the use of antibodies to the cancer protein and standard immunoassays. Proteomics and separation techniques also allow for quantification of expression.
[0527] Expression Monitoring to Identify Compounds that Modify Gene Expression
[0528] In one embodiment, gene expression monitoring, i.e., an expression profile, is monitored simultaneously for a number of entities. Such profiles will typically involve one or more of the genes of FIG. 2. In this embodiment, e.g., cancer nucleic acid probes are attached to biochips to detect and quantify cancer sequences in a particular cell. Alternatively, PCR can be used. Thus, a series, e.g., wells of a microtiter plate, can be used with dispensed primers in desired wells. A PCR reaction can then be performed and analyzed for each well.
[0529] Expression monitoring is performed to identify compounds that modify the expression of one or more cancer-associated sequences, e.g., a polynucleotide sequence set out in FIG. 2. Generally, a test modulator is added to the cells prior to analysis. Moreover, screens are also provided to identify agents that modulate cancer, modulate cancer proteins of the invention, bind to a cancer protein of the invention, or interfere with the binding of a cancer protein of the invention and an antibody or other binding partner.
[0530] In one embodiment, high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such “combinatorial chemical libraries” are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional “lead compounds,” as compounds for screening, or as therapeutics.
[0531] In certain embodiments, combinatorial libraries of potential modulators are screened for an abilityito bind to a cancer polypeptide or to modulate activity. Conventionally, new chemical entities with useful properties are generated by identifying a chemical compound (called a “lead compound”) with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.
[0532] As noted above, gene expression monitoring is conveniently used to test candidate modulators (e.g., protein, nucleic acid or small molecule). After the candidate agent has been added and the cells allowed to incubate for a period, the sample containing a target sequence to be analyzed is, e.g., added to a biochip.
[0533] If required, the target sequence is prepared using known techniques. For example, a sample is treated to lyse the cells, using known lysis buffers, electroporation, etc., with purification and/or amplification such as PCR performed as appropriate. For example, an in vitro transcription with labels covalently attached to the nucleotides is performed. Generally, the nucleic acids are labeled with biotin-FITC or PE, or with cy3 or cy5.
[0534] The target sequence can be labeled with, e.g., a fluorescent, a chemiluminescent, a chemical, or a radioactive signal, to provide a means of detecting the target sequence's specific binding to a probe. The label also can be an enzyme, such as alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a product that is detected. Alternatively, the label is a labeled compound or small molecule, such as an enzyme inhibitor, that binds but is not catalyzed or altered by the enzyme. The label also can be a moiety or compound, such as, an epitope tag or biotin which specifically binds to streptavidin. For the example of biotin, the streptavidin is labeled as described above, thereby, providing a detectable signal for the bound target sequence. Unbound labeled streptavidin is typically removed prior to analysis.
[0535] As will be appreciated by those in the art, these assays can be direct hybridization assays or can comprise “sandwich assays”, which include the use of multiple probes, as is generally outlined in U.S. Pat. Nos. 5,681,702; 5,597,909; 5,545,730; 5,594,117; 5,591,584; 5,571,670; 5,580,731; 5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118; 5,359,100; 5,124,246; and 5,681,697. In this embodiment, in general, the target nucleic acid is prepared as outlined above, and then added to the biochip comprising a plurality of nucleic acid probes, under conditions that allow the formation of a hybridization complex.
[0536] A variety of hybridization conditions are used in the present invention, including high, moderate and low stringency conditions as outlined above. The assays are generally run under stringency conditions which allow formation of the label probe hybridization complex only in the presence of target. Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, etc. These parameters may also be used to control non-specific binding, as is generally outlined in U.S. Pat. No. 5,681,697. Thus, it can be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding.
[0537] The reactions outlined herein can be accomplished in a variety of ways. Components of the reaction can be added simultaneously, or sequentially, in different orders, with preferred embodiments outlined below. In addition, the reaction may include a variety of other reagents. These include salts, buffers, neutral proteins, e.g. albumin, detergents, etc. which can be used to facilitate optimal hybridization and detection, and/or reduce nonspecific or background interactions. Reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may also be used as appropriate, depending on the sample preparation methods and purity of the target. The assay data are analyzed to determine the expression levels of individual genes, and changes in expression levels as between states, forming a gene expression profile.
[0538] Biological Activity-related Assays
[0539] The invention provides methods identify or screen for a compound that modulates the activity of a cancer-related gene or protein of the invention. The methods comprise adding a test compound, as defined above, to a cell comprising a cancer protein of the invention. The cells contain a recombinant nucleic acid that encodes a cancer protein of the invention. In another embodiment, a library of candidate agents is tested on a plurality of cells.
[0540] In one aspect, the assays are evaluated in the presence or absence or previous or subsequent exposure of physiological signals, e.g. hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e., cell-cell contacts). In another example, the determinations are made at different stages of the cell cycle process. In this way, compounds that modulate genes or proteins of the invention are identified. Compounds with pharmacological activity are able to enhance or interfere with the activity of the cancer protein of the invention. Once identified, similar structures are evaluated to identify critical structural features of the compound.
[0541] In one embodiment, a method of modulating (e.g., inhibiting) cancer cell division is provided; the method comprises administration of a cancer modulator. In another embodiment, a method of modulating ( e.g., inhibiting) cancer is provided; the method comprises administration of a cancer modulator. In a further embodiment, methods of treating cells or individuals with cancer are provided; the method comprises administration of a cancer modulator.
[0542] In one embodiment, a method for modulating the status of a cell that expresses a gene of the invention is provided. As used herein status comprises such art-accepted parameters such as growth, proliferation, survival, function, apoptosis, senescence, location, enzymatic activity, signal transduction, etc. of a cell. In one embodiment, a cancer inhibitor is an antibody as discussed above. In another embodiment, the cancer inhibitor is an antisense molecule. A variety of cell growth, proliferation, and metastasis assays are known to those of skill in the art, as described herein.
[0543] High Throughput Screening to Identify Modulators
[0544] The assays to identify suitable modulators are amenable to high throughput screening. Preferred assays thus detect enhancement or inhibition of cancer gene transcription, inhibition or enhancement of polypeptide expression, and inhibition or enhancement of polypeptide activity.
[0545] In one embodiment, modulators evaluated in high throughput screening methods are proteins, often naturally occurring proteins or fragments of naturally occurring proteins. Thus, e.g., cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, are used. In this way, libraries of proteins are made for screening in the methods of the invention. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred. Particularly useful test compound will be directed to the class of proteins to which the target belongs, e.g., substrates for enzymes, or ligands and receptors.
[0546] Use of Soft Agar Growth and Colony Formation to Identify and Characterize Modulators
[0547] Normal cells require a solid substrate to attach and grow. When cells are transformed, they lose this phenotype and grow detached from the substrate. For example, transformed cells can grow in stirred suspension culture or suspended in semi-solid media, such as semi-solid or soft agar. The transformed cells, when transfected with tumor suppressor genes, can regenerate normal phenotype and once again require a solid substrate to attach to and grow. Soft agar growth or colony formation in assays are used to identify modulators of cancer sequences, which when expressed in host cells, inhibit abnormal cellular proliferation and transformation. A modulator reduces or eliminates the host cells' ability to grow suspended in solid or semisolid media, such as agar.
[0548] Techniques for soft agar growth or colony formation in suspension assays are described in Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed., 1994). See also, the methods section of Garkavtsev et al. (1996), supra.
[0549] Evaluation of Contact Inhibition and Growth Density Limitation to Identify and Characterize Modulators
[0550] Normal cells typically grow in a flat and organized pattern in cell culture until they touch other cells. When the cells touch one another, they are contact inhibited and stop growing. Transformed cells, however, are not contact inhibited and continue to grow to high densities in disorganized foci. Thus, transformed cells grow to a higher saturation density than corresponding normal cells. This is detected morphologically by the formation of a disoriented monolayer of cells or cells in foci. Alternatively, labeling index with (3H)-thymidine at saturation density is used to measure density limitation of growth, similarly an MTT or Alamar blue assay will reveal proliferation capacity of cells and the the ability of modulators to affect same. See Freshney (1994), supra. Transformed cells, when transfected with tumor suppressor genes, can regenerate a normal phenotype and become contact inhibited and would grow to a lower density.
[0551] In this assay, labeling index with 3H)-thymidine at saturation density is a preferred method of measuring density limitation of growth. Transformed host cells are transfected with a cancer-associated sequence and are grown for 24 hours at saturation density in non-limiting medium conditions. The percentage of cells labeling with (3H)-thymidine is determined by incorporated cpm.
[0552] Contact independent growth is used to identify modulators of cancer sequences, which had led to abnormal cellular proliferation and transformation. A modulator reduces or eliminates contact independent growth, and returns the cells to a normal phenotype.
[0553] Evaluation of Growth Factor or Serum Dependence to Identify and Characterize Modulators
[0554] Transformed cells have lower serum dependence than their normal counterparts (see, e.g., Temin, J. Natl. Cancer Inst. 37:167-175 (1966); Eagle et al., J. Exp. Med 131:836-879 (1970)); Freshney, supra. This is in part due to release of various growth factors by the transformed cells. The degree of growth factor or serum dependence of transformed host cells can be compared with that of control. For example, growth factor or serum dependence of a cell is monitored in methods to identify and characterize compounds that modulate cancer-associated sequences of the invention.
[0555] Use of Tumor-specific Marker Levels to Identify and Characterize Modulators
[0556] Tumor cells release an increased amount of certain factors (hereinafter “tumor specific markers”) than their normal counterparts. For example, plasminogen activator (PA) is released from human glioma at a higher level than from normal brain cells (see, e.g., Gullino, Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985)). Similarly, Tumor Angiogenesis Factor (TAF) is released at a higher level in tumor cells than their normal counterparts. See, e.g., Folkman, Angiogenesis and Cancer, Sem Cancer Biol. (1992)), while bFGF is released from endothelial tumors (Ensoli, B et al).
[0557] Various techniques which measure the release of these factors are described in Freshney (1994), supra. Also, see, Unkless et al., J. Biol. Chem. 249:4295-4305 (1974); Strickland & Beers, J. Biol. Chem. 251:5694-5702 (1976); Whur et al., Br. J. Cancer 42:305 312 (1980); Gullino, Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985); Freshney, Anticancer Res. 5:111-130 (1985). For example, tumor specific marker levels are monitored in methods to identify and characterize compounds that modulate cancer-associated sequences of the invention.
[0558] Invasiveness into Matrigel to Identify and Characterize Modulators
[0559] The degree of invasiveness into Matrigel or an extracellular matrix constituent can be used as an assay to identify and characterize compounds that modulate cancer associated sequences. Tumor cells exhibit a positive correlation between malignancy and invasiveness of cells into Matrigel or some other extracellular matrix constituent. In this assay, tumorigenic cells are typically used as host cells. Expression of a tumor suppressor gene in these host cells would decrease invasiveness of the host cells. Techniques described in Cancer Res. 1999; 59:6010; Freshney (1994), supra, can be used. Briefly, the level of invasion of host cells is measured by using filters coated with Matrigel or some other extracellular matrix constituent. Penetration into the gel, or through to the distal side of the filter, is rated as invasiveness, and rated histologically by number of cells and distance moved, or by prelabeling the cells with 1251 and counting the radioactivity on the distal side of the filter or bottom of the dish. See, e.g., Freshney (1984), supra.
[0560] Evaluation of Tumor Growth In Vivo to Identify and Characterize Modulators
[0561] Effects of cancer-associated sequences on cell growth are tested in transgenic or immune-suppressed organisms. Transgenic organisms are prepared in a variety of art-accepted ways. For example, knock-out transgenic organisms, e.g., mammals such as mice, are made, in which a cancer gene is disrupted or in which a cancer gene is inserted. Knock-out transgenic mice are made by insertion of a marker gene or other heterologous gene into the endogenous cancer gene site in the mouse genome via homologous recombination. Such mice can also be made by substituting the endogenous cancer gene with a mutated version of the cancer gene, or by mutating the endogenous cancer gene, e.g., by exposure to carcinogens.
[0562] To prepare transgenic chimeric animals, e.g., mice, a DNA construct is introduced into the nuclei of embryonic stem cells. Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is re-implanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells some of which are derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288 (1989)). Chimeric mice can be derived according to U.S. Pat. No. 6,365,797, issued Apr. 2, 2002; U.S. Pat. No. 6,107,540 issued Aug. 22, 2000; Hogan et al., Manipulating the Mouse Embryo: A laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington, D.C., (1987).
[0563] Alternatively, various immune-suppressed or immune-deficient host animals can be used. For example, a genetically athymic “nude” mouse (see, e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)), a SCID mouse, a thymectornized mouse, or an irradiated mouse (see, e.g., Bradley et al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer 41:52 (1980)) can be used as a host. Transplantable tumor cells (typically about 106 cells) injected into isogenic hosts produce invasive tumors in a high proportion of cases, while normal cells of similar origin will not. In hosts which developed invasive tumors, cells expressing cancer-associated sequences are injected subcutaneously or orthotopically. Mice are then separated into groups, including control groups and treated experimental groups) e.g. treated with a modulator). After a suitable length of time, preferably 4-8 weeks, tumor growth is measured (e.g., by volume or by its two largest dimensions, or weight) and compared to the control. Tumors that have statistically significant reduction (using, e.g., Student's T test) are said to have inhibited growth.
[0564] In Vitro Assays to Identify and Characterize Modulators
[0565] Assays to identify compounds with modulating activity can be performed in vitro. For example, a cancer polypeptide is first contacted with a potential modulator and incubated for a suitable amount of time, e.g., from 0.5 to 48 hours. In one embodiment, the cancer polypeptide levels are determined in vitro by measuring the level of protein or mRNA. The level of protein is measured using immunoassays such as Western blotting, ELISA and the like with an antibody that selectively binds to the cancer polypeptide or a fragment thereof. For measurement of mRNA, amplification, e.g., using PCR, LCR, or hybridization assays, e. g., Northern hybridization, RNAse protection, dot blotting, are preferred. The level of protein or mRNA is detected using directly or indirectly labeled detection agents, e.g., fluorescently or radioactively labeled nucleic acids, radioactively or enzymatically labeled antibodies, and the like, as described herein.
[0566] Alternatively, a reporter gene system can be devised using a cancer protein promoter operably linked to a reporter gene such as luciferase, green fluorescent protein, CAT, or P-gal. The reporter construct is typically transfected into a cell. After treatment with a potential modulator, the amount of reporter gene transcription, translation, or activity is measured according to standard techniques known to those of skill in the art (Davis GF, supra; Gonzalez, J. & Negulescu, P. Curr. Opin. Biotechnol. 1998: 9:624).
[0567] As outlined above, in vitro screens are done on individual genes and gene products. That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of the expression of the gene or the gene product itself is performed.
[0568] In one embodiment, screening for modulators of expression of specific gene(s) is performed. Typically, the expression of only one or a few genes is evaluated. In another embodiment, screens are designed to first find compounds that bind to differentially expressed proteins. These compounds are then evaluated for the ability to modulate differentially expressed activity. Moreover, once initial candidate compounds are identified, variants can be further screened to better evaluate structure activity relationships.
[0569] Binding Assays to Identify and Characterize Modulators
[0570] In binding assays in accordance with the invention, a purified or isolated gene product of the invention is generally used. For example, antibodies are generated to a protein of the invention, and immunoassays are run to determine the amount and/or location of protein. Alternatively, cells comprising the cancer proteins are used in the assays.
[0571] Thus, the methods comprise combining a cancer protein of the invention and a candidate compound such as a ligand, and determining the binding of the compound to the cancer protein of the invention. Preferred embodiments utilize the human cancer protein; animal models of human disease of can also be developed and used. Also, other analogous mammalian proteins also can be used as appreciated by those of skill in the art. Moreover, in some embodiments variant or derivative cancer proteins are used.
[0572] Generally, the cancer protein of the invention, or the ligand, is non-diffusibly bound to an insoluble support. The support can, e.g., be one having isolated sample receiving areas (a microtiter plate, an array, etc.). The insoluble supports can be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports can be solid or porous and of any convenient shape.
[0573] Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharide, nylon, nitrocellulose, or Teflon™, etc. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner of binding of the composition to the support is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable. Preferred methods of binding include the use of antibodies which do not sterically block either the ligand binding site or activation sequence when attaching the protein to the support, direct binding to “sticky” or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or ligand/binding agent to the support, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.
[0574] Once a cancer protein of the invention is bound to the support, and a test compound is added to the assay. Alternatively, the candidate binding agent is bound to the support and the cancer protein of the invention is then added. Binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc.
[0575] Of particular interest are assays to identify agents that have a low toxicity for human cells. A wide variety of assays can be used for this purpose, including proliferation assays, cAMP assays, labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.
[0576] A determination of binding of the test compound (ligand, binding agent, modulator, etc.) to a cancer protein of the invention can be done in a number of ways. The test compound can be labeled, and binding determined directly; e.g., by attaching all or a portion of the cancer protein of the invention to a solid support, adding a labeled candidate compound (e.g., a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support. Various blocking and washing steps can be utilized as appropriate.
[0577] In certain embodiments, only one of the components is labeled, e.g., a protein of the invention or ligands labeled. Alternatively, more than one component is labeled with different labels, e.g., I125, for the proteins and a fluorophor for the compound. Proximity reagents, e.g., quenching or energy transfer reagents are also useful.
[0578] Competitive Binding to Identify and Characterize Modulators
[0579] In one embodiment, the binding of the “test compound” is determined by competitive binding assay with a “competitor.” The competitor is a binding moiety that binds to the target molecule (e.g., a cancer protein of the invention). Competitors include compounds such as antibodies, peptides, binding partners, ligands, etc. Under certain circumstances, the competitive binding between the test compound and the competitor displaces the test compound. In one embodiment, the test compound is labeled. Either the test compound, the competitor, or both, is added to the protein for a time sufficient to allow binding. Incubations are performed at a temperature that facilitates optimal activity, typically between four and 40° C. Incubation periods are typically optimized, e.g., to facilitate rapid high throughput screening; typically between zero and one hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.
[0580] In one embodiment, the competitor is added first, followed by the test compound. Displacement of the competitor is an indication that the test compound is binding to the cancer protein and thus is capable of binding to, and potentially modulating, the activity of the cancer protein. In this embodiment, either component can be labeled. Thus, e.g., if the competitor is labeled, the presence of label in the post-test compound wash solution indicates displacement by the test compound. Alternatively, if the test compound is labeled, the presence of the label on the support indicates displacement.
[0581] In an alternative embodiment, the test compound is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor indicates that the test compound binds to the cancer protein with higher affinity than the competitor. Thus, if the test compound is labeled, the presence of the label on the support, coupled with a lack of competitor binding, indicates that the test compound binds to and thus potentially modulates the cancer protein of the invention.
[0582] Accordingly, the competitive binding methods comprise differential screening to identity agents that are capable of modulating the activity of the cancer proteins of the invention. In this embodiment, the methods comprise combining a cancer protein and a competitor in a first sample. A second sample comprises a test compound, the cancer protein, and a competitor. The binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the cancer protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the cancer protein.
[0583] Alternatively, differential screening is used to identify drug candidates that bind to the native cancer protein, but cannot bind to modified cancer proteins. For example the structure of the cancer protein is modeled and used in rational drug design to synthesize agents that interact with that site, agents which generally do not bind to site-modified proteins. Moreover, such drug candidates that affect the activity of a native cancer protein are also identified by screening drugs for the ability to either enhance or reduce the activity of such proteins.
[0584] Positive controls and negative controls can be used in the assays. Preferably control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples occurs for a time sufficient to allow for the binding of the agent to the protein. Following incubation, samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples can be counted in a scintillation counter to determine the amount of bound compound.
[0585] A variety of other reagents can be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc. which are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., can be used. The mixture of components is added in an order that provides for the requisite binding.
[0586] Use of Polynucleotides to Down-regulate or Inhibit a Protein of the Invention.
[0587] Polynucleotide modulators of cancer can be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand-binding molecule, as described in WO 91/04753. Suitable ligand-binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell. Alternatively, a polynucleotide modulator of cancer can be introduced into a cell containing the target nucleic acid sequence, e.g., by formation of a polynucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of treatment.
[0588] Inhibitory and Antisense Nucleotides
[0589] In certain embodiments, the activity of a cancer-associated protein is down-regulated, or entirely inhibited, by the use of antisense polynucleotide or inhibitory small nuclear RNA (snRNA), i.e., a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, e.g., a cancer protein of the invention, mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the mRNA reduces the translation and/or stability of the mRNA.
[0590] In the context of this invention, antisense polynucleotides can comprise naturally occurring nucleotides, or synthetic species formed from naturally occurring subunits or their close homologs. Antisense polynucleotides may also have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur containing species which are known for use in the art. Analogs are comprised by this invention so long as they function effectively to hybridize with nucleotides of the invention. See, e.g., Isis Pharmaceuticals, Carlsbad, Calif.; Sequitor, Inc., Natick, Mass.
[0591] Such antisense polynucleotides can readily be synthesized using recombinant means, or can be synthesized in vitro. Equipment for such synthesis is sold by several vendors, including Applied Biosystems. The preparation of other oligonucleotides such as phosphorothioates and alkylated derivatives is also well known to those of skill in the art.
[0592] Antisense molecules as used herein include antiserse or sense oligonucleotides. Sense oligonucleotides can, e.g., be employed to block transcription by binding to the anti-sense strand. The antisense and sense oligonucleotide comprise a single stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for cancer molecules. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment generally at least about 12 nucleotides, preferably from about 12 to 30 nucleotides. The ability to derive an antsense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, e.g., Stein & Cohen (Cancer Res. 48:2659 (1988 and van der Krol et al. (BioTechniques 6:958 (1988)).
[0593] Ribozymes
[0594] In addition to antisense polynucleotides, ribozymes can be used to target and inhibit transcription of cancer-associated nucleotide sequences. A ribozyme is an RNA molecule that catalytically cleaves other RNA molecules. Different kinds of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNase P, and axhead ribozymes (see, e.g., Castanollo et al., Adv. in Pharmacology 25: 289-317 (1994) for a general review of the properties of different ribozymes).
[0595] The general features of hairpin ribozymes are described, e.g., in Hampel et al., Nucl. Acids Res. 18:299-304 (1990); European Patent Publication No. 0360257; U.S. Pat. No. 5,254,678. Methods of preparing are well known to those of skill in the art (see, e.g., WO 94/26877; Ojwang et al., Proc. Natl. Acad. Sci. USA 90:6340-6344 (1993); Yamada et al., Human Gene Therapy 1:3945 (1994); Leavitt et al., Proc. Natl. Acad Sci. USA 92:699-703 (1995); Leavitt et al., Human Gene Therapy 5: 1151-120 (1994), and Yamada et al., Virology 205:121-126 (1994)).
[0596] Use of Modulators in Phenotypic Screening
[0597] In one embodiment, a test compound is administered to a population of cancer cells, which have an associated cancer expression profile. By “administration” or “contacting” herein is meant that the modulator is added to the cells in such a manner as to allow the modulator to act upon the cell, whether by uptake and intracellular action, or by action at the cell surface. In some embodiments, a nucleic acid encoding a proteinaceous agent (i.e., a peptide) is put into a viral construct such as an adenoviral or retroviral construct, and added to the cell, such that expression of the peptide agent is accomplished, e.g., PCT US97/01019. Regulatable gene therapy systems can also be used. Once the modulator has been administered to the cells, the cells are washed if desired and are allowed to incubate under preferably physiological conditions for some period. The cells are then harvested and a new gene expression profile is generated. Thus, e.g., cancer tissue is screened for agents that modulate, e.g., induce or suppress, the cancer phenotype. A change in at least one gene, preferably many, of the expression profile indicates that the agent has an effect on cancer activity. Similarly, altering a biological function or a signaling pathway is indicative of modulator activity. By defining such a signature for the cancer phenotype, screens for new drugs that alter the phenotype are devised. With this approach, the drug target need not be known and need not be represented in the original gene/protein expression screening platform, nor does the level of transcript for the target protein need to change. The modulator inhibiting function will serve as a surrogate marker
[0598] As outlined above, screens are done to assess genes or gene products. That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of either the expression of the gene or the gene product itself is performed.
[0599] Use of Modulators to Affect Peptides of the Invention
[0600] Measurements of cancer polypeptide activity, or of the cancer phenotype are performed using a variety of assays. For example, the effects of modulators upon the function of a cancer polypeptide(s) are measured by examining parameters described above. A physiological change that affects activity is used to assess the influence of a test compound on the polypeptides of this invention. When the functional outcomes are determined using intact cells or animals, a variety of effects can be assesses such as, in the case of a cancer associated with solid tumors, tumor growth, tumor metastasis, neovascularization, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., by Northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as cGNIP.
[0601] Methods of Identifying Characterizing Cancer-associated Sequences
[0602] Expression of various gene sequences is correlated with cancer. Accordingly, disorders based on mutant or variant cancer genes are determined. In one embodiment, the invention provides methods for identifying cells containing variant cancer genes, e.g., determining the presence of, all or part, the sequence of at least one endogenous cancer gene in a cell. This is accomplished using any number of sequencing techniques. The invention comprises methods of identifying the cancer genotype of an individual, e.g., determining all or part of the sequence of at least one gene of the invention in the individual. This is generally done in at least one tissue of the individual, e.g., a tissue set forth in Table I, and may include the evaluation of a number of tissues or different samples of the same tissue. The method may include comparing the sequence of the sequenced gene to a known cancer gene, i.e., a wild-type gene to determine the presence of family members, homologies, mutations or variants. The sequence of all or part of the gene can then be compared to the sequence of a known cancer gene to determine if any differences exist. This is done using any number of known homology programs, such as BLAST, Bestfit, etc. The presence of a difference in the sequence between the cancer gene of the patient and the known cancer gene correlates with a disease state or a propensity for a disease state, as outlined herein.
[0603] In a preferred embodiment, the cancer genes are used as probes to determine the number of copies of the cancer gene in the genome. The cancer genes are used as probes to determine the chromosomal localization of the cancer genes. Information such as chromosomal localization finds use in providing a diagnosis or prognosis in particular when chromosomal abnormalities such as translocations, and the like are identified in the cancer gene locus.
[0604] XIV.) Kits/Articles of Manufacture
[0605] 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 FIG. 2-related protein or a FIG. 2 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 sequences in FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acid molecules that encodes such amino acid sequences.
[0606] 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; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use.
[0607] A label can be present on the container to indicate that the composition is used for a specific therapy or non-therapeutic application, such as a diagnostic or laboratory application, and can also indicate directions for either in vivo or in vitro use, such as those described herein. Directions and or other information can also be included on an insert(s) or label(s) which is included with or on the kit.
[0608] The terms “kit” and “article of manufacture” can be used as synonyms.
[0609] In another embodiment of the invention, an article(s) of manufacture containing compositions, such as amino acid sequence(s), small molecule(s), nucleic acid sequence(s), and/or antibody(s), e.g., materials useful for the diagnosis, prognosis, prophylaxis and/or treatment of neoplasias of tissues such as those set forth in Table I is provided. The article of manufacture typically comprises at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass or plastic. The container can hold amino acid sequence(s), small molecule(s), nucleic acid sequence(s), and/or antibody(s), in one embodiment the container holds a polynucleotide for use in examining the mRNA expression profile of a cell, together with reagents used for this purpose.
[0610] The container can alternatively hold a composition which is effective for treating, diagnosis, prognosing or prophylaxing a condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agents in the composition can be an antibody capable of specifically binding 251P5G2 and modulating the function of 251P5G2.
[0611] The label can be on or associated with the container. A label a can be on a container when letters, numbers or other characters forming the label are molded or etched into the container itself; a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. The label can indicate that the composition is used for diagnosing, treating, prophylaxing or prognosing a condition, such as a neoplasia of a tissue set forth in Table I. The article of manufacture can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and/ordextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, stirrers, needles, syringes, and/or package inserts with indications and/or instructions for use.
EXAMPLES[0612] 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[0613] SSH-Generated Isolation of cDNA Fragment of the 251P5G2 Gene
[0614] To isolate genes that are over-expressed in prostate cancer we used the Suppression Subtractive Hybridization (SSH) procedure using cDNA derived from prostate cancer tissues. The 251P5G2 SSH cDNA sequence was derived from a prostate cancer metastasis minus cDNAs derived from a pool of 9 normal tissues. The 251P5G2 cDNA was identified as highly expressed in the prostate cancer metastasis.
[0615] Materials and Methods
[0616] Human Tissues:
[0617] 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.
[0618] RNA Isolation:
[0619] 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.
[0620] Oligonucleotides:
[0621] The following HPLC purified oligonucleotides were used. 2 DPNCDN (cDNA synthesis primer): 5′TTTTGATCAAGCTT303′ (SEQ ID NO: 41) Adaptor 1: 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ ID NO: 42) 3′GGCCCGTCCTAG5′ (SEQ ID NO: 43) Adaptor 2: 5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO: 44) 3′CGGCTCCTAG5′ (SEQ ID NO: 45) PCR primer 1: 5′CTAATACGACTCACTATAGGGC3′ (SEQ ID NO: 46) Nested primer (NP)1: 5′TCGAGCGGCCGCCCGGGCAGGA3′ (SEQ ID NO: 47) Nested primer (NP)2: 5′AGCGTGGTCGCGGCCGAGGA3′ (SEQ ID NO: 48)
[0622] Suppression Subtractive Hybridization:
[0623] Suppression Subtractive Hybridization (SSH) was used to identify cDNAs corresponding to genes that may be differentially expressed in prostate cancer. The SSH reaction utilized cDNA from prostate cancer and normal tissues.
[0624] The gene 251P5G2 sequence was derived from a prostate cancer metastasis minus normal tissue cDNA subtraction. The SSH DNA sequence (FIG. 1) was identified.
[0625] The cDNA derived from of pool of normal tissues was used as the source of the “driver” cDNA, while the cDNA from prostate cancer metastasis 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 at37° C. Digested cDNA was extracted with phenol/chloroform (1:1) and ethanol precipitated.
[0626] 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.
[0627] 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 &mgr;l of water. The diluted cDNA (2 &mgr;l, 160 ng) was then ligated to 2 &mgr;l of Adaptor 1 and Adaptor 2 (10 &mgr;M), in separate ligation reactions, in a total volume of 10 &mgr;l at 160° 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.
[0628] 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.
[0629] PCR Amplification, Cloning and Sequencing of Gene Fragments Generated from SSH:
[0630] 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.
[0631] 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 ul of bacterial culture using the conditions of PCR1 and NP1 and NP2 as primers. PCR products were analyzed using 2% agarose gel electrophoresis.
[0632] 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.
[0633] RT-PCR Expression Analysis:
[0634] 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.
[0635] Normalization of the first strand cDNAs from multiple tissues was performed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO: 49) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 50) 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, 1XPCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCl2, 50 mM KCl, pH8.3) and 1X 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.
[0636] To determine expression levels of the 251P5G2 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 251P5G2 SSH sequence and are listed below: 3 251P5G2.1 5′-AGTGATTCAAAGAGCTGTGGAGA-3′ (SEQ ID NO: 51) 251P5G2.2 5′-GGCCAGAGCGCACTTACCTACC-3′ (SEQ ID NO: 52)
[0637] 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), prostate cancer metastasis to lymph node, prostate cancer pool, bladder cancer pool, and cancer metastasis pool. Normalization was performed by PCR using primers to actin and GAPDH. Semiquantitative PCR, using primers to 251P5G2, was performed at 26 and 30 cycles of amplification. Results show strong expression of 251P5G2 in prostate cancer metastasis, prostate cancer pool, and cancer metastasis pool. Expression of 251P5G2 was also detected in bladder cancer pool, but not in vital pool 1 and vital pool 2.
Example 2[0638] Isolation of Full Length 251P5G2 Encoding cDNA
[0639] The 251P5G2 SSH cDNA sequence was derived from a substraction consisting of prostate cancer metastasis to lymph node minus a mixture of 9 normal tissues: stomach, skeletal muscle, lung, brain, liver, kidney, pancreas, small intestine and heart. The SSH cDNA sequence (FIG. 1) was designated 251P5G2.
[0640] The 251P5G2 SSH DNA sequence of 162 bp (FIG. 1) was novel and did not show homology to any known gene. 251P5G2 v.1 (clone 4.7) of 2157 bp was cloned from prostate cancer cDNA library, revealing an ORF of 255 amino acids (FIG. 2 and FIG. 3). Other variants of 251P5G2 were also identified and these are listed in FIGS. 2 and 3.
[0641] 251P5G2 v.1, v.2, v.3, and v.4 proteins are 255 amino acids in length and differ from each other by one amino acid as shown in FIG. 11. 251P5G2 v.5, v.6, v.7, v.8, v.9, and v.10 code for the same protein as 251P5G2 v.1. 251P5G2 v.12 codes for a protein of 1266 amino acids in length. 251P5G2 v.13 codes for the same protein as 251P5G2 v.12.
[0642] 251P5G2 v.1 and variants v.2 through v.11 are novel, and did not show significant homology to known human genes. The 251P5G2 v.1 protein showed homology to the mouse vomeronasal 1 receptor C3, 44% identity and 60% homology over 234 amino acids (FIG. 4A). 251P5G2 v.12 protein aligns with the protein XM—063686 at 100% identity over 1213 amino acids, ranging from position 54 to 1266 of 251P5G2 v.12 protein (FIG. 4B). XM—063686 protein is a hypothetical protein predicted by automated computational analysis using GenomeScan.
Example 3[0643] Chromosomal Mapping of 251P5G2
[0644] 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 Cornell Institute (Camden, N.J.), and genomic viewers utilizing BLAST homologies to sequenced and mapped genomic clones (NCBI, Bethesda, Md.).
[0645] 251P5G2 maps to chromosome 15q11.2 using 251P5G2 sequence and the NCBI BLAST tool located on the World Wide Web at (.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs).
Example 4[0646] Expression Analysis of 251P5G2 in Normal Tissues and Patient Specimens
[0647] Expression analysis by RT-PCR demonstrated that 251P5G2 is strongly expressed in prostate 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), prostate cancer metastasis to lymph node, prostate cancer pool, bladder cancer pool, and cancer metastasis pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 251P5G2, was performed at 26 and 30 cycles of amplification. Results show strong expression of 251P5G2 in prostate cancer metastasis, prostate cancer pool, and cancer metastasis pool. Expression of 251P5G2 was also detected in bladder cancer pool, but not in vital pool 1 and vital pool 2.
[0648] Northern blot analysis of 251P5G2 is a technique known to those skilled in the art to detect 251P5G2 protein production. Northern blotting detects relative levels of mRNA expressed from a 251P5G2 gene. Specific mRNA is measured using a nucleic acid hybridization technique and the signal is detected on an autoradiogram. The stronger the signal, the more abundant is the mRNA. For 251P5G2 genes that produce mRNA that contains an open reading frame flanked by a good Kozak translation initiation site and a stop codon, in the vast majority of cases the synthesized mRNA is expressed as a protein.
[0649] The level of expression of the 251P5G2 gene is determined in various normal tissues and in various tumor tissues and tumor cell lines using the technique of Northern blotting, which detects production of messenger RNA. It is well known in the art that the production of messenger RNA, that encodes the protein, is a necessary step in the production of the protein itself. Thus, detection of high levels of messenger RNA by, for example, Northern blot, is a way of determining that the protein itself is produced. The Northern blot technique is used as a routine procedure because it does not require the time delays (as compared to Western blotting, immunoblotting or immunohistochemistry) involved in isolating or synthesizing the protein, preparing an immunological composition of the protein, eliciting a humoral immune response, harvesting the antibodies, and verifying the specificity thereof.
[0650] The Kozak consensus sequence for translation initiation CCACCATGG, where the ATG start codon is noted, is the sequence with the highest established probability of initiating translation. This was confirmed by Peri and Pandey Trends in Genetics (2001) 17: 685-687. The conclusion is consistent with the general knowledge in the art that, with rare exceptions, expression of an mRNA is predictive of expression of its encoded protein. This is particularly true for mRNA with an open reading frame and a Kozak consensus sequence for translation initiation.
[0651] It is understood in the art that the absolute levels of messenger RNA present and the amounts of protein produced do not always provide a 1:1 correlation. In those instances where the Northern blot has shown mRNA to be present, it is almost always possible to detect the presence of the corresponding protein in the tissue which provided a positive result in the Northern blot. The levels of the protein compared to the levels of the mRNA may be differential, but generally, cells that exhibit detectable mRNA also exhibit detectable corresponding protein and vice versa. This is particularly true where the mRNA has an open reading frame and a good Kozak sequence (See, Peri and Pandey, supra.).
[0652] Occasionally those skilled in the art encounter a rare occurrence where there is no detectable protein in the presence of corresponding mRNA. (See, Fu, L., et al., Embo. Journal, 15:4392-4401 (1996)). In many cases, a reported lack of protein expression is due to technical limitations of the protein detection assay. These limitations are readily known to those skilled in the art. These limitations include but are not limited to, available antibodies that only detect denatured protein and not native protein present in a cell and unstable proteins with very short half-life. Short-lived proteins are still functional and have been previously described to induce tumor formation. (See, e.g., Reinstein, et al., Oncogene, 19: 5944-5950). In such situations, when more sensitive detection techniques are performed and/or other antibodies are generated, protein expression is detected. When studies fail to take these principles into account, they are likely to report artifactually lowered correlations of mRNA to protein. Outside of these rare exceptions the use of Northern blot analysis is recognized to those skilled in the art to be predictive and indicative of the detection of 251P5G2 protein production.
[0653] Extensive northern blot analysis of 251P5G2 in multiple human normal tissues is shown in FIG. 15. Two multiple tissue northern blots (Clontech) both with 2 &mgr;g of mRNA/lane were probed with the 251P5G2 SSH sequence. Expression of 251P5G2 was detected in normal prostate and testis but not in any other normal tissues tested.
[0654] Expression of 251P5G2 in prostate cancer metastasis patient specimens and human normal tissues is shown in FIG. 16. RNA was extracted from two prostate cancer metastasis to lymph node isolated from two different patients (Met1 and Met2), as well as from normal bladder (NB), normal kidney (NK), normal lung (NL), normal breast (NBr), normal ovary (NO), and normal pancreas (NPa). Northern blot with 10 &mgr;g of total RNA/lane was probed with 251P5G2 SSH sequence. Results show strong expression of 251P5G2 in the prostate cancer metastasis specimens but not in the normal tissues tested.
[0655] Expression of 251P5G2 was also detected in prostate cancer patient specimens and prostate cancer xenograft tissues (FIG. 17). RNA was extracted from prostate cancer xenografts (LAPC4AD, LAPCA4AI, LAPC-9AD, LAPC-9AI), prostate cancer cell lines (LNCaP and PC3), normal prostate (N), and prostate cancer patient tumors (T). Northern blots with 10 &mgr;g of total RNA were probed with the 251P5G2 SSH fragment. Results show expression of 251P5G2 in the LAPC-9AD xenograft and in prostate tumor tissues. Lower level expression was detected in the other xenograft tissues and LNCaP cell line but not in PC3. The lower panel represents ethidium-bromide staining of the gel confirming the quality of the RNA.
[0656] FIG. 18 shows expression of 251 p5g2 in human normal and cancer tissues. First strand cDNA was prepared from a panel of 13 normal tissues, prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, pancreas cancer pool, 2 different prostate cancer metastasis specimens to lymph node, and a pool of prostate cancer LAPC xenografts (LAPC-4AD, LAPC-4AI, LAPC-9AD, and LAPC-9AI). Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 251P5G2, was performed at 26 and 30 cycles of amplification. A standard curve was generated using plasmid DNA containing 251P5G2 of known copy number. The experiment was performed in duplicate. Results show strong expression of 251P5G2 in prostate cancer metastasis, prostate cancer pool, and cancer metastasis pool. Expression of 251P5G2 was also detected in bladder cancer pool. Amongst normal tissues, very weak expression was detected in hear, prostate, skeletal muscle and testis but not in any other normal tissue tested.
[0657] FIG. 19 shows expression of 251P5G2 in prostate cancer patient specimens. First strand cDNA was prepared from normal prostate, prostate cancer cell lines (PC3, DU145, LNCaP, 293T), and a panel of prostate cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 251P5G2, was performed at 26 and 30 cycles of amplification. Results show expression of 251P5G2 in 10 out of 19 patient specimens. Very strong expression was detected in 5 out of the 10 expressing tumors. Expression was also detected in LNCaP but not in the other cell lines tested nor in normal prostate.
[0658] Expression of 251P5G2 in bladdercancer patient specimens is shown in FIG. 20. First strand cDNA was prepared from normal bladder, bladder cancer cell lines (UM-UC-3, TCCSUP, J82), and a panel of bladder cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 251P5G2, was performed at 26 and 30 cycles of amplification. Results show expression of 251P5G2 in 5 out of 9 patient specimens, but not in the cell lines tested nor in normal bladder.
[0659] The restricted expression of 251P5G2 in normal tissues and the expression detected in prostate cancer, prostate cancer metastasis, and bladder cancer suggest that 251P5G2 is a potential therapeutic target and a diagnostic marker for human cancers.
Example 5[0660] Transcript Variants of 251P5G2
[0661] Transcript variants are variants of mature mRNA from the same gene which arise 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 can encode proteins with different functions, and can be expressed in the same tissue at the same time, or in different tissues at the same time, or in the same tissue at different times, or in different tissues at different times. Proteins encoded by transcript variants can have similar or different cellular or extracellular localizations, e.g., secreted versus intracellular.
[0662] Transcript variants are identified by a variety of art-accepted methods. For example, alternative transcripts and splice variants are identified by 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. 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.
[0663] 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. 2000 April; 10(4):516-22); Grail (URL compbio.ornl.gov/Grail-bin/EmptyGrailForm) and Gen Scan (URL 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.
[0664] 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 al., 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(s1)-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. 2001 April; 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 al., Organization of the murine reduced folate carrier gene and identification of variant splice forms, Biochem Biophys Acta. Aug. 7, 1997; 1353(2): 191-8).
[0665] 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 251P5G2 has a particular expression profile related to cancer. Alternative transcripts and splice variants of 251P5G2 may also be involved in cancers in the same or different tissues, thus serving as tumor-associated markers/antigens.
[0666] The exon composition of the original transcript, designated as 251P5G2 v.1, is shown in Table LI. Using the full-length gene and EST sequences, two transcript variants were identified, designated as 251P5G2 v.12 and v.13. Compared with 251P5G2 v.1, transcript variant 251P5G2 v.12 has spliced out two fragments from variant 251P5G2 v.1, as shown in FIG. 12. Theoretically, each different combination of exons in spatial order, e.g. exons 2 and 3, is a potential splice variant. FIG. 12 shows the schematic alignment of exons of the two transcript variants. Tables LII (a) and (b) through LV (a) and (b) are set forth on a variant by variant basis. LII (a) and (b) shows nucleotide sequence of the transcript variant. Table LIII (a) and (b) shows the alignment of the transcript variant with nucleic acid sequence of 251P5G2 v.1. Table LIV (a) and (b) lays out amino acid translation of the transcript variant for the identified reading frame orientation. Table LV (a) and (b) displays alignments of the amino acid sequence encoded by the splice variant with that of 251P5G2 v.1.
Example 6[0667] Single Nucleotide Polymorphisms of 251P5G2
[0668] A Single Nucleotide Polymorphism (SNP) is a single base pair variation in a nucleotide sequence at a specific location. At any given point of the genome, there are four possible nucleotide base pairs: A/T, C/G, G/C and T/A. Genotype refers to the specific base pair sequence of one or more locations in the genome of an individual. Haplotype refers to the base pair sequence of more than one location on the same DNA molecule (or the same chromosome in higher organisms), often in the context of one gene or in the context of several tightly linked genes. 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; 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 diseases, and analysis of the genetic relationship between individuals (P. Nowotny, J. M. Kwon and A. M. Goate, “SNP analysis to dissect human traits,” Curr. Opin. Neurobiol. 2001 October; 11(5):637-641; M. Pirmohamed and B. K. Park, “Genetic susceptibility to adverse drug reactions,” Trends Pharmacol. Sci. 2001 June; 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. 2000 February; 1(1):39-47; R. Judson, J. C. Stephens and A. Windemuth, “The predictive power of haplotypes in clinical response,” Pharmacogenomics. 2000 February; 1(1):15-26).
[0669] SNPs are identified by a variety of art-accepted methods (P. Bean, “The promising voyage of SNP target discovery,” Am. Clin. Lab. 2001 October-November; 20(9):18-20; K. M. Weiss, “In search of human variation,” Genome Res. 1998 July; 8(7):691-697; M. M. She, “Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies,” Clin. Chem. 2001 February; 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. 2000 December; 5(4):329-340). Using the methods described above, ten SNPs were identified in the original transcript, 251P5G2 v.1, at positions 768 (T/G), 975 (T/A), 1005 (G/A), 1270 (A/G), 1459 (A/G), 1921 (A/G), 540 (GfT), 481 (C/T), 280 (G/A) and 162 (A/T). The transcripts or proteins with alternative alleles were designated as variants 251P5G2 v.2, v.3, v.4, v.5, v.6, v.7, v.8, v.9, v.10 and v.11, respectively. FIG. 10 shows the schematic alignment of the SNP 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 (such as 251P5G2 v.12) that contains the sequence context of the SNPs.
Example 7[0670] Production of Recombinant 251P5G2 in Prokarvotic Systems
[0671] To express recombinant 251P5G2 and 251P5G2 variants in prokaryotic cells, the full or partial length 251P5G2 and 251P5G2 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 251P5G2 variants are expressed: the full length sequence presented in FIGS. 2 and 3, 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 251P5G2, variants, or analogs thereof.
[0672] A. In Vitro Transcription and Translation Constructs:
[0673] pCRII: To generate 251P5G2 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 251P5G2 cDNA. The PCRII vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 251P5G2 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of 251P5G2 at the RNA level. Transcribed 251P5G2 RNA representing the cDNA amino acid coding region of the 251P5G2 gene is used in in vitro translation systems such as the TnT™ Coupled Reticulolysate System (Promega, Corp., Madison, Wis.) to synthesize 251P5G2 protein.
[0674] B. Bacterial Constructs:
[0675] pGEX Constructs: To generate recombinant 251P5G2 proteins in bacteria that are fused to the Glutathione S-transferase (GST) protein, all or parts of the 251P5G2 cDNA protein coding sequence are cloned into the pGEX family of GST-fusion vectors (Amersham Pharmacia Biotech, Piscataway, N.J.). These constructs allow controlled expression of recombinant 251P5G2 protein sequences with GST fused at the amino-terminus and a six histidine epitope (6X His) at the carboxyl-terminus. The GST and 6X 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 6X 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 251P5G2-related protein. The ampicillin resistance gene and pBR322 origin permits selection and maintenance of the pGEX plasmids in E. coli.
[0676] pMAL Constructs: To generate, in bacteria, recombinant 251P5G2 proteins that are fused to maltose-binding protein (MBP), all or parts of the 251P5G2 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 251P5G2 protein sequences with MBP fused at the amino-terminus and a 6X His epitope tag at the carboxyl-terminus. The MBP and 6X 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 6X 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 251P5G2. 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.
[0677] pET Constructs: To express 251P5G2 in bacterial cells, all or parts of the 251P5G2 cDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, Wis.). These vectors allow tightly controlled expression of recombinant 251P5G2 protein in bacteria with and without fusion to proteins that enhance solubility, such as NusA and thioredoxin (Trx), and epitope tags, such as 6X 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 251P5G2 protein are expressed as amino-terminal fusions to NusA.
[0678] C. Yeast Constructs:
[0679] pESC Constructs: To express 251P5G2 in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of the 251P5G2 cDNA protein coding sequence are cloned into the pESC family of vectors each of which contain 1 of 4 selectable markers, HIS3, TRP1, 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 251P5G2. In addition, expression in yeast yields similar post-translational modifications, such as glycosylations and phosphorylations that are found when expressed in eukaryotic cells.
[0680] PESP Constructs: To express 251P5G2 in the yeast species Saccharomyces pombe, all or parts of the 251P5G2 cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 251P5G2 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[0681] Production of Recombinant 251P5G2 in Higher Eukarvotic Systems
[0682] A. Mammalian Constructs:
[0683] To express recombinant 251P5G2 in eukaryotic cells, the full or partial length 251P5G2 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 251P5G2 are expressed in these constructs, amino acids 1 to 255, 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 251P5G2 v.1 through v.11; amino acids 1 to 1266, 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 251P5G2 v.12 and v.13, variants, or analogs thereof.
[0684] 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-251P5G2 polyclonal serum, described herein.
[0685] pcDNA4/HisMax Constructs: To express 251P5G2 in mammalian cells, a 251P5G2 ORF, or portions thereof, of 251P5G2 are cloned into pcDNA41HisMax Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter and the SP16 translational enhancer. The recombinant protein has Xpress™ and six histidine (6X 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.
[0686] pcDNA3.1/MycHis Constructs: To express 251P5G2 in mammalian cells, a 251P5G2 ORF, or portions thereof, of 251P5G2 with a consensus Kozak translation initiation site was 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 6X 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.
[0687] pcDNA3.1/CT-GFP-TOPO Construct: To express 251P5G2 in mammalian cells and to allow detection of the recombinant proteins using fluorescence, a 251P5G2 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 pcDNA3.1CT-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 251P5G2 protein.
[0688] PAPtag: A 251P5G2 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 251P5G2 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 251P5G2 protein. The resulting recombinant 251P5G2 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 251P5G2 proteins. Protein expression is driven from the CMV promoter and the recombinant proteins also contain myc and 6X 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.
[0689] pTag5: A 251P5G2 ORF, or portions thereof, is cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generates 251P5G2 protein with an amino-terminal IgGK signal sequence and myc and 6X His epitope tags at the carboxyl-terminus that facilitate detection and affinity purification. The resulting recombinant 251P5G2 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 251P5G2 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.
[0690] PsecFc: A 251P5G2 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, California). This construct generates an IgG1 Fc fusion at the carboxyl-terminus of the 251P5G2 proteins, while fusing the IgGk signal sequence to N-terminus. 251P5G2 fusions utilizing the murine IgG1 Fc region are also used. The resulting recombinant 251P5G2 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 251P5G2 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.
[0691] pSR&agr; Constructs: To generate mammalian cell lines that express 251P5G2 constitutively, 251P5G2 ORF, or portions thereof, of 251P5G2 were cloned into pSR&agr; constructs. Amphotropic and ecotropic retroviruses were 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, 251P5G2, 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 ColE1 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-1 cells.
[0692] Additional pSR&agr; constructs are made that fuse an epitope tag such as the FLAG™ tag to the carboxyl-terminus of 251P5G2 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: 53) is added to cloning primer at the 3′ end of the ORF. Additional pSR&agr; constructs are made to produce both amino-terminal and carboxyl-terminal GFP and myc/6X His fusion proteins of the full-length 251P5G2 proteins.
[0693] Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of 251P5G2. High virus fiter leading to high level expression of 251P5G2 is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. A 251P5G2 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, 251P5G2 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.
[0694] Regulated Expression Systems: To control expression of 251P5G2 in mammalian cells, coding sequences of 251P5G2, or portions thereof, are cloned into regulated mammalian expression systems such as the T-Rex System (Invitrogen), the GeneSwitch System (Invitrogen) and the tightiy-regulated Ecdysone System (Sratagene). These systems allow the study of the temporal and concentration dependent effects of recombinant 251P5G2. These vectors are thereafter used to control expression of 251P5G2 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
[0695] B. Baculovirus Expression Systems
[0696] To generate recombinant 251P5G2 proteins in a baculovirus expression system, 251P5G2 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-251P5G2 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.
[0697] Recombinant 251P5G2 protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant 251P5G2 protein can be detected using anti-251P5G2 or anti-His-tag antibody. 251P5G2 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 251P5G2.
Example 9[0698] Antigenicity Profiles and Secondary Structure
[0699] FIGS. 5(A & B), FIGS. 6(A & B), FIGS. 7(A & B), FIGS. 8(A & B), and FIGS. 9(A & B) depict graphically five amino acid profiles of 251P5G2 variants 1 and 12, each assessment available by accessing the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) on the ExPasy molecular biology server.
[0700] 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 251P5G2 protein. Each of the above amino acid profiles of 251P5G2 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.
[0701] 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.
[0702] 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.
[0703] Antigenic sequences of the 251P5G2 variant proteins indicated, e.g., by the profiles set forth in FIGS. 5(A & B), FIGS. 6(A & B), FIGS. 7(A & B), FIGS. 8(A & B), and/or FIGS. 9(A & B) are used to prepare immunogens, either peptides or nucleic acids that encode them, to generate therapeutic and diagnostic anti-251P5G2 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 251P5G2 protein variants 1 and 12 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 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profiles of FIG. 5; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 6; a peptde region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profiles of FIG. 7; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profiles on FIG. 8; and, a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIG. 9. Peptide immunogens of the invention can also comprise nucleic acids that encode any of the forgoing.
[0704] 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.
[0705] The secondary structure of 251P5G2 protein variants 1 and 12, namely the predicted presence and location of alpha helices, extended strands, and random coils, is predicted from the primary amino acid sequence 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 located on the World Wide Web at (.expasy.ch/tools/). The analysis indicates that 251P5G2 variant 1 is composed of 40.39% alpha helix, 18.82% extended strand, and 40.78% random coil (FIG. 13A). Variant 12 is composed of 42.28% alpha helix, 8.33% extended strand, and 49.39% random coil (FIG. 13B). Analysis for the potential presence of transmembrane domains in the 251P5G2 variant proteins was carried out using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server located on the World Wide Web at (.expasy.ch/tools/). Shown graphically in FIGS. 13C and 13D are the results of analysis of variant 1 depicting the presence and location of 6 transmembrane domains using the TMpred program (FIG. 13C) and 5 transmembrane domains using the TMHMM program (FIG. 13D). Shown graphically in FIGS. 13E and 13F are the results of analysis of variant 12 depicting the presence and location of 6 transmembrane domains using the TMpred program (FIG. 13E) and 3 transmembrane domains using the TMHMM program (FIG. 13F). The results of each program, namely the amino acids encoding the transmembrane domains are summarized in Table VI.
Example 10[0706] Generation of 251P5G2 Polyclonal Antibodies
[0707] 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 a full length 251P5G2 protein variant, 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. 5(A & B), FIGS. 6(A & B), FIGS. 7(A & B), FIGS. 8(A & B), or FIGS. 9(A & B) for amino acid profiles that indicate such regions of 251P5G2 protein variants).
[0708] For example, recombinant bacterial fusion proteins or peptdes containing hydrophilic, flexible, beta-turn regions of 251P5G2 protein variants are used as antigens to generate polyconal antibodies in New Zealand White rabbits. For example, in 251P5G2 variant 1, such regions include, but are not limited to, amino acids 28-40, amino acids 65-85, and amino acids 200-222. In sequence specific for variant 12, such regions include, but are not limited to, amino acids 236-251, amino acids 540-598, amino acids 832-978, and amino acids 1151-1242 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 65-85 of 251P5G2 variant 1 is conjugated to KLH and used to immunize the rabbit. Alternatively the immunizing agent may include all or portions of the 251P5G2 variant proteins, analogs or fusion proteins thereof. For example, the 251P5G2 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.
[0709] In one embodiment, a GST-fusion protein encoding the whole cDNA of 251P5G2 variant 1, amino acids 1-255 fused to GST, 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 Recombinant 251P5G2 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).
[0710] 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 251P5G2 in Eukaryotic Systems”), and retain post-translational modifications such as glycosylations found in native protein. In one embodiment, amino acids 60-85 of variant 1, encoding a loop between transmembrane domains, is cloned into the TagS 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 251P5G2 protein is then used as immunogen.
[0711] 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).
[0712] 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.
[0713] To test reactivity and specificity of immune serum, such as the rabbit serum derived from immunization with the Tag5-251P5G2 variant 1 protein, the full-length 251P5G2 variant 1 cDNA is cloned into pCDNA 3.1 myc-his expression vector (Invitrogen, see the Example entitled “Production of Recombinant 251P5G2 in Eukaryotic Systems”). After transfection of the constructs into 293T cells, cell lysates are probed with the anti-251P5G2 serum and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.) to determine specific reactivity to denatured 251P5G2 protein using the Western blot technique. In addition, the immune serum is tested by fluorescence microscopy, flow cytometry and immunoprecipitation against 293T and other recombinant 251P5G2-expressing cells to determine specific recognition of native protein. Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometric techniques using cells that endogenously express 251P5G2 are also carried out to test reactivity and specificity.
[0714] Anti-serum from rabbits immunized with 251P5G2 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-251P5G2 variant 1 fusion protein is first purified by passage over a column of GST protein covalenily coupled to AffiGel matrix (BioRad, Hercules, Calif.). The antiserum is then affinity purified by passage over a column composed of a MBP-251P5G2 fusion protein covalenfy 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[0715] Generation of 251P5G2 Monoclonal Antibodies (mAbs)
[0716] In one embodiment, therapeutic mAbs to 251P5G2 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 251P5G2 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 251P5G2 protein variant sequence, regions of the 251P5G2 protein variants predicted to be antigenic from computer analysis of the amino acid sequence (see, e.g., FIGS. 5(A & B), FIGS. 6(A & B), FIGS. 7(A & B), FIGS. 8(A & B), or FIGS. 9(A & B), and the Example entitled “Antigenicity Profiles”). 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 251P5G2 variant, such as 293T-251P5G2 variant 1 or 300.19-251P5G2 variant 1murine Pre-B cells, are used to immunize mice.
[0717] To generate mAbs to a 251P5G2 variant, mice are first immunized intraperitoneally (IP) with, typically, 10-50 &mgr;g of protein immunogen or 107 251P5G2-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 251P5G2 variant sequence is used to immunize mice by direct injection of the plasmid DNA. For example, amino acids 60-85 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 251P5G2 variant 1 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 251P5G2 variant.
[0718] 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).
[0719] In one embodiment for generating 251P5G2 monoclonal antibodies, a Tag5-251P5G2 variant 1 antigen encoding amino acids 60-85, is expressed and purified from stably transfected 293T cells. Balb C mice are initially immunized intraperitoneally with 25 &mgr;g of the Tag5-251P5G2 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 251P5G2 variant 1 protein is monitored by Western blotting, immunoprecipitation and flow cytometry using 293T cells transfected with an expression vector encoding the 251P5G2 variant 1 cDNA (see e.g., the Example entitled “Production of Recombinant 251P5G2 in Eukaryotic Systems” and FIG. 20. Other recombinant 251P5G2 variant 1-expressing cells or cells endogenously expressing 251P5G2 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 251P5G2 specific antibody-producing clones.
[0720] In another embodiment, a Tag5 antigen encoding amino acids 800-1266 of variant 12 is produced, purified and used as immunogen to derive monoclonal antibodies specific to 251P5G2 variant 12. Hybridoma supernatants are then screened on both 251P5G2 variant 1- and 251P5G2 variant 12-expressing cells to identify specific anti-251P5G2 variant 12 monoclonal antibodies.
[0721] The binding affinity of a 251P5G2 monoclonal antibody is determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 251P5G2 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[0722] HLA Class I and Class II Binding Assays
[0723] 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 1251-radiolabeled probe peptides as described. Following incubation, MHG-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 radiolabefed peptdes 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.
[0724] 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.
[0725] Binding assays as outined above may be used to analyze HLA supenmotif and/or HLA motif-bearing peptides (see Table IV).
Example 13[0726] Identification of HLA Supermotif and Motif-Bearing CTL Candidate Epitopes
[0727] 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 supernotif- and motif-bearing epitopes for the inclusion in such a vaccine composition. Calculation of population coverage is performed using the strategy described below.
[0728] Computer Searches and Algorithms for Identification of Supermotif and/or Motif-Bearing Epitopes
[0729] The searches performed to identify the motif-bearing peptide sequences in the Example entitled “Antigenicity Profiles” and Tables VIII-XXI and XXII-XLIX employ the protein sequence data from the gene product of 251P5G2 set forth in FIGS. 2 and 3, the specific search peptides used to generate the tables are listed in Table VII.
[0730] Computer searches for epitopes bearing HLA Class I or Class II supermotfs or motifs are performed as follows. All translated 251P5G2 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.
[0731] 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:
“&Dgr;G”=a1i×a2i×a3i . . . ×ani
[0732] where aji is a coefficient which represents the effect of the presence of a given amino acid (j) 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 ji to the free energy of binding of the peptide irrespective of the sequence of the rest of the peptide.
[0733] 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 ji. 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.
[0734] Selection of HLA-A2 Supertype Cross-Reactive Peptides
[0735] Protein sequences from 251P5G2 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).
[0736] 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.
[0737] Selection of HLA-A3 Supermotif-Bearing Epitopes
[0738] The 251P5G2 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-reactvity 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.
[0739] Selection of HLA-B7 Supermotif Bearing Epitopes
[0740] The 251P5G2 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.
[0741] Selection of A1 and A24 Motif-Bearing Epitopes
[0742] To further increase population coverage, HLA-A1 and -A24 epitopes can also be incorporated into vaccine compositions. An analysis of the 251P5G2 protein can also be performed to identify HLA-A1- and A24-motif-containing sequences.
[0743] High affinity and/or cross-reactive binding epitopes that bear other motif and/or supermotifs are identified using analogous methodology.
Example 14[0744] Confirmation of Immunogenicity
[0745] 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:
[0746] Target Cell Lines for Cellular Screening:
[0747] The .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 pepide-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 pepfide-specific CTLs to recognize endogenous antigen.
[0748] Primary CTL Induction Cultures:
[0749] 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-essental amino acids, sodium pyruvate, L-glutamine and penicillin/streptomycin). The monocytes are purilied 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 supematants. 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 IL4 are then added to each well. TNF&agr; is added to the DCs on day 6 at 75 ng/ml and the cells are used for CTL induction cultures on day 7.
[0750] Induction of CTL with DC and Peptide: CD8+ T-cells are isolated by positive selection with Dynal immunomagnetic beads (Dynabeads® M450) and the detacha-bead® reagent. Typically about 200-250×106 PBMC are processed to obtain 24×106 CD8+ T-cells (enough fora 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 20×106 cells/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/ml (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.
[0751] 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.
[0752] 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 &bgr;2 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 50 IU/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 IFNY 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.
[0753] Measurement of CTL Lytic Activity by 51Cr Release
[0754] 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. Peptde-pulsed targets are prepared by incubating the cells with 10 &mgr;g/ml peptide overnight at 37° C.
[0755] 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.
[0756] 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.
[0757] In situ Measurement of Human IFN&ggr; Production as an Indicator of Peptide-specific and Endogenous Recognition
[0758] Immulon 2 plates are coated with mouse anti-human IFN&ggr; monoclonal antibody (4 &mgr;g/ml 0.1M NaHCO3, pH8.2) 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.
[0759] Recombinant human IFN-gamma 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.
[0760] CTL Expansion
[0761] 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 sftu IFN&ggr; assay using the same targets as before the expansion.
[0762] 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 M, sodium pyruvate, 25 mM 2-ME, L-glutamine and gentamicin.
[0763] Immunogenicity of A2 Supermotif-Bearing Peptides
[0764] 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.
[0765] Immunogenicity can also be confirmed using PBMCs isolated from patients bearing a tumor that expresses 251P5G2. 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.
[0766] Evaluation of A*03/A11 Immunogenicity
[0767] 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.
[0768] Evaluation of B7 Immunogenicity
[0769] 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.
[0770] Peptides bearing other supermotifs/motifs, e.g., HLA-A1, HLA-A24 etc. are also confirmed using similar methodology
Example 15[0771] Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creating Analogs
[0772] 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.
[0773] Analoging at Primary Anchor Residues
[0774] 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.
[0775] 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.
[0776] 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.
[0777] The selection of analogs for immunogenicity 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).
[0778] 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.
[0779] Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides
[0780] 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.
[0781] 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.
[0782] 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).
[0783] Analoging at primary anchor residues of other motif and/or supermotif-bearing epitopes is performed in a like manner.
[0784] 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.
[0785] Analoging at Secondary Anchor Residues
[0786] 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.
[0787] 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 251P5G2-expressing tumors.
[0788] Other Analoging Strategies
[0789] 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. Ahmed and I. Chen, John Wiley & Sons, England, 1999).
[0790] 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[0791] Identification and Confirmation of 251P5G2-Derived Sequences with HLA-DR Binding Motifs
[0792] 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.
[0793] Selection of HLA-DR-Supermotif-Bearing Epitopes
[0794] To identify 251P5G2derived, HLA class II HTL epitopes, a 251P5G2 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).
[0795] 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.
[0796] The 251P5G2-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. 251P5G2-derived peptides found to bind common HLA-DR alleles are of particular interest.
[0797] Selection of DR3 Motif Peptides
[0798] 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.
[0799] To efficiently identify peptides that bind DR3, target 251P5G2 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.
[0800] DR3 binding epitopes identified in this manner are included in vaccine compositions with DR supermotif-bearing peptide epitopes.
[0801] 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[0802] Immunogenicity of 251P5G2-Derived HTL Epitones
[0803] This example determines immunogenic DR supermotif- and DR3 motif-bearing epitopes among those identified using the methodology set forth herein.
[0804] 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 251P5G2-expressing tumors.
Example 18[0805] Calculation of Phenotypic Frequencies of HLA-Supertypes in Various Ethnic Backgrounds to Determine Breadth of Population Coverage
[0806] 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.
[0807] 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].
[0808] 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).
[0809] 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 Al and A24 are combined with the coverage of the A2-, A3- and B7-supertype alleles is >95%, see, e.g., Table IV (G). An analogous approach can be used to estimate population coverage achieved with combinations of class II motif-bearing epitopes.
[0810] 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.
[0811] 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[0812] CTL Recognition Of Endogenously Processed Antigens After Priming
[0813] 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.
[0814] 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 251P5G2 expression vectors.
[0815] The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized 251P5G2 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-A1 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[0816] Activity Of CTL-HTL Coniuqated Epitopes In Transgenic Mice
[0817] This example illustrates the induction of CTLs and HTLs in transgenic mice, by use of a 251P5G2-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a 251P5G2-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.
[0818] 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.
[0819] 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)
[0820] 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.
[0821] 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.
[0822] The results are analyzed to assess the magnitude of the CTL responses of animals injected with the immunogenic CTU/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[0823] Selection of CTL and HTL Epitopes for Inclusion in a 251P5G2-Specific Vaccine
[0824] 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.
[0825] 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.
[0826] Epitopes are selected which, upon administration, mimic immune responses that are correlated with 251P5G2 clearance. The number of epitopes used depends on observations of patients who spontaneously clear 251P5G2. For example, if it has been observed that patients who spontaneously clear 251P5G2-expressing cells generate an immune response to at least three (3) epitopes from 251P5G2 antigen, then at least three epitopes should be included for HLA class I. A similar rationale is used to determine HLA class II epitopes.
[0827] 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/.
[0828] 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.
[0829] 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 251P5G2, 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.
[0830] 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 251P5G2.
Example 22[0831] Construction of “Minigene” Multi-Epitope DNA Plasmids
[0832] 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.
[0833] 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 251P5G2, are selected such that multiple supermofifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from 251P5G2 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.
[0834] Such a construct may additionally include sequences that direct the HTL epitopes to the endoplasmic reticulum. For example, the li protein may be fused to one or more HTL epitopes as described in the art, wherein the CLIP sequence of the li 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.
[0835] 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.
[0836] The minigene DNA plasmid of this example contains a consensus Kozak sequence and a consensus murine kappa lg-light chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disdosed 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.
[0837] 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.
[0838] 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 (1x=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 dones are screened by sequencing.
Example 23[0839] The Plasmid Construct and the Degree to Which it Induces Immunogenicity
[0840] 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).
[0841] 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.
[0842] 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-A2.1/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.
[0843] 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.
[0844] 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.
[0845] 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.
[0846] 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).
[0847] 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-specitic activity in an alpha, beta and/or gamma IFN ELISA.
[0848] 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-A11 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[0849] Peptide Compositions for Prophylactic Uses
[0850] Vaccine compositions of the present invention can be used to prevent 251P5G2 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 251P5G2-associated tumor.
[0851] 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 251P5G2-associated disease.
[0852] 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[0853] Polyepitopic Vaccine Compositions Derived from Native 251P5G2 Sequences
[0854] A native 251P5G2 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 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, ie., 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.
[0855] The vaccine composition will include, for example, multiple CTL epitopes from 251P5G2 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.
[0856] 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 251P5G2, 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.
[0857] 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[0858] Polyepitopic Vaccine Compositions from Multiple Antigens
[0859] The 251P5G2 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 251P5G2 and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from 251P5G2 as well as tumor-associated antigens that are often expressed with a target cancer associated with 251P5G2 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[0860] Use of Peptides to Evaluate an Immune Response
[0861] Peptides of the invention may be used to analyze an immune response for the presence of specific antibodies, CTL or HTL directed to 251P5G2. 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.
[0862] In this example highly sensitive human leukocyte antigen tetrameric complexes (“tetramers”) are used for a cross-sectional analysis of, for example, 251P5G2 HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of disease or following immunization comprising a 251P5G2 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 &bgr;2-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, P2-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.
[0863] 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 251P5G2 epitope, and thus the status of exposure to 251P5G2, or exposure to a vaccine that elicits a protective or therapeutic response.
Example 28[0864] Use of Peptide Epitopes to Evaluate Recall Responses
[0865] 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 251P5G2-associated disease or who have been vaccinated with a 251P5G2 vaccine.
[0866] For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any 251P5G2 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.
[0867] 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 (50 U/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.
[0868] 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).
[0869] 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).
[0870] 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.
[0871] 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 effectoritarget (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.
[0872] The results of such an analysis indicate the extent to which HLA-restricted CTL populations have been stimulated by previous exposure to 251P5G2 or a 251P5G2 vaccine.
[0873] 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 251P5G2 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[0874] Induction Of Specific CTL Response In Humans
[0875] 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:
[0876] A total of about 27 individuals are enrolled and divided into 3 groups:
[0877] Group I: 3 subjects are injected with placebo and 6 subjects are injected with 5 &mgr;g of peptide composition;
[0878] Group II: 3 subjects are injected with placebo and 6 subjects are injected with 50 &mgr;g peptide composition;
[0879] Group III: 3 subjects are injected with placebo and 6 subjects are injected with 500 &mgr;g of peptide composition.
[0880] After 4 weeks following the first injection, all subjects receive a booster inoculation at the same dosage.
[0881] 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.
[0882] Safety: The incidence of adverse events is monitored in the placebo and drug treatment group and assessed in terms of degree and reversibility.
[0883] 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.
[0884] The vaccine is found to be both safe and efficacious.
Example 30[0885] Phase II Trials In Patients Expressing 251P5G2
[0886] Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses 251P5G2. The main objectives of the trial are to determine an effective dose and regimen for inducing CTLs in cancer patients that express 251P5G2, 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:
[0887] 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.
[0888] 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 251P5G2.
[0889] 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 251P5G2-associated disease.
Example 31[0890] Induction of CTL Responses Using a Prime Boost Protocol
[0891] 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.
[0892] 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.
[0893] Analysis of the results indicates that a magnitude of response sufficient to achieve a therapeutic or protective immunity against 251P5G2 is generated.
Example 32[0894] Administration of Vaccine Compositions Using Dendritic Cells (DC)
[0895] 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 251P5G2 protein from which the epitopes in the vaccine are derived.
[0896] 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.
[0897] 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.
[0898] 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.
[0899] Ex Vivo Activation of CTL/HTL Responses
[0900] Alternatively, ex vivo CTL or HTL responses to 251P5G2 antigens can be induced by incubating, in tissue culture, the patients, 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[0901] An Alternative Method of Identifying and Confirming Motif-Bearing Peptides
[0902] 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. 251P5G2. 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.
[0903] 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 251P5G2 to isolate peptides corresponding to 251P5G2 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.
[0904] 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[0905] Complementary Polynucleotides
[0906] Sequences complementary to the 251P5G2-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring 251P5G2. 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 251P5G2. 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 251P5G2-encoding transcript.
Example 35[0907] Purification of Naturally-occurring or Recombinant 251P5G2 Using 251P5G2-Specific Antibodies
[0908] Naturally occurring or recombinant 251P5G2 is substantially purified by immunoaffinity chromatography using antibodies specific for 251P5G2. An immunoaffinity column is constructed by covalently coupling anti-251P5G2 antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturers instructions.
[0909] Media containing 251P5G2 are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of 251P5G2 (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/251P5G2 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[0910] Identification of Molecules Which Interact with 251P5G2
[0911] 251P5G2, 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 251P5G2, washed, and any wells with labeled 251P5G2 complex are assayed. Data obtained using different concentrations of 251P5G2 are used to calculate values for the number, affinity, and association of 251P5G2 with the candidate molecules.
Example 37[0912] In Vivo Assay for 251P5G2 Tumor Growth Promotion
[0913] The effect of the 251P5G2 protein on tumor cell growth is evaluated in vivo by evaluating tumor development and growth of cells expressing or lacking 251P5G2. For example, SCID mice are injected subcutaneously on each flank with 1×106 of either 3T3, prostate or bladder cancer cell lines (e.g. PC3 or J82 cells) containing tkNeo empty vector or 251P5G2. At least two strategies may be used: (1) Constitutive 251P5G2 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), adenovinus (such as Adenovinus 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, tetracycline, etc., provided such promoters are compatible with the host cell systems. Tumor volume is then monitored by caliper measurement at the appearance of palpable tumors and followed over time to determine if 251P5G2-expressing cells grow at a faster rate and whether tumors produced by 251P5G2-expressing cells demonstrate characteristics of altered aggressiveness (e.g. enhanced metastasis, vascularization, reduced responsiveness to chemotherapeutic drugs).
[0914] Additionally, mice can be implanted with 1×105 of the same cells orthotopically to determine if 251P5G2 has an effect on local growth in the bladder or prostate, and whether 251P5G2 affects the ability of the cells to metastasize, specifically to lymph nodes and bone (Fu. X. et al, Int J Cancer. 1992, 52:987; Fu. X. et al, Int J Cancer. 1991, 49:938). The assay is also useful to determine the 251P5G2 inhibitory effect of candidate therapeutic compositions, such as for example, 251P5G2 intrabodies, 251P5G2 antisense molecules and ribozymes.
Example 38[0915] 251P5G2 Monoclonal Antibody-mediated Inhibition of Bladder, and Prostate Tumors In Vivo
[0916] The significant expression of 251P5G2 in cancer tissues, together with its restrictive expression in normal tissues makes 251P5G2 a good target for antibody therapy. Similarly, 251P5G2 is a target for T cell-based immunotherapy. Thus, the therapeutic efficacy of anti-251P5G2 mAbs in human prostate cancer xenograft mouse models is evaluated by using recombinant cell lines such as PC3-251P5G2, and 3T3-251P5G2 (see, e.g., Kaighn, M. E., et al., Invest Urol, 1979. 17(1): p. 16-23), as well as human prostate xenograft models such as LAPC9 (Saffran et al, Proc Natl Acad Sci USA. 2001, 98:2658). Similarly, anti-251P5G2 mAbs are evaluated in human bladder cancer xenograft models using recombinant cell lines such as J82-251P5G2.
[0917] Antibody efficacy on tumor growth and metastasis formation is studied, e.g., in a mouse orthotopic bladder cancer xenograft model, and a mouse prostate cancer xenograft model. The antibodies can be unconjugated, as discussed in this Example, or can be conjugated to a therapeutic modality, as appreciated in the art. Anti-251P5G2 mAbs inhibit formation of prostate and bladder xenografts. Anti-251P5G2 mAbs also retard the growth of established orthotopic tumors and prolonged survival of tumor-bearing mice. These results indicate the utility of anti-251P5G2 mAbs in the treatment of local and advanced stages of prostate and bladder cancer. (See, e.g., Saffran, D., et al., PNAS 10:1073-1078 or on the World Wide Web at (.pnas.org/cgi/doi/10.1073/pnas.051624698).
[0918] Administration of the anti-251P5G2 mAbs led to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies indicate that 251P5G2 as an attractive target for immunotherapy and demonstrate the therapeutic potential of anti-251P5G2 mAbs for the treatment of local and metastatic cancer. This example demonstrates that unconjugated 251P5G2 monoclonal antibodies are effective to inhibit the growth of human bladder and prostate tumor xenografts grown in SCID mice; accordingly a combination of such efficacious monoclonal antibodies is also effective.
[0919] Tumor Inhibition Using Multiple Unconjugated 251P5G2 mAbs
[0920] Materials and Methods
[0921] 251P5G2 Monoclonal Antibodies:
[0922] Monoclonal antibodies are raised against 251P5G2 as described in the Example entitled “Generation of 251P5G2 Monoclonal Antibodies (mAbs).” The antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation for their capacity to bind 251P5G2. Epitope mapping data for the anti-251P5G2 mAbs, as determined by ELISA and Western analysis, recognize epitopes on the 251P5G2 protein. Immunohistochemical analysis of prostate cancer tissues and cells with these antibodies is performed.
[0923] 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 SCABER, J82, A498, 769P, CaOv1 or PA1 tumor xenografts.
[0924] Cell Lines
[0925] The bladder and prostate carcinoma cell lines, J82 and PC3 as well as the fibroblast line NIH 3T3 (American Type Culture Collection) are maintained in media supplemented with L-glutamine and 10% FBS.
[0926] A J82-251P5G2, PC3-251P5G2 and 3T3-251P5G2 cell populations are generated by retroviral gene transfer as described in Hubert, R. S., et al., Proc Natl Acad Sci USA, 1999. 96(25): 14523.
[0927] Xenograft Mouse Models.
[0928] Subcutaneous (s.c.) tumors are generated by injection of 1×106 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. Tumor sizes are determined by 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.
[0929] Orthotopic injections are performed under anesthesia by using ketamine/xylazine. For bladder orthotopic studies, an incision is made through the abdomen to expose the bladder, and tumor cells (5×105) mixed with Matrigel are injected into the bladder wall in a 10-&mgr;l volume. To monitor tumor growth, mice are palpated and blood is collected on a weekly basis to measure BTA levels. For prostate orthopotic models, an incision is made through the abdominal muscles to expose the bladder and seminal vesicles, which then are delivered through the incision to expose the dorsal prostate. Tumor cells e.g. LAPC-9 cells (5×105) mixed with Matrigel are injected into the prostate in a 10-&mgr;l volume (Yoshida Y et al, Anticancer Res. 1998, 18:327; Ahn et al, Tumour Biol. 2001, 22:146). To monitor tumor growth, blood is collected on a weekly basis measuring PSA levels. The mice are segregated into groups for the appropriate treatments, with anti-251P5G2 or control mAbs being injected i.p.
[0930] Anti-251P5G2 mAbs Inhibit Growth of 251P5G2-Expressing Xenograft-Cancer Tumors
[0931] The effect of anti-251P5G2 mAbs on tumor formation is tested on the growth and progression of bladder, and prostate cancer xenografts using J82-251P5G2, and PC3-251P5G2 orthotopic models. As compared with the s.c. tumor model, the orthotopic model, which requires injection of tumor cells directly in the mouse bladder, and prostate, respectively, results in a local tumor growth, development of metastasis in distal sites, deterioration of mouse health, and subsequent death (Saffran, D., et al., PNAS supra; Fu, X, et al., Int J Cancer, 1992. 52(6): p. 987-90; Kubota, T., J Cell Biochem, 1994. 56(1): p. 4-8). The features make the orthotopic model more representative of human disease progression and allowed us to follow the therapeutic effect of mAbs on clinically relevant end points.
[0932] Accordingly, tumor cells are injected into the mouse bladder, or prostate, and 2 days later, the mice are segregated into two groups and treated with either: a) 200-500 &mgr;g, of anti-251P5G2 Ab, or b) PBS three times per week for two to five weeks.
[0933] A major advantage of the orthotopic cancer models is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is studies by IHC analysis on lung sections using an antibody against a tumor-specific cell-surface protein such as anti-CK20 for bladder cancer, anti-STEAP-1 for prostate cancer models (Lin S et al, Cancer Detect Prev. 2001; 25:202; Saffran, D., et al., PNAS supra).
[0934] Mice bearing established orthotopic tumors are administered 1000 &mgr;g injections of either anti-251P5G2 mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden, to ensure a high frequency of metastasis formation in mouse lungs. Mice then are killed and their bladders, livers, bone and lungs are analyzed for the presence of tumor cells by IHC analysis.
[0935] These studies demonstrate a broad anti-tumor efficacy of anti-251P5G2 antibodies on initiation and progression of prostate and kidney cancer in xenograft mouse models. Anti-251P5G2 antibodies inhibit tumor formation of tumors as well as retarding the growth of already established tumors and prolong the survival of treated mice. Moreover, anti-251P5G2 mAbs demonstrate a dramatic inhibitory effect on the spread of local bladder and prostate tumor to distal sites, even in the presence of a large tumor burden. Thus, anti-251P5G2 mAbs are efficacious on major clinically relevant end points (tumor growth), prolongation of survival, and health.
Example 39[0936] Therapeutic and Diagnostic use of Anti-251P5G2 Antibodies in Humans
[0937] Anti-251P5G2 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-251P5G2 mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. Detection of 251P5G2 in carcinoma and in metastatic disease demonstrates the usefulness of the mAb as a diagnostic and/or prognostic indicator. Anti-251P5G2 antibodies are therefore used in diagnostic applications such as immunohistochemistry of kidney biopsy specimens to detect cancer from suspect patients.
[0938] As determined by flow cytometry, anti-251P5G2 mAb specifically binds to carcinoma cells. Thus, anti-251P5G2 antibodies are used in diagnostic whole body imaging applications, such as radioimmunosdintigraphy 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 251P5G2. Shedding or release of an extracellular domain of 251P5G2 into the extracellular milieu, such as that seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology 27:563-568 (1998)), allows diagnostic detection of 251P5G2 by anti-251P5G2 antibodies in serum and/or urine samples from suspect patients.
[0939] Anti-251P5G2 antibodies that specifically bind 251P5G2 are used in therapeutic applications for the treatment of cancers that express 251P5G2. Anti-251P5G2 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-251P5G2 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 “251P5G2 Monoclonal Antibody-mediated Inhibition of Bladder and Lung Tumors In Vivo”). Either conjugated and unconjugated anti-251P5G2 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[0940] Human Clinical Trials for the Treatment and Diagnosis of Human Carcinomas through Use of Human Anti-251P5G2 Antibodies In vivo
[0941] Antibodies are used in accordance with the present invention which recognize an epitope on 251P5G2, and are used in the treatment of certain tumors such as those listed in Table I. Based upon a number of factors, including 251P5G2 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.
[0942] I.) Adjunctive therapy: In adjunctive therapy, patients are treated with anti-251P5G2 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-251P5G2 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-251P5G2 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).
[0943] II.) Monotherapy: In connection with the use of the anti-251P5G2 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.
[0944] III.) Imaging Agent: Through binding a radionuclide (e.g., iodine or yttrium (I131, Y90) to anti-251P5G2 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 251P5G2. In connection with the use of the anti-251P5G2 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)-251P5G2 antibody is used as an imaging agent in a Phase I human clinical trial in patients having a carcinoma that expresses 251P5G2 (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.
[0945] Dose and Route of Administration
[0946] 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-251P5G2 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-251P5G2 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-251P5G2 antibodies that are fully human antibodies, as compared to the chimeric antibody, have slower clearance; accordingly, dosing in patients with such fully human anti-251P5G2 antibodies can be lower, perhaps in the range of 50 to 300 mg/m2, 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.
[0947] Three distinct delivery approaches are useful for delivery of anti-251P5G2 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.
[0948] Clinical Development Plan (CDP)
[0949] Overview: The CDP follows and develops treatments of anti-251P5G2 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-251P5G2 antibodies. As will be appreciated, one criteria that can be utilized in connection with enrollment of patients is 251P5G2 expression levels in their tumors as determined by biopsy.
[0950] 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 HAHA response); and, (iii) toxicity to normal cells that express 251P5G2. Standard tests and follow-up are utilized to monitor each of these safety concerns. Anti-251P5G2 antibodies are found to be safe upon human administration.
Example 41[0951] Human Clinical Trial Adiunctive Therapy with Human Anti-251P5G2 Antibody and Chemotherapeutic Agent
[0952] A phase I human clinical trial is initiated to assess the safety of six intravenous doses of a human anti-251P5G2 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-251P5G2 antibodies when utilized as an adjunctive therapy to an antineoplastic or chemotherapeutic agent as defined herein, such as, without limitation: cisplatin, topotecan, doxorubicin, adriamycin, taxol, or the like, is assessed. The trial design includes delivery of six single doses of an anti-251P5G2 antibody with dosage of antibody escalating from approximately about 25 mg/m2to about 275 mg/m2 over the course of the treatment in accordance with the following schedule: 4 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)
[0953] 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 251P5G2. 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.
[0954] The anti-251P5G2 antibodies are demonstrated to be safe and elfacious, Phase II trials confirm the efficacy and refine optimum dosing.
Example 42[0955] Human Clinical Trial: Monotherapy with Human Anti-251P5G2 Antibody
[0956] Anti-251P5G2 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-251P5G2 antibodies.
Example 43[0957] Human Clinical Trial: Diagnostic Imaging with Anti-251P5G2 Antibody
[0958] 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-251P5G2 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[0959] Homology Comparison of 251P5G2 to Known Sequences
[0960] The 251P5G2 protein of FIG. 3 has 255 amino acids with calculated molecular weight of 29.3 kDa, and pl of 9.4. Three variants of 251P5G2 have been identified, 251P5G2 v.1, v.2 and v 3, which differ from variant 1 by 1 amino acid each at aa positions 16 and 95 respectively. The 251P5G2 protein exhibits homology to a previously cloned murine gene, namely vomeronasal 1 receptor, C3 (gi 20821692), and shows 44% identity and 60% homology to that gene over the length of the protein (FIG. 4). 251P5G2 is a multi-transmembrane protein, predicted to carry 5 or 6 transmembrane domains. Bioinformatic analysis indicates that the 251P5G2 protein may localize to the endoplasmic reticulum or peroxisome (see Table L). However, based on it topology and similarity to vomeronasal receptor suggest that 251P5G2 may also localize to the plasma membrane. Motif analysis revealed the presence of a vomeronasal organ pheromone receptor family signature, a rhodopsin-like GPCR superfamily signature, and iodothyronine deiodinase motif (see Table VI).
[0961] The vomeronasal receptors share sequence homology to other families of G protein-coupled receptors, and are distantly related to the T2R bitter taste receptors and rhodopsin-like GPCRs. G-protein coupled receptors are seven-transmembrane receptors that exhibit an extracellular amino-terminus, three extracellular loops, three intracellular loops and an intracellular carboxyl terminus. G-protein coupled receptors are stimulated by a variety of stimuli, including polypeptide hormones, neurotransmitters, chemokines and phospholipids (Civelli O et al, Trends Neurosci. 2001, 24:230; Vrecl M et al Mol Endocrinol. 1998, 12:1818). Ligand binding traditionally occurs between the first and second extracellular loops of the GPCR. Upon ligand binding GPCRs transduce signals across the cell surface membrane by associating with trimeric G proteins. Vomeronasal receptors are expressed in the apical regions of the vomeranasal organs, often in neurons expressing Gi2 subunits of G proteins. Vomeronasal receptors are activated by pheromones and signal by activating the ERK-MAPK and inositol trisphosphate pathways (Dudley C A et al, Brain Res. 2001, 915:32; Wekesa K S et al, Endocrinology. 1997, 138:3497). Although the function of vomeronasal receptors is not well understood, they appear to be associated with motility, cell proliferation and programmed cell death (Riccardi D. Cell Calcium. 1999, 26:77) all of which have a direct effect on tumor growth and progression. This is further supported by recent studies associating GPCRs with cellular transformation. In particular, KSHV G protein-coupled receptor was found to transform NIH 3T3 cells in vitro and induces multifocal KS-like lesions in KSHV-GPCR-transgenic mice (Schwarz M, Murphy P M. J Immunol 2001, 167:505). KSHV-GPCR was capable of producing its effect on endothelial cells and fibroblasts by activating defined signaling pathways, including the AKT survival pathway (Montaner S et al, Cancer Res 2001, 61:2641). In addition, KSHV-GPCR induced the activation of mitogenic pathways such as AP-1 and NFkB, resulting in the expression of pro-inflammatory genes (Schwarz M, Murphy P M. J Immunol 2001, 167:505).
[0962] Accordingly, when 251P5G2 functions as a regulator of tumor formation, cell proliferation, invasion or cell signaling, 251P5G2 is used for therapeutic, diagnostic, prognostic and/or preventative purposes.
Example 45[0963] Identification and Confirmation of Potential Signal Transduction Pathways
[0964] Many mammalian proteins have been reported to interact with signaling molecules and to participate in regulating signaling pathways. (J Neurochem. 2001; 76:217-223). In particular, GPCRs have been reported to activate MAK cascades as well as G proteins, and been associated with the EGFR pathway in epithelial cells (Naor, Z., et al, Trends Endocrinol Metab. 2000, 11:91; Vacate Fetal, Cancer Res. 2000, 60:5310; Della Rocca G. J., et al, J Biol Chem. 1999, 274:13978). In addition, GPCRs transmit their signals by activating the protein kinase A or the phospholipase C pathways, generating inositol 1,4,5-trisphosphate (IP3) and diacyl-glycerol (DAG) (Breer, 1993, Ciba Found Symp 179: 97; Bruch, 1996, Comp Biochem Physiol B Biochem Mol Biol 113:451).
[0965] Using immunoprecipitation and Western blotting techniques, proteins are identified that associate with 251P5G2 and mediate signaling events. Several pathways known to play a role in cancer biology can be regulated by 251P5G2, including phospholipid pathways such as P13K, AKT, 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). Using Western blotting and other techniques, the ability of 251P5G2 to regulate these pathways is confirmed. Cells expressing or lacking 251P5G2 are either left untreated or stimulated with cytokines, androgen 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, PI3K, PLC and other signaling molecules.
[0966] To confirm that 251P5G2 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.
[0967] 1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress
[0968] 2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation
[0969] 3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress
[0970] 4. ARE-luc, androgen receptor; steroids/MAPK; growth/differentiation/apoptosis
[0971] 5. p53-luc, p53; SAPK; growth/differentiation/apoptosis
[0972] 6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress
[0973] 7. TCF-luc, TCF/Lef; -catenin, Adhesion/invasion
[0974] 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.
[0975] Signaling pathways activated by 251P5G2 are mapped and used for the identification and validation of therapeutic targets. When 251P5G2 is involved in cell signaling, it is used as target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 46[0976] 251P5G2 Functions as a GPCR Inhibitors
[0977] Sequence and homology analysis of 251P5G2 indicated that 251P5G2 is a member of the pheromone GPCR family. Vomeronasal receptors are known to regulate biological-responses by activating PLC. In order to confirm that 251P5G2 functions as a GPCR and mediates the activation PLC, phosphorylation of PLC is investigated in PC3 and PC3-251P5G2 cells. Control PC3 and PC3-1251P5G2 cells are grown in a low concentration of fetal bovine serum (FBS) and stimulated with 10% FBS, pheromones and LPA. PLC phosphorylation is investigated by western blotting.
[0978] GPCRs are activated by ligand binding to the extracellular loops, resulting in the binding of trimeric G proteins to GPCR and their activation. Using this information, several therapeutic and small molecule strategies are utilized to inhibit GPCR activation or downstream signaling events.
[0979] One strategy inhibits receptor and ligand binding. Recent studies using several types of GPCRs, have demonstrated the effectiveness of this strategy (Fawzi A B, et al. 2001, Mol. Pharmacol., 59:30). Using a compound named SCH-202676, they inhibited agonist and antagonist binding to GPCRs by allosterically hindering ligand-GPCR interaction. Using this and even more specific allosteric (small molecule) inhibitors, signal transduction through 251P5G2 can be inhibited, thereby providing therapeutic, prognostic, diagnostic and/or prophylactic benefit.
[0980] A second approach is to inhibit G alpha subunit activation. Activation of GPCRs results in the exchange of GTP for GDP on the G alpha subunit of the trimeric G protein. Inhibition of Ga activation prevents the activation of downstream signaling cascades and therefore biological effects of GPCR. One molecule used to inhibit GDP exchange on G&agr; subunits is Suranim (Freissmuth M et al, 1996, Mol. Pharmacol, 49:602). Since suranim functions as a universal G&agr; inhibitor, it prevents the activation of most G&agr; subunits.
[0981] A third approach is to inhibit G&agr; subunit association with GPCR. In order for trimeric G proteins to be activated following GPCR/ligand interaction, it is necessary for them to associate with their corresponding GPCR. Mutational analysis has mapped the interaction of G&agr; to the first and third intracellular loops of GPCRs (Heller R at al. 1996, Biochem. Biophys. Res. Commun). Several studies have used synthetic (small molecule) peptides corresponding to the intracellular sequence of loops 1 and 3 as inhibitors (Mukherjee, S., et al. 1999, J. Biol. Chem.). Using such short peptides that serve as receptor mimics, they are used to compete for binding of Ga subunits to 251P5G2 and thereby provide therapeutic, prognostic, diagnostic and/or prophylactic benefit.
[0982] Thus, compounds and small molecules designed to inhibit 251P5G2 function and downstream signaling events are used for therapeutic diagnostic, prognostic and/or preventative purposes.
Example 47[0983] Regulation of Transcription
[0984] The cell surface localization of 251P5G2 and its similarity to GPCRs indicate that it is effectively used as a modulator of the transcriptional regulation of eukaryotic genes. Regulation of gene expression is confirmed, e.g., by studying gene expression in cells expressing or lacking 251P5G2. For this purpose, two types of experiments are performed.
[0985] In the first set of experiments, RNA from parental and 251P5G2-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, pheromones, or growth factors 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.).
[0986] 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.
[0987] Thus, 251P5G2 plays a role in gene regulation, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 48[0988] Involvement in Tumor Progression
[0989] The 251P5G2 gene can contribute to the growth of cancer cells. The role of 251P5G2 in tumor growth is confirmed in a variety of primary and transfected cell lines including prostate, and bladder cell lines, as well as NIH 3T3 cells engineered to stably express 251P5G2. Parental cells lacking 251P5G2 and cells expressing 251P5G2 are evaluated for cell growth using a well-documented proliferation assay (Fraser S P, et al., Prostate 2000; 44:61, Johnson D E, Ochieng J, Evans S L. Anticancer Drugs. 1996, 7:288). The effect of 251P5G2 can also observed on cell cycle progression. Control and 251P5G2-expressing cells are grown in low serum overnight, and treated with 10% FBS for 48 and 72 hrs. Cells are analyzed for BrdU and propidium iodide incorporation by FACS analysis.
[0990] To confirm the role of 251P5G2 in the transformation process, its effect in colony forming assays is investigated. Parental NIH-3T3 cells lacking 251P5G2 are compared to NIH-3T3 cells expressing 251P5G2, using a soft agar assay under stringent and more permissive conditions (Song Z. et al. Cancer Res. 2000; 60:6730).
[0991] To confirm the role of 251P5G2 in invasion and metastasis of cancer cells, a well-established assay is used. A non-limiting example is the use of an assay which provides a basement membrane or an analog thereof used to detect whether cells are invasive (e.g., a Transwell Insert System assay (Becton Dickinson) (Cancer Res. 1999; 59:6010)). Control cells, including prostate, and bladder cell lines lacking 251P5G2 are compared to cells expressing 251P5G2. Cells are loaded with the fluorescent dye, calcein, and plated in the top well of a support structure coated with a basement membrane analog (e.g. the Transwell insert) and used in the assay. Invasion is determined by fluorescence of cells in the lower chamber relative to the fluorescence of the entire cell population.
[0992] 251P5G2 can also play a role in cell cycle and apoptosis. Parental cells and cells expressing 251P5G2 are compared for differences in cell cycle regulation using a well-established BrdU assay (Abdel-Malek Z A. 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 251P5G2, including normal and tumor prostate, colon and lung cells. Engineered and parental cells are treated with various chemotherapeutic agents, such as etoposide, flutamide, 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 251P5G2 can play a critical role in regulating tumor progression and tumor load.
[0993] When 251P5G2 plays a role in cell growth, transformation, invasion or apoptosis, it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 49[0994] Involvement in Angiogenesis
[0995] 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). Based on the effect of phsophodieseterase inhibitors on endothelial cells, 251P5G2 plays a role in angiogenesis (DeFouw L et al, Microvasc Res 2001, 62:263). 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 role of 251P5G2 in angiogenesis, enhancement or inhibition, is confirmed.
[0996] For example, endothelial cells engineered to express 251P5G2 are evaluated using tube formation and proliferation assays. The effect of 251P5G2 is also confirmed in animal models in vivo. For example, cells either expressing or lacking 251P5G2 are implanted subcutaneously in immunocompromised mice. Endothelial cell migration and angiogenesis are evaluated 5-15 days later using immunohistochemistry techniques. 251P5G2 affects angiogenesis, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 50[0997] Involvement in Protein-Protein Interactions
[0998] Several GPCRs have been shown to interact with other proteins, thereby regulating signal transduction, gene transcription, transformation and cell adhesion (Sexton P M et al, Cell Signal. 2001, 13:73; Turner C E, J Cell Sci. 2000, 23:4139). Using immunoprecipitation techniques as well as two yeast hybrid systems, proteins are identified that associate with 251P5G2. Immunoprecipitates from cells expressing 251P5G2 and cells lacking 251P5G2 are compared for specific protein-protein associations.
[0999] Studies are performed to confirm the extent of association of 251P5G2 with effector molecules, such as nuclear proteins, transcription factors, kinases, phsophates etc. Studies comparing 251P5G2 positive and 251P5G2 negative cells as well as studies comparing unstimulated/resting cells and cells treated with epithelial cell activators, such as cytokines, growth factors and anti-integrin Ab reveal unique interactions.
[1000] In addition, protein-protein interactions are confirmed using two yeast hybrid methodology (Curr Opin Chem Biol. 1999, 3:64). A vector carrying a library of proteins fused to the activation domain of a transcription factor is introduced into yeast expressing a 251P5G2-DNA-binding domain fusion protein and a reporter construct. Protein-protein interaction is detected by colorimetric reporter activity. Specific association with effector molecules and transcription factors directs one of skill to the mode of action of 251P5G2, and thus identifies therapeutic, prognostic, preventative and/or diagnostic targets for cancer. This and similar assays are also used to identify and screen for small molecules that interact with 251P5G2.
[1001] Thus it is found that 251P5G2 associates with proteins and small molecules. Accordingly, 251P5G2 and these proteins and small molecules are used for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 51[1002] Involvement in Adhesion
[1003] Cell adhesion plays a critical role in tissue colonization and metastasis. The presence of link motif in 251P5G2 is indicative of its role in cell adhesion. To confirm that 251P5G2 plays a role in cell adhesion, control cells lacking 251P5G2 are compared to cells expressing 251P5G2, using techniques previously described (see, e.g., Haier et al, Br. J. Cancer. 1999, 80:1867; Lehr and Pienta, J. Natl. Cancer Inst. 1998, 90:118). Briefly, in one embodiment, cells labeled with a fluorescent indicator, such as calcein, are incubated on tissue culture wells coated with media alone or with matrix proteins. Adherent cells are detected by fluorimetric analysis and percent adhesion is calculated. This experimental system can be used to identify proteins, antibodies and/or small molecules that modulate cell adhesion to extracellular matrix and cell-cell interaction. Since cell adhesion plays a critical role in tumor growth, progression, and, colonization, the gene involved in this process can serves as a diagnostic, preventative and therapeutic modality.
[1004] 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.
[1005] 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. 5 TABLE I Tissues that Express 251P5G2: a. Malignant Tissues Prostate Bladder
[1006] 6 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 methionine 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
[1007] 7 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 world wide web URL 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
[1008] TABLE IV:
[1009] HLA Class I/II Motifs/Supermotifs 8 TABLE IV A HLA Class I Supermotifs/Motifs POSITION POSITION POSITION 3 (Primary C Terminus 2 (Primary Anchor) Anchor) (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 FWYLIVMA 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
[1010] 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. 9 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
[1011] 10 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 Supermotif MFLIVWY VMSTACPLI Italicized residues indicate less preferred or “tolerated” residues
[1012] 11 TABLE IV (D) HLA Class I Supermotifs SUPER- POSITION: MOTIFS 1 2 3 4 5 6 7 8 C-terminus A1 1° Anchor 1° Anchor TILVMS FWY A2 1° Anchor 1° Anchor {overscore (LIVMATQ)} LIVMAT A3 Preferred 1° Anchor YFW YFW YFW P 1° Anchor VSMATLI (4/5) (3/5) (4/5) (4/5) RK deleterious DE(3/5); DE P(5/5) (4/5) A24 1° Anchor 1° Anchor {overscore (YFWIVLMT)} FIYWLM B7 Preferred FWY(5/5) 1° Anchor FWY FWY 1° Anchor LIVM(3/5) P (4/5) (3/5) {overscore (VILFMWYA)} deleterious DE(3/5); DE G QN DE P(5/5); (3/5) (4/5) (4/5) (4/5) G(4/5); A(3/5); QN(3/5) B27 1° Anchor 1° Anchor RHK {overscore (FYLWMIVA)} B44 1° Anchor 1° Anchor ED {overscore (FWYLIMVA)} B58 1° Anchor 1° Anchor ATS {overscore (FWYLIVMA)} B62 1° Anchor 1° Anchor QLIVMP {overscore (FWYMIVLA)} Italicized residues indicate less preferred or “tolerated” residues
[1013] 12 TABLE IV (E) HLA Class I Motifs POSITION 9 or C- C- 1 2 3 4 5 6 7 8 terminus terminus A1 preferred GFYW 1° Anchor DEA YFW P DEQN YFW 1° Anchor 9-mer 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 RHKDEPYFW DE PQN RHK PG GP 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 RHKDEPYFW P G PRHK QN A2.1 preferred YFW 1° Anchor YFW STC YFW A P 1° Anchor 9-mer LMIVQAT VLIMAT deleterious DEP DERKH RKH DERKH POSITION: C- 1 2 3 4 5 6 7 8 9 terminus A2.1 pre- AYFW 1° Anchor LVIM G G FYWLVIM 1° Anchor 10-mer ferred LMIVQAT VLIMAT dele- DEP DE RKHA P RKH DERKH RKH te- rious A3 pre- RHK 1° Anchor YFW PRHKYFW A YFW P 1° Anchor ferred {overscore (LMVISATFCGD)} KYRHFA dele- DEP DE te- rious A11 pre- A 1° Anchor YFW YFW A YFW YFW P 1° Anchor ferred VTLMISAGNCDF KRYH dele- DEP A G te- rious A24 pre- YFWRHK 1° Anchor STC YFW YFW 1° Anchor 9-mer ferred YFWM FLIW dele- DEG DE G QNP DERHK G AQN te- rious A24 Pre- 1° Anchor P YFWP P 1° Anchor 10-mer ferred YFWM FLIW Dele- GDE QN RHK DE A QN DEA te- rious A3101 Pre- RHK 1° Anchor YFW P YFW YFW AP 1° Anchor ferred MVTALIS RK Dele- DEP DE ADE DE DE DE te- rious A3301 Pre- 1° Anchor YFW AYFW 1° Anchor ferred {overscore (MVALFIST)} RK Dele- GP DE te- rious A6801 Pre- YFWSTC 1° Anchor YFW YFW P 1° Anchor ferred AVTMSLI LIVM RK Dele- GP DEG RHK A te- rious B0702 Pre- RHKFWY 1° Anchor RHK RHK RHK RHK PA 1° Anchor ferred P {overscore (LMFWY)} AIV Dele- DEQNP DEP DE DE GDE QN DE te- rious B3501 Pre- FWYLIVM 1° Anchor FWY FWY 1° Anchor ferred P {overscore (LMFWYIVA)} dele- AGP G G te- rious POSITION: 9 or C- C- 1 2 3 4 5 6 7 8 terminus terminus A1 pre- GFYW 1° Anchor DEA YFW P DEQN YFW 1° Anchor 9-mer ferred STM Y dele- DE RHKLIVMP A G A te- rious A1 pre- GRHK ASTCLIVM 1° Anchor GSTC A LIVM DE 1° Anchor 9-mer ferred DEAS STC Y dele- A RHKDEP DE PQN RHK PG GP te- YFW rious dele- AGP G G te- rious B51 Pre- LIVMFWY 1° Anchor FWY STC FWY G FWY 1° Anchor ferred P {overscore (LIVFWYAM)} dele- AGPDERHKSTC DE G DEQN GDE te- rious B5301 pre- LIVMFWY 1° Anchor FWY STC FWY LIVM FWY 1° Anchor ferred P FWY {overscore (IMFWYALV)} dele- AGPQN G RHKQN DE te- rious B5401 pre- FWY 1° Anchor FWYLIVM LIVM ALIVM FWYAP 1° Anchor ferred P {overscore (ATIVLMFWY)} dele- GPQNDE GDESTC RHKDE DE QNDGE DE te- rious
[1014] 13 TABLE IV (F) Summary of HLA-supertypes Overall phenotypic frequencies of HLA-supertypes in different ethnic populations Specificity Phenotypic frequency Supertype Position 2 C-Terminus Caucasian N.A. Black Japanese Chinese Hispanic Average B7 P AILMVFWY 43.2 55.1 57.1 43.0 49.3 49.5 A3 AILMVST RK 37.5 42.1 45.8 52.7 43.1 44.2 A2 AILMVT AILMVT 45.8 39.0 42.4 45.9 43.0 42.2 A24 YF (WIVLMT) FI (YWLM) 23.9 38.9 58.6 40.1 38.3 40.0 B44 E (D) FWYLIMVA 43.0 21.2 42.9 39.1 39.0 37.0 A1 TI (LVMS) FWY 47.1 16.1 21.8 14.7 26.3 25.2 B27 RHK FYL (WMI) 28.4 26.1 13.3 13.9 35.3 23.4 B62 QL (IVMP) FWY (MIV) 12.6 4.8 36.5 25.4 11.1 18.1 B58 ATS FWY (LIV) 10.0 25.1 1.6 9.0 5.9 10.3
[1015] 14 TABLE IV (G) Calculated population coverage afforded by different HLA-supertype combinations HLA-supertypes Phenotypic frequency Caucasian N.A Blacks Japanese Chinese Hispanic Average A2, A3 and B7 83.0 86.1 87.5 88.4 86.3 86.2 A2, A3, B7, A24, B44 99.5 98.1 100.0 99.5 99.4 99.3 and A1 99.9 99.6 100.0 99.8 99.9 99.8 A2, A3, B7, A24 B44, A1, B27, B62, and B 58 Motifs indicate the residues defining supertype specificites. The motifs incorporate residues determined on the basis of published data to be recognized by multiple alleles within the supertype. Residues within brackets are additional residues also predicted to be tolerated by multiple alleles within the supertype.
[1016] 15 TABLE V Frequently Occurring Motifs avrg. % Name identity Description Potential Function zf-C2H2 34% Zinc finger, C2H2 type Nucleic acid-binding protein functions as tanscription factor, nuclear location probable cytochrome_b_N 68% Cytochrome b(N- membrane bound oxidase, generate terminal)/b6/petB superoxide Ig 19% Immunoglobulin domain domains are one hundred amino acids long and include a conserved intradomain disulfide bond. WD40 18% WD domain, G-beta repeat tandem repeats of 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 containing an ATP binding site and a catalytic site PH 16% PH domain pleckstrin homology involved in intracellular signaling or as constituents of the cytoskeleton 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_q1 32% NADH- membrane associated. Involved in Ubiquinone/plastoquinone proton translocation across the (complex I), various chains membrane Efhand 24% 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 proteases, centered on protease a catalytic aspartyl residue Collagen 42% Collagen triple helix repeat extracellular structural proteins involved (20 copies) 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 domain Located in the extracellular 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 receptor seven hydrophobic transmembrane (rhodopsin family) regions, with the N-terminus located extracellularly while the C-terminus is cytoplasmic. Signal through G proteins
[1017] 16 TABLE VI Motifs and Post-translational Modifications of 251P5G2 N-glycosylation site 145-148 NVTQ (SEQ ID NO: 54) N-myristoylation site 168-173 GLFFTL (SEQ ID NO: 55) Leucine zipper pattern 31-52 LRPERTYLPVCHVALIHMVVLL (SEQ ID NO: 56)
[1018] 17 TABLE VII Search Peptides 251P5G2 Variant 1, original sequence, nonamers, decamers. MPFISKINLA SQPTLFSFFS ASSPFLLFLD LRPERTYLPV CHVALIHMVV LLTMVFLSPQ (SEQ ID NO: 57) LFESLNFQND FKYEASFYLR RVIRVLSTCT TCLLGMLQVV NISPSISWLV RFKWKSTIFT FHLFSWSLSF PVSSSLIFYT VASSNVTQJM LHVSKYCSLF PTNSIIRGLF FTLSLFRDVF LKQIMLFSSV YMMTLTQELQ EILVPSQPQP LPKDLCRGKS HQHILLPVSF SVGMYKMDFI ISTSSTLPWA YDRGV 251P5G2 Variant 2 nonamers VLA SQPTLCSFFS ASSP. (SEQ ID NO: 58) decamers LVLA SQPTLCSFFS ASSPF (SEQ ID NO: 59) 251P5G2 Variant 3 nonamers FYLR RVIRDLSICT TCL. (SEQ ID NO: 60) decamers SFYLR RVIRDLSICT TCLL. (SEQ ID NO: 61) 251P5G2 Variant 4 nonamers SICT TCLLDMLQVV NIS. (SEQ ID NO: 62) decamers LSICT TCLLDMLQVV NISP. (SEQ ID NO: 63) 251P5G2 Variant 12 251P5G2 Variant 12a Nonamers ISPSISWLIMLFSSVY. (SEQ ID NO: 64) Decamers NISPSTSWLIMLFSSVYM. (SEQ ID NO: 65) 15-mers MLQVVNISPSISWLIMLFSSVYMMTLIQ. (SEQ ID NO: 66) 251P5G2 Variant 12b Nonamers. SHQHILLPTQATFAAATGLWAALTTVSNPSRADPVTWRKEPAVLPCCNLEKGSWLSFPGTAARKEFSTTLTGHSA (SEQ ID NO: 67) LSLSSSRALPGSLPAFADLPRSCPESEQSATPAGAFLLGWERVVQRRLEVPRPQAAPATSATPSRDPSPPCHQRR DAACLRAQGLTRAFQVVHLAPTAPDGGAGCPPSRNSYRLTHVRCAQGLEAASANLPGAPGRSSSCALRYRSGPSV SSAPSPAEPPAHQRLLFLPRAPQAVSGPQEQPSEEALGVGSLSVFQLHLIQCIPNLSYPLVLRHIPEILKFSEKE TGGGILGLELPATAARLSGLNSIMQIKEFEELVKLHSLSHKVIQCVFAKKKNVDKWDDFCLSECYGHSFLTMKET STKISGLIQEMGSGKSNVGTWGDYDDSAFMEPRYHVRREDLDKLHRAAWWGKVPRKDLIVMLRDTDMNKRDKQKR TALHLASANGNSEVVQLLLDRRCQLNVLDNKKRTALIKAVQCQEDECVLMLLEHGADGNIQDEYGNTALHYAIYN EDKLMAKALLLYGADIESKNKCGLTPLLLGVHEQKQEVVKFLIKKKANLNALDRYGRTALILAVCCGSASIVNLL LEQNVDVSSQDLSGQTAREYAVSSHHHVICELLSDY1EKQMLKISSENSNPVTTILNIKLPLKVEEEIKKGGSNP VGLPENLTNGASAGNGDDGLIPQRKSRKPENQQFPDTENEEYHSDEQNDTQKQLSEEQNTGISQDEILTNKQKQI EVAEKEMNSELSLSHKKEEDLLRENSMLREEIAKLRLELDETKHQNQLRENKILEEIESVKEKLLKTIQLNEEAL TKTKVAGFSLRQLGLAQHAQASVQQLCYKWNHTEKTEQQAQEQEVAGFSLRQLGLAQHAQASVQQLCYKWGHTEK TEQQAQEQGAALRSQIGDPCGVPLSEGGTAAGDQGPGTHLPPREPPASPGTPSLVRLASGAPAAALPPPTGKNGR SPTKQKSVCDSSGWILPVPTFSSGSFLGRRCPMFDVSPAMRLKSDSNRETHQAFRDKDDLPFFKTQQSPRHTKDL GQDDRAGVLAPKCRPGTLCHTDTPPHRNADTPPHRHTTTLPHRDTTTSLPHFHVSAGGVCPTTLGSNREIT Decamers: KSHQHILLPTQATFAAATGLWAALTTVSNPSRADPVTWRKEPAVLPCCNLEKGSWLSFPGTAARKEFSTTLTGHS (SEQ ID NO: 68) ALSLSSSRALPGSLPAFADLPRSCPESEQSATPAGAFLLCWERVVQRRLEVPRPQAAPATSATPSRDPSPPCHQR RDAACLRAQGLTRAFQVVHLAPTAPDGGAGCPPSRNSYRLTHVRCAQGLEAASANLPGAPGRSSSCCARYRSGPS VSSAPSPAEPPAHQRLLFLPPAPQAVSGPQEQPSEEALGVGSLSVFQLHLIQCIPNLSYPLVLRHIPEILKFSEK ETGGGILGLELPATAARLSGLNSIMQIKEFEELVKLHSLSHKVIQCVFAKKKNVDKWDDFCLSEGYCHSFLIMKE TSTKISGLIQEMGSGKSNVGTWGDYDDSAFMEPRYHVRREDLDKLHRAAWWGKVPRKDLIVMLRDTDMNKRDKQK RTALHLASANGNSEVVQLLLDRRCQLNVLDNKKRTALTKAVQCQEDECVLMLLEHGADGNIQDEYGNTALHYAIY NEDKLMAKALLLYGADIESKNKCGLTPLLLGVHEQKQEVVKFLIKKKANLNALDRYGRTALILAVCCGSASIVNL LLEQNVDVSSQDLSGQTAREYAVSSHHHVICELLSDYKEKQMLKISSENSNPVITILNIKLPLKVEEEIKKHGSN PVGLPENLThGASAGNGDDGLIPQRKSRKPENQQFPDTENEEYHSDEQNDTQKQLSEEQNTGISQDEILTNKQKQ IEVAEKEMNSELSLSHKKEEDLLRENSMLREEIAKLRLELDETKHQHQLRENKILEETESVKEKLLKTIQLNEEA LTKTKVAGFSLRQLGLAQHAQASVQQLCYKWNHTEKTEQQAQEQEVAGFSLRQLGLAQHAQASVQQLCYKWGHTE KTEQQAQEQGAALRSQTGDPGGVPLSEGGTAAGDQGPGTHLPPREPRASPGTPSLVRLASGARAAALPPPTGKNG RSPTKQKSVCDSSGWILPVPTFSSGSFLGRRCPMFDVSPAMRLKSDSNRETHQAFRDKDDLPFFKTQQSPRHTKD LGQDDRAGVLAPKCRPGTLCHTDTPPHRNADTPPHRHTTTLPHRDTTTSLPHFHVSAGGVGPTTLGSNREIT 15-mers. DLCRGKSHQHILLPTQATFAAATGLWAALTTVSNPSRADPVTWRKEPAVLPCCNLEKGSWLSFPGTAARKEFSTT (SEQ ID NO: 69) LTGHSALSLSSSRALPGSLPAFADLPRSCPESEQSATPAGAFLLGWERVVQRRLEVPRPQAAPATSATPSRDPSP PCHQRRDAACLRAQGLTRAFQVVHLAPTAPDGGAGCPPSRNSYRLTHVRCAQGLEAASANLPGAPGRSSSCALRY RSGPSVSSAPSPAEPPAHQRLLFLPRAPQAVSGPQEQPSEEALGVGSLSVFQLHLIQCIPNLSYPLVLRHIPEIL KFSEKETGGGILGLELPATAARLSGLNSTMQIKEFEELVKLHSLSHKVIQCVFAKKKNVDKWDDFCLSEGYGHSF LIMKETSTKISGLIQEMGSGKSNVGTWGDYDDSAFMEPRYHVRREDLDKLHRAAWWGKVPRKDLIVMLRDTDMNK RDKQKRTALHLASANGNSEVVQLLLDRRCQLNVLDNKKRTALIKAVQCQEDECVLMLLEHGADGHIQDEYGNTAL HYAIYNEDKLMAKALLLYGADIESKNKCGLTPLLLGVHEQKQEVVKFLIKKKANLNALDRYGRTALILAVCCGSA SIVNLLLEQNDVSSQDLSGQTAREYAVSSHHIIVICELLSDYKEKQMLKISSENSNPVITILNIKLPLKVEEEIK KHGSNPVGLPENLTHGASAGNGDDGLIPQRKSRKPENQQFPDTENEEYHSDEQNDTQKQLSEEQNTGISQDEILT HKQKQIEVAEKEMMSELSLSHKKEEDLLRENSMLREEIAKLRLELDETKHQNQLREHKILEEIESVKEKLLKTIQ LNEEALTKTKVAGFSLRQLGLAQHAQASVQQLCYKWNHTEKTEQQAQEQEVAGFSLRQLGLAQHAQASVQQLCYK WGHTEKTEQQAQEQGAALRSQIGDPGGVPLSEGGTAAGDQGPGTHLPPREPRASPGTPSLVRLASGARAAALPPP TGKNGRSPTKQKSVCDSSGWILPVPTFSSGSFLGRRCPMFDVSPAMRLKSDSHRETHQAFRDKDDLPFFKTQQSP RHTKDLGQDDRAGVLAPKCRPGTLCHTDTPPHRNADTPPHRHTTTLPHRDTTTSLPHFHVSAGGVGPTTLGSNRE IT
[1019] 18 TABLE VIII V1-A1-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Pos 123456789 Score 10 ASQPTLFSF 7.500 245 STLPWAYDR 5.000 205 PSQPQPLPK 1.500 228 VSFSVGMYK 1.500 209 QPLPKDLCR 1.250 72 KYEASFYLR 0.900 243 TSSTLPWAY 0.750 163 NSIIRGLFF 0.750 21 ASSPFLLFL 0.750 196 IQELQEILV 0.675 65 LNFQNDFKY 0.625 48 MVVLLTMVF 0.500 28 FLDLRPERT 0.500 148 QINLHVSKY 0.500 56 FLSPQLFES 0.500 122 HLFSWSLSF 0.500 231 SVGMYKMDF 0.500 224 ILLPVSFSV 0.500 183 QIMLFSSVY 0.500 20 SASSPFLLF 0.500 199 LQEILVPSQ 0.270 131 PVSSSLIFY 0.250 236 KMDFIISTS 0.250 116 STIFTFHLF 0.250 61 LFESLNFQN 0.225 105 SISWLVRFK 0.200 64 SLNFQNDFK 0.200 128 LSFPVSSSL 0.150 63 ESLNFQNDF 0.150 90 TTCLLGMLQ 0.125 68 QNDFKYEAS 0.125 103 SPSISWLVR 0.125 130 FPVSSSLIF 0.125 32 RPERTYLPV 0.113 54 MVFLSPQLF 0.100 168 GLFFTLSLF 0.100 172 TLSLFRDVF 0.100 174 SLFRDVFLK 0.100 101 NISPSISWL 0.100 158 SLFPINSII 0.100 8 VLASQPTLF 0.100 115 KSTIFTFHL 0.075 132 VSSSLIFYT 0.075 187 FSSVYMMTL 0.075 19 FSASSPFLL 0.075 241 ISTSSTLPW 0.075 143 SSNVTQINL 0.075 147 TQINLHVSK 0.060 40 VCHVALIHM 0.050 42 HVALIHMVV 0.050 117 TIFTFHLFS 0.050 9 LASQPTLFS 0.050 156 YCSLFPINS 0.050 242 STSSTLPWA 0.050 189 SVYMMTLIQ 0.050 88 ICTTCLLGM 0.050 87 SICTTCLLG 0.050 145 NVTQINLHV 0.050 227 PVSFSVGMY 0.050 178 DVFLKQIML 0.050 91 TCLLGMLQV 0.050 45 LIHMVVLLT 0.050 50 VLLTMVFLS 0.050 104 PSISWLVRF 0.030 173 LSLFRDVFL 0.030 133 SSSLIFYTV 0.030 126 WSLSFPVSS 0.030 159 LFPINSIIR 0.025 35 RTYLPVCHV 0.025 52 LTMVFLSPQ 0.025 73 YEASFYLRR 0.025 146 VTQINLHVS 0.025 162 INSIIRGLF 0.025 207 QPQPLPKDL 0.025 176 FRDVFLKQI 0.025 119 FTFHLFSWS 0.025 89 CTTCLLGML 0.025 139 YTVASSNVT 0.025 169 LFFTLSLFR 0.025 171 FTLSLFRDV 0.025 7 LVLASQPTL 0.020 140 TVASSNVTQ 0.020 136 LIFYTVASS 0.020 185 MLFSSVYMM 0.020 43 VALIHMVVL 0.020 93 LLGMLQVVN 0.020 150 NLHVSKYCS 0.020 202 ILVPSQPQP 0.020 98 QVVNISPSI 0.020 37 YLPVCHVAL 0.020 44 ALIHMVVLL 0.020 135 SLIFYTVAS 0.020 49 VVLLTMVFL 0.020 198 ELQEILVPS 0.020 188 SSVYMMTLI 0.015 86 LSICTTCLL 0.015 142 ASSNVTQIN 0.015 102 ISPSISWLV 0.015 57 LSPQLFESL 0.015 157 CSLFPINSI 0.015 V2-A1-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 3 ASQPTLCSF 1.500 2 LASQPTLCS 0.050 8 LCSFFSASS 0.020 4 SQPTLCSFF 0.015 5 QPTLCSFFS 0.013 6 PTLCSFFSA 0.013 7 TLCSFFSAS 0.010 1 VLASQPTLC 0.010 9 CSFFSASSP 0.002 V3-A1-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 7 IRDLSICTT 0.025 9 DLSICTTCL 0.010 6 VIRDLSICT 0.005 4 RRVIRDLSI 0.003 5 RVIRDLSIC 0.001 8 RDLSICTTC 0.001 2 YLRRVIRDL 0.000 3 LRRVIRDLS 0.000 1 FYLRRVIRD 0.000 V4-A1-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 7 LLDMLQVVN 1.000 4 TTCLLDMLQ 0.125 5 TCLLDMLQV 0.050 2 ICTTCLLDM 0.050 3 CTTCLLDML 0.025 6 CLLDMLQVV 0.010 9 DMLQVVNIS 0.005 1 SICTTCLLD 0.005 8 LDMLQVVNI 0.001 V12A-A1-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 8 LIMLFSSVY 0.500 4 SISWLIMLF 0.500 1 ISPSISWLI 0.015 2 SPSISWLIM 0.013 7 WLIMLFSSV 0.010 3 PSISWLIML 0.008 5 ISWLIMLFS 0.008 6 SWLIMLFSS 0.003 V12B-A1-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 31 RADPVTWRK 100.000 404 FMEPRYHVR 45.000 758 NSELSLSHK 27.000 439 DTDMNKRDK 25.000 524 YNEDKLMAK 22.500 629 ICELLSDYK 18.000 803 ILEEIESVK 18.000 361 LSEGYGHSF 13.500 538 GADIESKNK 10.000 231 PAEPPAHQR 9.000 461 NSEVVQLLL 6.750 709 FPDTENEEY 6.250 179 GCPPSRNSY 5.000 711 DTENEEYHS 4.500 326 IKEFEELVK 4.500 211 RSSSCALRY 3.750 55 LSFPGTAAR 3.000 905 AQEQGAALR 2.700 865 AQEQEVAGF 2.700 645 SSENSNPVI 2.700 924 LSEGGTAAG 2.700 916 IGDPGGVPL 2.500 845 QASVQQLCY 2.500 1031 DKDDLPFFK 2.500 885 QASVQQLCY 2.500 516 NTALHYAIY 2.500 860 KTEQQAQEQ 2.250 38 RKEPAVLPC 2.250 900 KTEQQAQEQ 2.250 964 AALPPPTGK 2.000 5 ILLPTQATF 2.000 1010 DVSPAMRLK 2.000 197 GLEAASANL 1.800 122 RLEVPRPQA 1.800 738 SQDEILTNK 1.500 401 DSAFMEPRY 1.500 729 LSEEQNTGI 1.350 100 ESEQSATPA 1.350 430 RKDLIVMLR 1.250 413 REDLDKLHR 1.250 1070 HTDTPPHRN 1.250 396 WGDYDDSAF 1.250 819 QLNEEALTK 1.000 43 VLPCCNLEK 1.000 341 KVIQCVFAK 1.000 694 GLIPQRKSR 1.000 540 DIESKNKCG 0.900 719 SDEQNDTQK 0.900 663 KVEEEIKKH 0.900 1022 NRETHQAFR 0.900 786 RLELDETKH 0.900 501 LLEHGADGN 0.900 821 NEEALTKTK 0.900 749 QIEVAEKEM 0.900 806 EIESVKEKL 0.900 600 LLEQNVDVS 0.900 307 GLELPATAA 0.900 1018 KSDSNRETH 0.750 612 LSGQTAREY 0.750 281 LSYPLVLRH 0.750 718 HSDEQNDTQ 0.750 996 FSSGSFLGR 0.750 633 LSDYKEKQM 0.750 867 EQEVAGFSL 0.675 560 KQEVVKFLI 0.675 690 NGDDGLIPQ 0.625 82 RALPGSLPA 0.500 23 LTTVSNPSR 0.500 691 GDDGLIPQR 0.500 1108 GVGPTTLGS 0.500 548 GLTPLLLGV 0.500 139 SRDPSPPCH 0.500 1094 DTTTSLPHF 0.500 1078 NADTPPHRH 0.500 604 NVDVSSQDL 0.500 983 VCDSSGWIL 0.500
[1020] 19 TABLE IX Pos 1234567890 Score V1-A1-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 28 FLDLRPERTY 25.000 158 SLFPINSIIR 5.000 64 SLNFQNDFKY 2.500 68 QNDFKYEASF 2.500 72 KYEASFYLRR 2.250 173 LSLFRDVFLK 1.500 10 ASQPTLFSFF 1.500 242 STSSTLPWAY 1.250 146 VTQINLHVSK 1.000 230 FSVGMYKMDF 0.750 19 FSASSPFLLF 0.750 102 ISPSISWLVR 0.750 130 FPVSSSLIFY 0.625 168 GLFFTLSLFR 0.500 178 DVFLKQIMLF 0.500 9 LASQPTLFSF 0.500 244 SSTLPWAYDR 0.300 75 ASFYLRRVIR 0.300 63 ESLNFQNDFK 0.300 171 FTLSLFRDVF 0.250 245 STLPWAYDRG 0.250 236 KMDFIISTSS 0.250 204 VPSQPQPLPK 0.250 47 HMVVLLTMVF 0.250 26 LLFLDLRPER 0.200 128 LSFPVSSSLI 0.150 115 KSTIFTFHLF 0.150 16 FSFFSASSPF 0.150 90 TTCLLGMLQV 0.125 116 STIFTFHLFS 0.125 162 INSIIRGLFF 0.125 89 CTTCLLGMLQ 0.125 58 SPQLFESLNF 0.125 226 LPVSFSVGMY 0.125 56 FLSPQLFESL 0.100 224 ILLPVSFSVG 0.100 7 LVLASQPTLF 0.100 202 ILVPSQPQPL 0.100 101 NISPSISWLV 0.100 227 PVSFSVGMYK 0.100 219 KSHQHILLPV 0.075 188 SSVYMMTLIQ 0.075 163 NSIIRGLFFT 0.075 22 SSPFLLFLDL 0.075 21 ASSPFLLFLD 0.075 142 ASSNVTQINL 0.075 182 KQIMLFSSVY 0.075 147 TQINLHVSKY 0.075 86 LSICTTCLLG 0.075 196 IQELQEILVP 0.068 53 TMVFLSPQLF 0.050 185 MLFSSVYMMT 0.050 87 SICTTCLLGM 0.050 49 VVLLTMVFLS 0.050 105 SISWLVRFKW 0.050 60 QLFESLNFQN 0.050 117 TIFTFHLFSW 0.050 8 VLASQPTLFS 0.050 240 IISTSSTLPW 0.050 45 LIHMVVLLTM 0.050 52 LTMVFLSPQL 0.050 103 SPSISWLVRF 0.050 44 ALIHMVVLLT 0.050 99 VVNISPSISW 0.050 195 LIQELQEILV 0.050 139 YTVASSNVTQ 0.050 223 HILLPVSFSV 0.050 20 SASSPFLLFL 0.050 32 RPERTYLPVC 0.045 104 PSISWLVRFK 0.030 106 ISWLVRFKWK 0.030 221 HQHILLPVSF 0.030 241 ISTSSTLPWA 0.030 132 VSSSLIFYTV 0.030 228 VSFSVGMYKM 0.030 134 SSLIFYTVAS 0.030 167 RGLFFTLSLF 0.025 23 SPFLLFLDLR 0.025 129 SFPVSSSLIF 0.025 209 QPLPKDLCRG 0.025 207 QPQPLPKDLC 0.025 193 MTLIQELQEI 0.025 119 FTFHLFSWSL 0.025 35 RTYLPVCHVA 0.025 176 FRDVFLKQIM 0.025 121 FHLFSWSLSF 0.025 14 TLFSFFSASS 0.020 42 HVALIHMVVL 0.020 198 ELQEILVPSQ 0.020 6 KLVLASQPTL 0.020 93 LLGMLQVVNI 0.020 135 SLIFYTVASS 0.020 225 LLPVSFSVGM 0.020 43 VALIHMVVLL 0.020 92 CLLGMLQVVN 0.020 48 MVVLLTMVFL 0.020 37 YLPVCHVALI 0.020 183 QIMLFSSVYM 0.020 172 TLSLFRDVFL 0.020 57 LSPQLFESLN 0.015 V2-A1-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 4 ASQPTLCSFF 1.500 10 CSFFSASSPF 0.150 3 LASQPTLCSF 0.100 2 VLASQPTLCS 0.050 8 TLCSFFSASS 0.020 6 QPTLCSFFSA 0.013 1 LVLASQPTLC 0.010 5 SQPTLCSFFS 0.007 7 PTLCSFFSAS 0.003 9 LCSFFSASSP 0.001 V3-A1-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 8 IRDLSICTTC 0.025 10 DLSICTTCLL 0.010 6 RVIRDLSICT 0.005 7 VIRDLSICTT 0.001 9 RDLSICTTCL 0.001 5 RRVIRDLSIC 0.001 4 LRRVIRDLSI 0.000 1 SFYLRRVIRD 0.000 3 YLRRVIRDLS 0.000 2 FYLRRVIRDL 0.000 V4-A1-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 8 LLDMLQVVNI 1.000 5 TTCLLDMLQV 0.125 4 CTTCLLDMLQ 0.125 2 SICTTCLLDM 0.050 7 CLLDMLQVVN 0.020 3 ICTTCLLDML 0.010 6 TCLLDMLQVV 0.010 1 LSICTTCLLD 0.007 10 DMLQVVNISP 0.003 9 LDMLQVVNIS 0.001 V12A-A1-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 8 WLIMLFSSVY 0.500 1 NISPSISWLI 0.100 4 PSISWLIMLF 0.075 2 ISPSISWLIM 0.075 5 SISWLIMLFS 0.050 9 LIMLFSSVYM 0.020 3 SPSISWLIML 0.013 6 ISWLIMLFSS 0.008 7 SWLIMLFSSV 0.001 V12B-A1-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 405 FMEPRYHVRR 90.000 759 NSELSLSHKK 27.000 506 GADGNIQDEY 25.000 308 GLELPATAAR 18.000 719 HSDEQNDTQK 15.000 561 KQEVVKFLIK 13.500 43 AVLPCCNLEK 10.000 56 LSFPGTAARK 6.000 821 LNEEALTKTK 4.500 291 IPEILKFSEK 4.500 49 NLEKGSWLSF 4.500 101 ESEQSATPAG 2.700 646 SSENSNPVIT 2.700 529 KLMAKALLLY 2.500 691 NGDDGLIPQR 2.500 917 IGDPGGVPLS 2.500 275 LIQCIPNLSY 2.500 413 RREDLDKLHR 2.250 901 KTEQQAQEQG 2.250 695 GLIPQRKSRK 2.000 83 RALPGSLPAF 2.000 601 LLEQNVDVSS 1.800 557 VHEQKQEVVK 1.800 776 NSMLREEIAK 1.500 362 LSEGYGHSFL 1.350 868 EQEVAGFSLR 1.350 730 LSEEQNTGIS 1.350 1071 HTDTPPHRNA 1.250 357 WDDFCLSEGY 1.250 440 DTDMNKRDKQ 1.250 179 AGCPPSRNSY 1.250 180 GCPPSRNSYR 1.000 787 RLELDETKHQ 0.900 502 LLEHGADGNI 0.900 805 LEEIESVKEK 0.900 740 QDEILTNKQK 0.900 664 KVEEEIKKHG 0.900 807 EIESVKEKLL 0.900 232 PAEPPAHQRL 0.900 39 RKEPAVLPCC 0.900 750 QIEVAEKEMN 0.900 541 DIESKNKCGL 0.900 123 RLEVPRPQAA 0.900 391 KSNVGTWGDY 0.750 948 ASPGTPSLVR 0.750 610 SQDLSGQTAR 0.750 649 NSNPVITILN 0.750 634 LSDYKEKQML 0.750 1019 KSDSNRETHQ 0.750 462 NSEVVQLLLD 0.675 906 AQEQGAALRS 0.675 493 CQEDECVLML 0.675 628 HVICELLSDY 0.500 990 WILPVPTFSS 0.500 105 SATPAGAFLL 0.500 605 NVDVSSQDLS 0.500 32 RADPVTWRKE 0.500 789 ELDETKHQNQ 0.500 572 KANLNALDRY 0.500 1011 DVSPAMRLKS 0.500 1040 KTQQSPRHTK 0.500 612 DLSGQTAREY 0.500 88 SLPAFADLPR 0.500 692 GDDGLIPQRK 0.500 478 VLDNKKRTAL 0.500 1073 DTPPHRNADT 0.500 1079 NADTPPHRHT 0.500 539 GADIESKNKC 0.500 704 KPENQQFPDT 0.450 525 YNEDKLMAKA 0.450 342 KVIQCVFAKK 0.400 655 TILNIKLPLK 0.400 845 AQASVQQLCY 0.375 885 AQASVQQLCY 0.375 1098 TSLPHFH 0.300 673 GSNPVGLPEN 0.300 738 ISQDEILTNK 0.300 258 PSEEALGVGS 0.270 925 LSEGGTAAGD 0.270 397 WGDYDDSAFM 0.250 967 LPPPTGKNGR 0.250 1089 TTLPHRDTTT 0.250 1081 DTPPHRHTTT 0.250 984 VCDSSGWILP 0.250 516 GNTALHYAIY 0.250 142 DPSPPCHQRR 0.250 938 GTHLPPREPR 0.250 400 YDDSAFMEPR 0.250 353 NVDKWDDFCL 0.250 140 SRDPSPPCHQ 0.250 469 LLDRRCQLNV 0.250 712 DTENEEYHSD 0.225 679 LPENLTNGAS 0.225 299 EKETGGGILG 0.225 329 EFEELVKLHS 0.225 373 MKETSTKISG 0.225 861 KTEQQAQEQE 0.225 779 LREEIAKLRL 0.225 858 HTEKTEQQAQ 0.225 731 SEEQNTGISQ 0.225
[1021] 20 TABLE X Pos 123456789 Score V1-A2-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 224 ILLPVSFSV 2537.396 191 YMMTLIQEL 603.960 92 CLLGMLQVV 242.674 49 VVLLTMVFL 148.730 184 IMLFSSVYM 124.127 124 FSWSLSFPV 100.540 37 YLPVCHVAL 98.267 164 SIIRGLFFT 75.179 185 MLFSSVYMM 71.872 233 GMYKMDFII 57.337 71 FKYEASFYL 57.122 44 ALIHMVVLL 49.134 182 KQIMLFSSV 46.894 101 NISPSISWL 37.157 85 VLSICTTCL 36.316 67 FQNDFKYEA 36.099 6 KLVLASQPT 26.082 194 TLIQELQEI 23.995 158 SLFPINSII 15.827 53 TMVFLSPQL 15.428 239 FIISTSSTL 13.512 7 LVLASQPTL 11.757 115 KSTIFTFHL 10.757 108 WLVRFKWKS 9.770 35 RTYLPVCHV 7.110 28 FLDLRPERT 6.719 21 ASSPFLLFL 6.703 235 YKMDFIIST 6.312 50 VLLTMVFLS 6.253 171 FTLSLFRDV 6.248 145 NVTQINLHV 6.086 132 VSSSLIFYT 6.067 102 ISPSISWLV 5.789 173 LSLFRDVFL 4.824 56 FLSPQLFES 4.573 165 IIRGLFFTL 4.182 195 LIQELQEIL 4.113 45 LIHMVVLLT 4.006 47 HMVVLLTMV 3.928 91 TCLLGMLQV 3.864 19 FSASSPFLL 3.720 203 LVPSQPQPL 3.178 60 QLFESLNFQ 2.860 232 VGMYKMDFI 2.655 112 FKWKSTIFT 2.173 14 TLFSFFSAS 1.991 167 RGLFFTLSL 1.961 43 VALIHMVVL 1.760 84 RVLSICTTC 1.608 187 FSSVYMMTL 1.475 78 YLRRVIRVL 1.409 247 LPWAYDRGV 1.281 149 INLHVSKYC 1.122 94 LGMLQVVNI 0.985 23 SPFLLFLDL 0.980 98 QVVNISPSI 0.913 128 LSFPVSSSL 0.877 242 STSSTLPWA 0.873 133 SSSLIFYTV 0.863 117 TIFTFHLFS 0.792 196 IQELQEILV 0.767 174 SLFRDVFLK 0.736 30 DLRPERTYL 0.670 168 GLFFTLSLF 0.634 154 SKYCSLFPI 0.619 157 CSLFPINSI 0.580 141 VASSNVTQI 0.567 178 DVFLKQIML 0.519 120 TFHLFSWSL 0.423 81 RVIRVLSIC 0.410 77 FYLRRVIRV 0.378 18 FFSASSPFL 0.375 1 MPFISKLVL 0.360 134 SSLIFYTVA 0.280 38 LPVCHVALI 0.266 25 FLLFLDLRP 0.254 86 LSICTTCLL 0.237 57 LSPQLFESL 0.221 226 LPVSFSVGM 0.209 139 YTVASSNVT 0.195 95 GMLQVVNIS 0.188 119 FTFHLFSWS 0.184 236 KMDFIISTS 0.173 127 SLSFPVSSS 0.171 188 SSVYMMTLI 0.157 180 FLKQIMLFS 0.152 136 LIFYTVASS 0.148 109 LVRFKWKST 0.143 207 QPQPLPKDL 0.139 143 SSNVTQINL 0.139 82 VIRVLSICT 0.132 88 ICTTCLLGM 0.127 40 VCHVALIHM 0.127 8 VLASQPTLF 0.127 220 SHQHILLPV 0.111 41 CHVALIHMV 0.111 3 FISKLVLAS 0.108 225 LLPVSFSVG 0.099 89 CTTCLLGML 0.089 42 HVALIHMVV 0.085 V2-A2-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 1 VLASQPTLC 8.446 7 TLCSFFSAS 0.538 6 PTLCSFFSA 0.062 4 SQPTLCSFF 0.042 5 QPTLCSFFS 0.016 8 LCSFFSASS 0.003 2 LASQPTLCS 0.002 3 ASQPTLCSF 0.001 9 CSFFSASSP 0.000 V3-A2-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 2 YLRRVIRDL 3.435 9 DLSICTTCL 1.602 6 VIRDLSICT 0.543 5 RVIRDLSIC 0.410 8 RDLSICTTC 0.026 7 IRDLSICTT 0.002 4 RRVIRDLSI 0.001 1 FYLRRVIRD 0.000 3 LRRVIRDLS 0.000 V4-A2-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 6 CLLDMLQVV 994.963 5 TCLLDMLQV 3.864 3 CTTCLLDML 0.334 8 LDMLQVVNI 0.210 2 ICTTCLLDM 0.127 7 LLDMLQVVN 0.021 9 DMLQVVNIS 0.014 1 SICTTCLLD 0.002 4 TTCLLDMLQ 0.000 V12A-A2-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 7 WLIMLFSSV 607.884 1 ISPSISWLI 0.868 8 LIMLFSSVY 0.100 5 ISWLIMLFS 0.087 4 SISWLIMLF 0.024 2 SPSISWLIM 0.023 3 PSISWLIML 0.007 6 SWLIMLFSS 0.001 V12B-A2-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 599 LLLEQNVDV 1793.677 18 GLWAALTTV 1327.748 802 KILEEIESV 572.255 467 LLLDRRCQL 550.915 242 FLPRAPQAV 319.939 528 KLMAKALLL 276.643 334 KLHSLSHKV 243.432 548 GLTPLLLGV 159.970 566 FLIKKKANL 98.267 111 FLLGWERVV 97.070 266 SLSVFQLHL 81.177 261 ALGVGSLSV 69.552 159 GLTRAFQVV 54.181 327 KEFEELVKL 50.726 6 LLPTQATFA 46.451 270 FQLHLIQCI 41.407 533 ALLLYGADI 38.601 369 FLIMKETST 34.279 498 VLMLLEHGA 31.249 305 ILGLELPAT 29.137 777 MLREEIAKL 26.027 555 GVHEQKQEV 24.952 54 WLSFPGTAA 22.853 345 CVFAKKKNV 22.517 1098 SLPHFHVSA 18.878 742 ILTNKQKQI 17.736 35 VTWRKEPAV 13.630 285 LVLRHIPEI 13.206 850 QLCYKWNHT 12.668 47 CNLEKGSWL 11.635 337 SLSHKVIQC 11.426 654 TILNIKLPL 10.868 418 KLHRAAWWG 10.759 476 NVLDNKKRT 9.892 727 KQLSEEQNT 9.784 158 QGLTRAFQV 9.743 427 KVPRKDLIV 8.733 164 FQVVHLAPT 7.994 1089 TLPHRDTTT 7.452 614 GQTAREYAV 7.052 117 RVVQRRLEV 6.086 770 DLLRENSML 5.928 490 VQCQEDECV 5.874 1096 TTSLPHFHV 5.603 378 KISGLIQEM 5.499 755 KEMNSELSL 5.379 652 VITILNIKL 4.993 953 SLVRLASGA 4.968 75 ALSLSSSRA 4.968 818 IQLNEEALT 4.752 620 YAVSSHHHV 4.444 385 EMGSGKSNV 3.767 324 MQIKEFEEL 3.428 796 NQLRENKIL 3.286 644 ISSENSNPV 3.165 1050 LGQDDRAGV 3.165 264 VGSLSVFQL 3.162 277 CIPNLSYPL 2.937 9 TQATFAAAT 2.871 597 VNLLLEQNV 2.856 434 IVMLRDTDM 2.734 982 SVCDSSGWI 2.676 325 QIKEFEELV 2.555 304 GILGLELPA 2.527 362 SEGYGHSFL 2.285 784 KLRLELDET 2.234 813 KLLKTIQLN 2.220 522 AIYNEDKLM 2.186 316 RLSGLNSIM 2.037 500 MLLEHGADG 1.922 514 YGNTALHYA 1.887 836 RQLGLAQHA 1.864 876 RQLGLAQHA 1.864 256 QPSEEALGV 1.861 371 IMKETSTKI 1.838 338 LSHKVIQCV 1.775 13 FAAATGLWA 1.746 105 ATPAGAFLL 1.721 273 HLIQCIPNL 1.671 890 QLCYKWGHT 1.647 840 LAQHAQASV 1.642 880 LAQHAQASV 1.642 104 SATPAGAFL 1.632 309 ELPATAARL 1.602 493 QEDECVLML 1.567 559 QKQEVVKFL 1.539 1113 TLGSNREIT 1.497 817 TIQLNEEAL 1.439 510 IQDEYGNTA 1.404 560 KQEVVKFLI 1.374 197 GLEAASANL 1.367 460 GNSEVVQLL 1.315 904 QAQEQGAAL 1.216 1007 PMFDVSPAM 1.197 14 AAATGLWAA 1.190 278 IPNLSYPLV 1.158 586 ILAVCCGSA 1.098 68 TTLTGHSAL 1.098 11 ATFAAATGL 1.098 184 RNSYRLTHV 1.044
[1022] 21 TABLE XI Pos 123456789 Score V1-A2-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 119 FTFHLFSWSL 143.920 172 TLSLFRDVFL 117.493 37 YLPVCHVALI 110.379 56 FLSPQLFESL 91.487 6 KLVLASQPTL 74.768 101 NISPSISWLV 71.726 185 MLFSSVYMMT 70.310 195 LIQELQEILV 66.657 108 WLVRFKWKST 58.275 184 IMLFSSVYMM 51.908 93 LLGMLQVVNI 40.792 48 MVVLLTMVFL 40.197 85 VLSICTTCLL 36.316 202 ILVPSQPQPL 36.316 164 SIIRGLFFTL 32.369 225 LLPVSFSVGM 32.093 150 NLHVSKYCSL 32.044 127 SLSFPVSSSL 21.362 44 ALIHMVVLLT 17.140 20 SASSPFLLFL 14.262 246 TLPWAYDRGV 13.910 183 QIMLFSSVYM 13.901 223 HILLPVSFSV 6.978 84 RVLSICTTCL 6.916 60 QLFESLNFQN 6.557 231 SVGMYKMDFI 5.658 43 VALIHMVVLL 4.292 194 TLIQELQEIL 4.292 148 QINLHVSKYC 3.757 219 KSHQHILLPV 3.655 163 NSIIRGLFFT 3.569 114 WKSTIFTFHL 3.008 100 VNISPSISWL 2.999 73 YEASFYLRRV 2.862 45 LIHMVVLLTM 2.671 90 TTCLLGMLQV 2.222 206 SQPQPLPKDL 2.166 140 TVASSNVTQI 2.100 193 MTLIQELQEI 2.096 52 LTMVFLSPQL 1.866 97 LQVVNISPSI 1.798 40 VCHVALIHMV 1.775 91 TCLLGMLQVV 1.584 87 SICTTCLLGM 1.571 132 VSSSLIFYTV 1.466 131 PVSSSLIFYT 1.052 14 TLFSFFSASS 1.048 29 LDLRPERTYL 1.026 232 VGMYKMDFII 1.019 11 SQPTLFSFFS 0.916 187 FSSVYMMTLI 0.721 156 YCSLFPINSI 0.721 46 IHMVVLLTMV 0.699 241 ISTSSTLPWA 0.697 8 VLASQPTLFS 0.697 81 RVIRVLSICT 0.652 49 VVLLTMVFLS 0.547 117 TIFTFHLFSW 0.506 123 LFSWSLSFPV 0.476 1 MPFISKLVLA 0.469 228 VSFSVGMYKM 0.469 144 SNVTQINLHV 0.454 64 SLNFQNDFKY 0.432 128 LSFPVSSSLI 0.428 18 FFSASSPFLL 0.396 224 ILLPVSFSVG 0.365 12 QPTLFSFFSA 0.357 76 SFYLRRVIRV 0.355 181 LKQIMLFSSV 0.312 82 VIRVLSICTT 0.304 168 GLFFTLSLFR 0.303 17 SFFSASSPFL 0.302 160 FPINSIIRGL 0.295 96 MLQVVNISPS 0.291 22 SSPFLLFLDL 0.265 137 IFYTVASSNV 0.263 51 LLTMVFLSPQ 0.221 50 VLLTMVFLSP 0.178 135 SLIFYTVASS 0.171 180 FLKQIMLFSS 0.160 142 ASSNVTQINL 0.139 233 GMYKMDFIIS 0.134 109 LVRFKWKSTI 0.118 25 FLLFLDLRPE 0.117 92 CLLGMLQVVN 0.113 174 SLFRDVFLKQ 0.105 67 FQNDFKYEAS 0.105 157 CSLFPINSII 0.103 31 LRPERTYLPV 0.101 26 LLFLDLRPER 0.094 35 RTYLPVCHVA 0.091 191 YMMTLIQELQ 0.090 170 FFTLSLFRDV 0.084 133 SSSLIFYTVA 0.076 236 KMDFIISTSS 0.075 88 ICTTCLLGML 0.071 27 LFLDLRPERT 0.065 237 MDFIISTSST 0.065 136 LIFYTVASSN 0.064 42 HVALIHMVVL 0.060 V2-A2-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 1 LVLASQPTLC 2.734 6 QPTLCSFFSA 0.357 8 TLCSFFSASS 0.283 5 SQPTLCSFFS 0.241 2 VLASQPTLCS 0.127 3 LASQPTLCSF 0.004 4 ASQPTLCSFF 0.003 10 CSFFSASSPF 0.002 7 PTLCSFFSAS 0.001 9 LCSFFSASSP 0.000 V3-A2-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 10 DLSICTTCLL 1.602 7 VIRDLSICTT 1.248 6 RVIRDLSICT 0.652 9 RDLSICTTCL 0.110 2 FYLRRVIRDL 0.023 3 YLRRVIRDLS 0.013 5 RRVIRDLSIC 0.001 8 IRDLSICTTC 0.000 4 LRRVIRDLSI 0.000 1 SFYLRRVIRD 0.000 V4-A2-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 8 LLDMLQVVNI 16.317 5 TTCLLDMLQV 2.222 6 TCLLDMLQVV 1.584 2 SICTTCLLDM 1.571 7 CLLDMLQVVN 0.463 3 ICTTCLLDML 0.267 10 DMLQVVNISP 0.003 9 LDMLQVVNIS 0.001 4 CTTCLLDMLQ 0.000 1 LSICTTCLLD 0.000 V12A-A2-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 9 LIMLFSSVYM 23.632 1 NISPSISWLI 10.759 8 WLIMLFSSVY 0.534 3 SPSISWLIML 0.321 5 SISWLIMLFS 0.130 6 ISWLIMLFSS 0.092 7 SWLIMLFSSV 0.068 2 ISPSISWLIM 0.038 4 PSISWLIMLF 0.000 V12B-A2-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 599 NLLLEQNVDV 257.342 6 ILLPTQATFA 171.868 338 SLSHKVIQCV 159.970 777 SMLREEIAKL 131.296 656 ILNIKLPLKV 118.238 467 QLLLDRRCQL 79.041 820 QLNEEALTKT 70.272 880 GLAQHAQASV 69.552 840 GLAQHAQASV 69.552 7 LLPTQATFAA 48.984 469 LLDRRCQLNV 47.295 729 QLSEEQNTGI 42.774 158 AQGLTRAFQV 40.900 633 LLSDYKEKQM 34.627 644 KISSENSNPV 33.472 241 LLFLPRAPQA 31.249 84 ALPGSLPAFA 27.324 264 GVGSLSVFQL 24.935 324 IMQIKEFEEL 24.419 267 SLSVFQLHLI 23.995 983 SVCDSSGWIL 23.566 325 MQIKEFEELV 22.322 70 TLTGHSALSL 21.362 678 GLPENLTNGA 20.369 660 KLPLKVEEEI 17.892 335 KLHSLSHKVI 14.971 478 VLDNKKRTAL 14.526 278 CIPNLSYPLV 14.345 1059 VLAPKCRPGT 12.668 916 QIGDPGGVPL 12.043 763 SLSHKKEEDL 10.468 597 IVNLLLEQNV 10.346 867 QEQEVAGFSL 9.878 371 LIMKETSTKI 9.023 305 GILGLELPAT 8.720 577 ALDRYGRTAL 8.545 306 ILGLELPATA 8.446 749 KQIEVAEKEM 7.228 511 IQDEYGNTAL 6.039 1096 TTTSLPHFHV 5.603 14 FAAATGLWAA 5.475 217 ALRYRSGPSV 5.286 1050 DLGQDDRAGV 5.216 556 GVHEQKQEVV 5.013 385 QEMGSGKSNV 5.004 878 QLGLAQHAQA 4.968 957 RLASGARAAA 4.968 838 QLGLAQHAQA 4.968 850 QQLCYKWNHT 4.752 353 NVDKWDDFCL 4.337 76 ALSLSSSRAL 4.272 18 TGLWAALTTV 3.864 548 CGLTPLLLGV 3.864 269 SVFQLHLIQC 3.699 403 SAFMEPRYHV 3.574 300 KETGGGILGL 3.344 374 KETSTKISGL 3.344 830 KVAGFSLRQL 3.009 490 AVQCQEDECV 2.982 110 GAFLLGWERV 2.977 510 NIQDEYGNTA 2.801 309 LELPATAARL 2.613 589 AVCCGSASIV 2.495 286 LVLRHIPEIL 2.362 165 FQVVHLAPTA 2.317 529 KLMAKALLLY 2.220 523 AIYNEDKLMA 2.186 904 QQAQEQGAAL 2.166 1042 QQSPRHTKDL 2.166 197 QGLEAASANL 2.115 266 GSLSVFQLHL 1.961 468 LLLDRRCQLN 1.922 533 KALLLYGADI 1.876 105 SATPAGAFLL 1.721 36 VTWRKEPAVL 1.716 744 LTNKQKQIEV 1.642 456 LASANGNSEV 1.642 498 CVLMLLEHGA 1.608 494 QEDECVLMLL 1.567 491 VQCQEDECVL 1.510 428 KVPRKDLIVM 1.435 862 TEQQAQEQEV 1.352 477 NVLDNKKRTA 1.319 159 QGLTRAFQVV 1.309 493 CQEDECVLML 1.307 994 VPTFSSGSFL 1.304 352 KNVDKWDDFC 1.254 363 SEGYGHSFLI 1.177 434 LIVMLRDTDM 1.161 530 LMAKALLLYG 1.157 818 TIQLNEEALT 1.025 990 WILPVPTFSS 1.011 947 RASPGTPSLV 0.966 501 MLLEHGADGN 0.942 600 LLLEQNVDVS 0.888 155 CLRAQGLTRA 0.868 307 LGLELPATAA 0.836 1052 GQDDRAGVLA 0.826 1003 LGRRCPMFDV 0.783 555 LGVHEQKQEV 0.772
[1023] 22 TABLE XII Pos 123456789 Score V1-A3-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 174 SLFRDVFLK 900.000 122 HLFSWSLSF 60.000 168 GLFFTLSLF 45.000 233 GMYKMDFII 27.000 64 SLNFQNDFK 20.000 185 MLFSSVYMM 9.000 158 SLFPINSII 6.750 172 TLSLFRDVF 6.000 245 STLPWAYDR 4.050 44 ALIHMVVLL 2.700 224 ILLPVSFSV 2.025 8 VLASQPTLF 2.000 183 QIMLFSSVY 1.800 14 TLFSFFSAS 1.800 228 VSFSVGMYK 1.500 191 YMMTLIQEL 1.350 194 TLIQELQEI 1.350 148 QINLHVSKY 1.200 231 SVGMYKMDF 1.200 54 MVFLSPQLF 1.000 147 TQINLHVSK 0.900 53 TMVFLSPQL 0.900 165 IIRGLFFTL 0.810 95 GMLQVVNIS 0.810 92 CLLGMLQVV 0.675 152 HVSKYCSLF 0.600 37 YLPVCHVAL 0.600 105 SISWLVRFK 0.600 85 VLSICTTCL 0.600 48 MVVLLTMVF 0.600 108 WLVRFKWKS 0.540 50 VLLTMVFLS 0.540 47 HMVVLLTMV 0.450 116 STIFTFHLF 0.450 6 KLVLASQPT 0.450 184 IMLFSSVYM 0.300 56 FLSPQLFES 0.270 236 KMDFIISTS 0.270 30 DLRPERTYL 0.270 60 QLFESLNFQ 0.225 35 RTYLPVCHV 0.225 178 DVFLKQIML 0.180 23 SPFLLFLDL 0.180 20 SASSPFLLF 0.180 180 FLKQIMLFS 0.180 135 SLIFYTVAS 0.180 127 SLSFPVSSS 0.180 51 LLTMVFLSP 0.180 11 SQPTLFSFF 0.180 209 QPLPKDLCR 0.180 78 YLRRVIRVL 0.135 101 NISPSISWL 0.135 49 VVLLTMVFL 0.135 98 QVVNISPSI 0.135 117 TIFTFHLFS 0.120 131 PVSSSLIFY 0.120 65 LNFQNDFKY 0.120 150 NLHVSKYCS 0.120 72 KYEASFYLR 0.108 28 FLDLRPERT 0.100 239 FIISTSSTL 0.090 7 LVLASQPTL 0.090 195 LIQELQEIL 0.090 45 LIHMVVLLT 0.090 115 KSTIFTFHL 0.081 182 KQIMLFSSV 0.081 103 SPSISWLVR 0.080 73 YEASFYLRR 0.072 164 SIIRGLFFT 0.068 81 RVIRVLSIC 0.068 10 ASQPTLFSF 0.068 42 HVALIHMVV 0.060 145 NVTQINLHV 0.060 1 MPFISKLVL 0.060 136 LIFYTVASS 0.060 130 FPVSSSLIF 0.060 225 LLPVSFSVG 0.060 243 TSSTLPWAY 0.060 25 FLLFLDLRP 0.060 96 MLQVVNISP 0.060 203 LVPSQPQPL 0.060 67 FQNDFKYEA 0.054 107 SWLVRFKWK 0.045 84 RVLSICTTC 0.045 202 ILVPSQPQP 0.045 198 ELQEILVPS 0.041 21 ASSPFLLFL 0.041 169 LFFTLSLFR 0.040 227 PVSFSVGMY 0.036 128 LSFPVSSSL 0.034 205 PSQPQPLPK 0.030 119 FTFHLFSWS 0.030 192 MMTLIQELQ 0.030 163 NSIIRGLFF 0.030 69 NDFKYEASF 0.030 113 KWKSTIFTF 0.027 223 HILLPVSFS 0.027 187 FSSVYMMTL 0.027 38 LPVCHVALI 0.027 3 FISKLVLAS 0.024 V2-A3-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 7 TLCSFFSAS 0.360 1 VLASQPTLC 0.200 4 SQPTLCSFF 0.060 3 ASQPTLCSF 0.022 6 PTLCSFFSA 0.013 8 LCSFFSASS 0.001 5 QPTLCSFFS 0.001 2 LASQPTLCS 0.001 9 CSFFSASSP 0.001 V3-A3-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 9 DLSICTTCL 0.180 2 YLRRVIRDL 0.135 5 RVIRDLSIC 0.045 6 VIRDLSICT 0.020 4 RRVIRDLSI 0.002 8 RDLSICTTC 0.000 1 FYLRRVIRD 0.000 7 IRDLSICTT 0.000 3 LRRVIRDLS 0.000 V4-A3-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 6 CLLDMLQVV 0.450 9 DMLQVVNIS 0.081 3 CTTCLLDML 0.045 7 LLDMLQVVN 0.020 5 TCLLDMLQV 0.009 2 ICTTCLLDM 0.006 1 SICTTCLLD 0.004 8 LDMLQVVNI 0.003 4 TTCLLDMLQ 0.002 V12A-A3-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 8 LIMLFSSVY 1.800 7 WLIMLFSSV 0.900 4 SISWLIMLF 0.600 1 ISPSISWLI 0.013 5 ISWLIMLFS 0.005 3 PSISWLIML 0.004 2 SPSISWLIM 0.004 6 SWLIMLFSS 0.000 V12B-A3-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 319 GLNSIMQIK 135.000 341 KVIQCVFAK 81.000 552 LLLGVHEQK 67.500 819 QLNEEALTK 60.000 776 SMLREEIAK 60.000 803 ILEEIESVK 45.000 436 MLRDTDMNK 40.000 43 VLPCCNLEK 40.000 655 ILNIKLPLK 30.000 474 QLNVLDNKK 20.000 694 GLIPQRKSR 13.500 771 LLRENSMLR 12.000 529 LMAKALLLY 12.000 342 VIQCVFAKK 9.000 280 NLSYPLVLR 9.000 562 EVVKFLIKK 8.100 154 CLRAQGLTR 8.000 404 FMEPRYHVR 6.000 528 KLMAKALLL 5.400 5 ILLPTQATF 4.500 631 ELLSDYKEK 4.500 83 ALPGSLPAF 4.500 18 GLWAALTTV 4.500 410 HVRREDLDK 4.000 266 SLSVFQLHL 3.600 441 DMNKRDKQK 3.000 382 LIQEMGSGK 3.000 370 LIMKETSTK 3.000 533 ALLLYGADI 2.700 548 GLTPLLLGV 2.700 473 CQLNVLDNK 2.025 738 SQDEILTNK 2.025 695 LIPQRKSRK 2.000 1001 FLGRRCPMF 2.000 661 PLKVEEEIK 2.000 159 GLTRAFQVV 1.800 197 GLEAASANL 1.800 109 GAFLLGWER 1.800 563 VVKFLIKKK 1.500 273 HLIQCIPNL 1.350 31 RADPVTWRK 1.350 777 MLREEIAKL 1.350 337 SLSHKVIQC 1.200 307 GLELPATAA 0.900 467 LLLDRRCQL 0.900 286 VLRHIPEIL 0.900 566 FLIKKKANL 0.900 1040 TQQSPRHTK 0.900 628 VICELLSDY 0.900 371 IMKETSTKI 0.900 343 IQCVFAKKK 0.900 237 HQRLLFLPR 0.720 651 PVITILNIK 0.675 964 AALPPPTGK 0.675 535 LLYGADIES 0.600 1098 SLPHFHVSA 0.600 794 HQNQLRENK 0.600 464 VVQLLLDRR 0.600 334 KLHSLSHKV 0.600 463 EVVQLLLDR 0.540 561 QEVVKFLIK 0.540 599 LLLEQNVDV 0.450 784 KLRLELDET 0.450 423 AWWGKVPRK 0.450 261 ALGVGSLSV 0.400 275 IQCIPNLSY 0.360 939 HLPPREPRA 0.300 825 LTKTKVAGF 0.300 498 VLMLLEHGA 0.300 538 GADIESKNK 0.300 316 RLSGLNSIM 0.300 54 WLSFPGTAA 0.300 742 ILTNKQKQI 0.300 122 RLEVPRPQA 0.300 953 SLVRLASGA 0.300 304 GILGLELPA 0.270 770 DLLRENSML 0.270 827 KTKVAGFSL 0.270 654 TILNIKLPL 0.270 560 KQEVVKFLI 0.243 571 KANLNALDR 0.240 886 ASVQQLCYK 0.225 846 ASVQQLCYK 0.225 802 KILEEIESV 0.203 422 AAWWGKVPR 0.200 516 NTALHYAIY 0.200 75 ALSLSSSRA 0.200 23 LTTVSNPSR 0.200 786 RLELDETKH 0.200 242 FLPRAPQAV 0.200 347 FAKKKNVDK 0.200 6 LLPTQATFA 0.200 629 ICELLSDYK 0.200 365 YGHSFLIMK 0.180 418 KLHRAAWWG 0.180 990 ILPVPTFSS 0.180 309 ELPATAARL 0.180 659 KLPLKVEEE 0.180 935 GPGTHLPPR 0.180 179 GCPPSRNSY 0.180
[1024] 23 TABLE XIII Pos 1234567890 Score V1-A3-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 168 GLFFTLSLFR 120.000 158 SLFPINSIIR 60.000 26 LLFLDLRPER 20.000 64 SLNFQNDFKY 12.000 47 HMVVLLTMVF 6.000 233 GMYKMDFIIS 3.600 53 TMVFLSPQLF 3.000 184 IMLFSSVYMM 2.700 56 FLSPQLFESL 2.700 6 KLVLASQPTL 2.700 37 YLPVCHVALI 1.800 93 LLGMLQVVNI 1.800 182 KQIMLFSSVY 1.620 185 MLFSSVYMMT 1.500 44 ALIHMVVLLT 1.350 119 FTFHLFSWSL 1.350 202 ILVPSQPQPL 1.350 173 LSLFRDVFLK 1.350 146 VTQINLHVSK 1.000 178 DVFLKQIMLF 0.900 127 SLSFPVSSSL 0.900 194 TLIQELQEIL 0.900 23 SPFLLFLDLR 0.900 174 SLFRDVFLKQ 0.900 164 SIIRGLFFTL 0.810 106 ISWLVRFKWK 0.750 85 VLSICTTCLL 0.600 242 STSSTLPWAY 0.600 28 FLDLRPERTY 0.600 150 NLHVSKYCSL 0.600 225 LLPVSFSVGM 0.600 172 TLSLFRDVFL 0.600 227 PVSFSVGMYK 0.600 14 TLFSFFSASS 0.600 147 TQINLHVSKY 0.540 117 TIFTFHLFSW 0.450 171 FTLSLFRDVF 0.450 60 QLFESLNFQN 0.450 204 VPSQPQPLPK 0.400 7 LVLASQPTLF 0.300 95 GMLQVVNISP 0.270 50 VLLTMVFLSP 0.270 71 FKYEASFYLR 0.270 210 PLPKDLCRGK 0.200 236 KMDFIISTSS 0.180 135 SLIFYTVASS 0.180 180 FLKQIMLFSS 0.180 244 SSTLPWAYDR 0.180 140 TVASSNVTQI 0.180 109 LVRFKWKSTI 0.180 130 FPVSSSLIFY 0.180 122 HLFSWSLSFP 0.150 223 HILLPVSFSV 0.135 224 ILLPVSFSVG 0.135 230 FSVGMYKMDF 0.135 48 MVVLLTMVFL 0.135 101 NISPSISWLV 0.135 8 VLASQPTLFS 0.120 75 ASFYLRRVIR 0.100 115 KSTIFTFHLF 0.090 9 LASQPTLFSF 0.090 231 SVGMYKMDFI 0.090 19 FSASSPFLLF 0.090 84 RVLSICTTCL 0.090 42 HVALIHMVVL 0.090 103 SPSISWLVRF 0.090 51 LLTMVFLSPQ 0.090 105 SISWLVRFKW 0.090 45 LIHMVVLLTM 0.090 35 RTYLPVCHVA 0.075 108 WLVRFKWKST 0.075 72 KYEASFYLRR 0.072 193 MTLIQELQEI 0.068 195 LIQELQEILV 0.060 96 MLQVVNISPS 0.060 87 SICTTCLLGM 0.060 V2-A3-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 2 VLASQPTLCS 0.120 8 TLCSFFSASS 0.120 10 CSFFSASSPF 0.050 1 LVLASQPTLC 0.030 3 LASQPTLCSF 0.030 6 QPTLCSFFSA 0.018 4 ASQPTLCSFF 0.015 5 SQPTLCSFFS 0.004 7 PTLCSFFSAS 0.003 9 LCSFFSASSP 0.000 V3-A3-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 10 DLSICTTCLL 0.180 3 YLRRVIRDLS 0.060 6 RVIRDLSICT 0.030 7 VIRDLSICTT 0.015 4 LRRVIRDLSI 0.001 9 RDLSICTTCL 0.001 1 SFYLRRVIRD 0.001 5 RRVIRDLSIC 0.000 8 IRDLSICTTC 0.000 2 FYLRRVIRDL 0.000 V4-A3-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 8 LLDMLQVVNI 1.800 2 SICTTCLLDM 0.060 5 TTCLLDMLQV 0.030 7 CLLDMLQVVN 0.030 10 DMLQVVNISP 0.027 3 ICTTCLLDML 0.009 6 TCLLDMLQVV 0.005 4 CTTCLLDMLQ 0.002 1 LSICTTCLLD 0.000 9 LDMLQVVNIS 0.000 V12A-A3-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 8 WLIMLFSSVY 18.000 1 NISPSISWLI 0.405 3 SPSISWLIML 0.054 9 LIMLFSSVYM 0.030 5 SISWLIMLFS 0.018 4 PSISWLIMLF 0.005 6 ISWLIMLFSS 0.005 2 ISPSISWLIM 0.002 7 SWLIMLFSSV 0.001 V12B-A3-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine 536 LLYGADIESK 225.000 419 KLHRAAWWGK 180.000 695 GLIPQRKSRK 135.000 382 GLIQEMGSGK 90.000 785 KLRLELDETK 60.000 436 VMLRDTDMNK 60.000 287 VLRHIPEILK 60.000 529 KLMAKALLLY 54.000 342 KVIQCVFAKK 40.500 370 FLIMKETSTK 30.000 803 KILEEIESVK 20.250 405 FMEPRYHVRR 18.000 113 LLGWERVVQR 12.000 308 GLELPATAAR 12.000 574 NLNALDRYGR 12.000 561 KQEWKFLIK 10.800 43 AVLPCCNLEK 9.000 361 CLSEGYGHSF 9.000 88 SLPAFADLPR 8.000 825 ALTKTKVAGF 6.000 326 QIKEFEELVK 6.000 437 MLRDTDMNKR 6.000 747 KQKQIEVAEK 5.400 1040 KTQQSPRHTK 4.500 655 TILNIKLPLK 4.500 552 PLLLGVHEQK 4.500 778 MLREEIAKLR 4.500 49 NLEKGSWLSF 4.000 55 WLSFPGTAAR 4.000 662 PLKVEEEIKK 4.000 1015 AMRLKSDSNR 4.000 23 ALTTVSNPSR 4.000 771 DLLRENSMLR 3.600 629 VICELLSDYK 3.000 262 ALGVGSLSVF 3.000 423 AAWWGKVPRK 3.000 333 LVKLHSLSHK 3.000 343 VIQCVFAKKK 3.000 660 KLPLKVEEEI 2.700 1008 PMFDVSPAMR 2.000 475 QLNVLDNKKR 2.000 954 SLVRLASGAR 1.800 324 IMQIKEFEEL 1.800 70 TLTGHSALSL 1.800 828 KTKVAGFSLR 1.800 160 GLTRAFQVVH 1.800 819 IQLNEEALTk 1.800 264 GVGSLSVFQL 1.620 777 SMLREEIAKL 1.350 275 LIQCIPNLSY 1.200 281 NLSYPLVLRH 1.200 442 DMNKRDKQKR 1.200 852 LCYKWNHTEK 1.000 892 LCYKWGHTEK 1.000 241 LLFLPRAPQA 1.000 729 QLSEEQNTGI 0.900 678 GLPENLTNGA 0.900 938 GTHLPPREPR 0.900 628 HVICELLSDY 0.900 335 KLHSLSHKVI 0.900 267 SLSVFQLHLI 0.900 474 CQLNVLDNKK 0.900 467 QLLLDRRCQL 0.900 562 QEVVKFLIKK 0.810 56 LSFPGTAARK 0.750 563 EVVKFLIKKK 0.675 651 NPVITILNIK 0.675 395 GTWGDYDDSA 0.675 840 GLAQHAQASV 0.600 291 IPEILKFSEK 0.600 7 LLPTQATFAA 0.600 478 VLDNKKRTAL 0.600 880 GLAQHAQASV 0.600 19 GLWAALTTVS 0.600 577 ALDRYGRTAL 0.600 763 SLSHKKEEDL 0.600 365 GYGHSFLIMK 0.540 473 RCQLNVLDNK 0.450 6 ILLPTQATFA 0.450 338 SLSHKVIQCV 0.450 549 GLTPLLLGVH 0.405 469 LLDRRCQLNV 0.400 656 ILNIKLPLKV 0.400 119 VVQRRLEVPR 0.400 520 LHYAIYNEDK 0.300 84 ALPGSLPAFA 0.300 875 SLRQLGLAQH 0.300 155 CLRAQGLTRA 0.300 776 NSMLREEIAK 0.300 5 HILLPTQATF 0.300 661 LPLKVEEEIK 0.300 964 AAALPPPTGK 0.300 886 QASVQQLCYK 0.300 599 NLLLEQNVDV 0.300 846 QASVQQLCYK 0.300 835 SLRQLGLAQH 0.300 269 SVFQLHLIQC 0.300 341 HKVIQCVFAK 0.270 506 GADGNIQDEY 0.270 464 EVVQLLLDRR 0.270
[1025] 24 TABLE XIV Pos 123456789 Score V1-A11-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 174 SLFRDVFLK 2.400 147 TQINLHVSK 0.900 245 STLPWAYDR 0.600 72 KYEASFYLR 0.480 64 SLNFQNDFK 0.400 169 LFFTLSLFR 0.160 209 QPLPKDLCR 0.120 159 LFPINSIIR 0.080 103 SPSISWLVR 0.080 76 SFYLRRVIR 0.080 228 VSFSVGMYK 0.080 233 GMYKMDFII 0.072 35 RTYLPVCHV 0.060 27 LFLDLRPER 0.060 48 MVVLLTMVF 0.060 231 SVGMYKMDF 0.040 54 MVFLSPQLF 0.040 42 HVALIHMVV 0.040 105 SISWLVRFK 0.040 145 NVTQINLHV 0.040 98 QVVNISPSI 0.030 49 VVLLTMVFL 0.030 7 LVLASQPTL 0.030 182 KQIMLFSSV 0.027 178 DVFLKQIML 0.024 168 GLFFTLSLF 0.024 73 YEASFYLRR 0.024 203 LVPSQPQPL 0.020 152 HVSKYCSLF 0.020 211 LPKDLCRGK 0.020 224 ILLPVSFSV 0.018 122 HLFSWSLSF 0.016 185 MLFSSVYMM 0.016 107 SWLVRFKWK 0.015 116 STIFTFHLF 0.015 165 IIRGLFFTL 0.012 77 FYLRRVIRV 0.012 67 FQNDFKYEA 0.012 196 IQELQEILV 0.012 32 RPERTYLPV 0.012 242 STSSTLPWA 0.010 89 CTTCLLGML 0.010 81 RVIRLVSIC 0.009 84 RVLSICTTC 0.009 23 SPFLLFLDL 0.008 158 SLFPINSII 0.008 189 SVYMMTLIQ 0.008 183 QIMLFSSVY 0.008 1 MPFISKLVL 0.008 191 YMMTLIQEL 0.008 24 PFLLFLDLR 0.006 92 CLLGMLQVV 0.006 44 ALIHMVVLL 0.006 184 IMLFSSVYM 0.006 118 IFTFHLFSW 0.006 36 TYLPVCHVA 0.006 194 TLIQELQEI 0.006 91 TCLLGMLQV 0.006 239 FIISTSSTL 0.006 111 RFKWKSTIF 0.006 11 SQPTLFSFF 0.006 47 HMVVLLTMV 0.006 179 VFLKQIMLF 0.006 130 FPVSSSLIF 0.006 53 TMVFLSPQL 0.006 13 PTLFSFFSA 0.005 205 PSQPQPLPK 0.004 148 QINLHVSKY 0.004 40 VCHVALIHM 0.004 39 PVCHVALIH 0.004 172 TLSLFRDVF 0.004 131 PVSSSLIFY 0.004 229 SFSVGMYKM 0.004 120 TFHLFSWSL 0.004 37 YLPVCHVAL 0.004 88 ICTTCLLGM 0.004 8 VLASQPTLF 0.004 195 LIQELQEIL 0.004 138 FYTVASSNV 0.004 85 VLSICTTCL 0.004 17 SFFSASSPF 0.004 20 SASSPFLLF 0.004 101 NISPSISWL 0.004 226 LPVSFSVGM 0.003 38 LPVCHVALI 0.003 43 VALIHMVVL 0.003 193 MTLIQELQE 0.003 65 LNFQNDFKY 0.002 99 VVNISPSIS 0.002 52 LTMVFLSPQ 0.002 90 TTCLLGMLQ 0.002 119 FTFHLFSWS 0.002 141 VASSNVTQI 0.002 215 LCRGKSHQH 0.002 140 TVASSNVTQ 0.002 18 FFSASSPFL 0.002 227 PVSFSVGMY 0.002 129 SFPVSSSLI 0.002 80 RRVIRVLSI 0.002 59 PQLFESLNF 0.002 V2-A11-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 4 SQPTLCSFF 0.006 6 PTLCSFFSA 0.005 5 QPTLCSFFS 0.001 7 TLCSFFSAS 0.000 1 VLASQPTLC 0.000 2 LASQPTLCS 0.000 8 LCSFFSASS 0.000 3 ASQPTLCSF 0.000 9 CSFFSASSP 0.000 V3-A11-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 5 RVIRDLSIC 0.009 4 RRVIRDLSI 0.002 1 FYLRRVIRD 0.001 9 DLSICTTCL 0.001 6 VIRDLSICT 0.001 2 YLRRVIRDL 0.000 8 RDLSICTTC 0.000 7 IRDLSICTT 0.000 3 LRRVIRDLS 0.000 V4-A11-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 3 CTTCLLDML 0.010 5 TCLLDMLQV 0.006 6 CLLDMLQVV 0.006 2 ICTTCLLDM 0.004 4 TTCLLDMLQ 0.002 1 SICTTCLLD 0.001 7 LLDMLQVVN 0.000 8 LDMLQVVNI 0.000 9 DMLQVVNIS 0.000 V12A-A11-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 8 LIMLFSSVY 0.008 4 SISWLIMLF 0.008 7 WLIMLFSSV 0.006 2 SPSISWLIM 0.004 1 ISPSISWLI 0.000 6 SWLIMLFSS 0.000 5 ISWLIMLFS 0.000 3 PSISWLIML 0.000 V12B-A11-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 341 KVIQCVFAK 27.000 410 HVRREDLDK 4.000 562 EVVKFLIKK 1.800 31 RADPVTWRK 1.200 776 SMLREEIAK 1.200 319 GLNSIMQIK 1.200 563 VVKFLIKKK 1.000 473 CQLNVLDNK 0.900 370 LIMKETSTK 0.800 436 MLRDTDMNK 0.800 819 QLNEEALTK 0.800 43 VLPCCNLEK 0.800 738 SQDEILTNK 0.600 794 HQNQLRENK 0.600 1040 TQQSPRHTK 0.600 552 LLLGVHEQK 0.600 109 GAFLLGWER 0.480 803 ILEEIESVK 0.400 852 CYKWNHTEK 0.400 474 QLNVLDNKK 0.400 464 VVQLLLDRR 0.400 954 LVRLASGAR 0.400 382 LIQEMGSGK 0.400 695 LIPQRKSRK 0.400 342 VIQCVFAKK 0.400 520 HYAIYNEDK 0.400 655 ILNIKLPLK 0.400 892 CYKWGHTEK 0.400 536 LYGADIESK 0.400 463 EVVQLLLDR 0.360 651 PVITILNIK 0.300 343 IQCVFAKKK 0.300 964 AALPPPTGK 0.300 538 GADIESKNK 0.300 237 HQRLLFLPR 0.240 571 KANLNALDR 0.240 635 DYKEKQMLK 0.240 23 LTTVSNPSR 0.200 347 FAKKKNVDK 0.200 56 SFPGTAARK 0.200 629 ICELLSDYK 0.200 117 RVVQRRLEV 0.180 694 GLIPQRKSR 0.180 561 QEVVKFLIK 0.180 154 CLRAQGLTR 0.160 771 LLRENSMLR 0.160 935 GPGTHLPPR 0.120 119 VQRRLEVPR 0.120 1110 GPTTLGSNR 0.120 905 AQEQGAALR 0.120 427 KVPRKDLIV 0.120 827 KTKVAGFSL 0.090 631 ELLSDYKEK 0.090 759 SELSLSHKK 0.090 422 AAWWGKVPR 0.080 404 FMEPRYHVR 0.080 948 SPGTPSLVR 0.080 88 LPAFADLPR 0.080 280 NLSYPLVLR 0.080 524 YNEDKLMAK 0.080 413 REDLDKLHR 0.072 662 LKVEEEIKK 0.060 971 GKNGRSPTK 0.060 1010 DVSPAMRLK 0.060 441 DMNKRDKQK 0.060 74 SALSLSSSR 0.060 555 GVHEQKQEV 0.060 557 HEQKQEVVK 0.060 791 ETKHQNQLR 0.060 560 KQEVVKFLI 0.054 528 KLMAKALLL 0.048 816 KTIQLNEEA 0.045 887 SVQQLCYKW 0.040 423 AWWGKVPRK 0.040 332 LVKLHSLSH 0.040 326 IKEFEELVK 0.040 1008 MFDVSPAMR 0.040 480 NKKRTALIK 0.040 180 CPPSRNSYR 0.040 847 SVQQLCYKW 0.040 287 LRHIPEILK 0.040 1037 FFKTQQSPR 0.040 434 IVMLRDTDM 0.040 419 LHRAAWWGK 0.040 661 PLKVEEEIK 0.040 365 YGHSFLIMK 0.040 614 GQTAREYAV 0.036 447 KQKRTALHL 0.036 304 GILGLELPA 0.036 785 LRLELDETK 0.030 886 ASVQQLCYK 0.030 1024 ETHQAFRDK 0.030 439 DTDMNKRDK 0.030 333 VKLHSLSHK 0.030 846 ASVQQLCYK 0.030 1096 TTSLPHFHV 0.030 105 ATPAGAFLL 0.030 663 KVEEEIKKH 0.030 821 NEEALTKTK 0.030 450 RTALHLASA 0.030
[1026] 25 TABLE XV Pos 1234567890 Score V1-A11-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 146 VTQINLHVSK 1.000 168 GLFFTLSLFR 0.960 72 KYEASFYLRR 0.480 204 VPSQPQPLPK 0.400 227 PVSFSVGMYK 0.400 158 SLFPINSIIR 0.320 26 LLFLDLRPER 0.160 173 LSLFRDVFLK 0.090 84 RVLSICTTCL 0.090 23 SPFLLFLDLR 0.080 35 RTYLPVCHVA 0.060 99 VVNISPSISW 0.040 119 FTFHLFSWSL 0.040 7 LVLASQPTLF 0.030 48 MVVLLTMVFL 0.030 182 KQIMLFSSVY 0.027 208 PQPLPKDLCR 0.024 117 TIFTFHLFSW 0.024 178 DVFLKQIMLF 0.024 109 LVRFKWKSTI 0.020 140 TVASSNVTQI 0.020 231 SVGMYKMDFI 0.020 242 STSSTLPWAY 0.020 90 TTCLLGMLQV 0.020 42 HVALIHMVVL 0.020 106 ISWLVRFKWK 0.020 52 LTMVFLSPQL 0.020 164 SIIRGLFFTL 0.018 6 KLVLASQPTL 0.018 81 RVIRVLSICT 0.018 223 HILLPVSFSV 0.018 71 FKYEASFYLR 0.016 171 FTLSLFRDVF 0.015 193 MTLIQELQEI 0.015 47 HMVVLLTMVF 0.012 64 SLNFQNDFKY 0.012 184 IMLFSSVYMM 0.012 105 SISWLVRFKW 0.012 63 ESLNFQNDFK 0.009 49 VVLLTMVFLS 0.009 97 LQVVNISPSI 0.009 147 TQINLHVSKY 0.009 190 VYMMTLIQEL 0.008 87 SICTTCLLGM 0.008 240 IISTSSTLPW 0.008 183 QIMLFSSVYM 0.008 101 NISPSISWLV 0.008 244 SSTLPWAYDR 0.008 54 MVFLSPQLFE 0.008 45 LIHMVVLLTM 0.008 189 SVYMMTLIQE 0.008 75 ASFYLRRVIR 0.008 102 ISPSISWLVR 0.008 195 LIQELQEILV 0.008 76 SFYLRRVIRV 0.008 202 ILVPSQPQPL 0.006 9 LASQPTLFSF 0.006 53 TMVFLSPQLF 0.006 130 FPVSSSLIFY 0.006 36 TYLPVCHVAL 0.006 221 HQHILLPVSF 0.006 194 TLIQELQEIL 0.006 38 LPVCHVALIH 0.006 123 LFSWSLSFPV 0.006 18 FFSASSPFLL 0.006 12 QPTLFSFFSA 0.006 233 GMYKMDFIIS 0.005 210 PLPKDLCRGK 0.004 1 MPFISKLVLA 0.004 66 NFQNDFKYEA 0.004 172 TLSLFRDVFL 0.004 152 HVSKYCSLFP 0.004 129 SPFVSSSLIF 0.004 56 FLSPQLFESL 0.004 93 LLGMLQVVNI 0.004 17 SFFSASSPFL 0.004 37 YLPVCHVALI 0.004 39 PVCHVALIHM 0.004 20 SASSPFLLFL 0.004 85 VLSICTTCLL 0.004 127 SLSFPVSSSL 0.004 58 SPQLFESLNF 0.004 225 LLPVSFSVGM 0.004 150 NLHVSKYCSL 0.004 186 LFSSVYMMTL 0.004 137 IFYTVASSNV 0.004 95 GMLQVVNISP 0.004 116 STIFTFHLFS 0.003 226 LPVSFSVGMY 0.003 43 VALIHMVVLL 0.003 91 TCLLGMLQVV 0.003 206 SQPQPLPKDL 0.003 98 QVVNISPSIS 0.003 60 QLFESLNFQN 0.002 155 KYCSLFPINS 0.002 156 YCSLFPINSI 0.002 88 ICTTCLLGML 0.002 40 VCHVALIHMV 0.002 215 LCRGKSHQHI 0.002 203 LVPSQPQPLP 0.002 V2-A11-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 6 QPTLCSFFSA 0.006 1 LVLASQPTLC 0.003 3 LASQPTLCSF 0.002 5 SQPTLCSFFS 0.002 2 VLASQPTLCS 0.001 10 CSFFSASSPF 0.000 8 TLCSFFSASS 0.000 9 LCSFFSASSP 0.000 4 ASQPTLCSFF 0.000 7 PTLCSFFSAS 0.000 V3-A11-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 6 RVIRDLSICT 0.018 10 DLSICTTCLL 0.001 9 RDLSICTTCL 0.001 1 SFYLRRVIRD 0.001 2 FYLRRVIRDL 0.001 7 VIRDLSICTT 0.000 4 LRRVIRDLSI 0.000 3 YLRRVIRDLS 0.000 5 RRVIRDLSIC 0.000 8 IRDLSICTTC 0.000 V4-A11-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 5 TTCLLDMLQV 0.020 2 SICTTCLLDM 0.008 8 LLDMLQVVNI 0.004 6 TCLLDMLQVV 0.003 3 ICTTCLLDML 0.002 4 CTTCLLDMLQ 0.002 7 CLLDMLQVVN 0.001 10 DMLQVVNISP 0.000 1 LSICTTCLLD 0.000 9 LDMLQVVNIS 0.000 V12A-A11-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 9 LIMLFSSVYM 0.008 1 NISPSISWLI 0.008 8 WLIMLFSSVY 0.006 3 SPSISWLIML 0.004 5 SISWLIMLFS 0.001 2 ISPSISWLIM 0.000 7 SWLIMLFSSV 0.000 6 ISWLIMLFSS 0.000 4 PSISWLIMLF 0.000 V12B-A11-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 342 KVIQCVFAKK 9.000 43 AVLPCCNLEK 6.000 561 KQEVVKFLIK 3.600 1040 KTQQSPRHTK 3.000 365 GYGHSFLIMK 2.400 419 KLHRAAWWGK 2.400 333 LVKLHSLSHK 2.000 819 IQLNEEALTK 1.800 695 GLIPQRKSRK 1.800 803 KILEEIESVK 1.800 747 KQKQIEVAEK 1.800 382 GLIQEMGSGK 1.800 785 KLRLELDETK 1.200 436 VMLRDTDMNK 1.200 474 CQLNVLDNKK 0.900 287 VLRHIPEILK 0.800 524 IYNEDKLMAK 0.800 536 LLYGADIESK 0.800 326 QIKEFEELVK 0.800 828 KTKVAGFSLR 0.600 655 TILNIKLPLK 0.600 370 FLIMKETSTK 0.600 473 RCQLNVLDNK 0.600 938 GTHLPPREPR 0.600 563 EVVKFLIKKK 0.450 852 LCYKWNHTEK 0.400 119 VVQRRLEVPR 0.400 629 VICELLSDYK 0.400 423 AAWWGKVPRK 0.400 892 LCYKWGHTEK 0.400 661 LPLKVEEEIK 0.300 651 NPVITILNIK 0.300 308 GLELPATAAR 0.240 964 AAALPPPTGK 0.200 291 IPEILKFSEK 0.200 1047 HTKDLGQDDR 0.200 343 VIQCVFAKKK 0.200 347 VFAKKKNVDK 0.200 886 QASVQQLCYK 0.200 846 QASVQQLCYK 0.200 1031 RDKDDLPFFK 0.180 464 EVVQLLLDRR 0.180 264 GVGSLSVFQL 0.180 562 QEVVKFLIKK 0.180 88 SLPAFADLPR 0.160 574 NLNALDRYGR 0.160 154 ACLRAQGLTR 0.120 610 SQDLSGWTAR 0.120 422 RAAWWGKVPR 0.120 954 SLVRLASGAR 0.120 148 HQRRDAACLR 0.120 180 GCPPSRNSYR 0.120 428 KVPRKDLIVM 0.120 808 IESVKEKLLK 0.120 341 HKVIQCVFAK 0.090 635 SDYKEKQMLK 0.080 405 FMEPRYHVRR 0.080 23 ALTTVSNPSR 0.080 55 WLSFPGTAAR 0.080 404 AFMEPRYHVR 0.080 662 PLKVEEEIKK 0.080 1015 AMRLKSDSNR 0.080 113 LLGWERVVQR 0.080 996 TFSSGSFLGR 0.080 437 MLRDTDMNKR 0.080 776 NSMLREEIAK 0.080 771 DLLRENSMLR 0.072 794 KHQNQLRENK 0.060 973 KNGRSPTKQK 0.060 692 GDDGLIPQRK 0.060 410 YHVRREDLDK 0.060 552 PLLLGVHEQK 0.060 1056 RAGVLAPKCR 0.060 353 NVDKWDDFCL 0.060 395 GTWGDYDDSA 0.060 556 GVHEQKQEVV 0.060
[1027] 26 TABLE XVI Pos 123456789 Score V1-A24-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 120 TFHLFSWSL 20.000 111 RFKWKSTIF 20.000 18 FFSASSPFL 20.000 179 VFLKQIMLF 15.000 155 KYCSLFPIN 14.400 36 TYLPVCHVA 12.600 167 RGLFFTLSL 12.000 17 SFFSASSPF 10.000 191 YMMTLIQEL 9.504 207 QPQPLPKDL 8.640 57 LSPQLFESL 8.640 195 LIQELQEIL 8.6401 115 KSTIFTFHL 8.000 217 RGKSHQHIL 8.000 77 FYLRRVIRV 7.500 129 SFPVSSSLI 7.500 203 LVPSQPQPL 7.200 53 TMVFLSPQL 7.200 128 LSFPVSSSL 6.720 37 YLPVCHVAL 6.000 44 ALIHMVVLL 6.000 49 VVLLTMVFL 6.000 86 LSICTTCLL 6.000 173 LSLFRDVFL 6.000 7 LVLASQPTL 6.000 239 FIISTSSTL 6.000 143 SSNVTQINL 6.000 234 MYKMDFIIS 6.000 43 VALIHMVVL 6.000 165 IIRGLFFTL 5.760 23 SPFLLFLDL 5.760 78 YLRRVIRVL 5.600 138 FYTVASSNV 5.000 30 DLRPERTYL 4.800 89 CTTCLLGML 4.800 101 NISPSISWL 4.800 21 ASSPFLLFL 4.800 11 SQPTLFSFF 4.320 113 KWKSTIFTF 4.000 19 FSASSPFLL 4.000 85 VLSICTTCL 4.000 187 FSSVYMMTL 4.000 178 DVFLKQIML 4.000 1 MPFISKLVL 4.000 116 STIFTFHLF 3.600 63 ESLNFQNDF 3.600 10 ASQPTLFSF 3.600 48 MVVLLTMVF 3.600 163 NSIIRGLFF 3.000 130 FPVSSSLIF 3.000 162 INSIIRGLF 2.800 229 SFSVGMYKM 2.750 20 SASSPFLLF 2.400 54 MVFLSPQLF 2.400 98 QVVNISPSI 2.100 168 GLFFTLSLF 2.000 172 TLSLFRDVF 2.000 231 SVGMYKMDF 2.000 8 VLASQPTLF 2.000 122 HLFSWSLSF 2.000 152 HVSKYGSLF 2.000 194 TLIQELQEI 1.980 72 KYEASFYLR 1.800 157 CSLFPINSI 1.800 158 SLFPINSII 1.680 188 SSVYMMTLI 1.500 94 LGMLQVVNI 1.500 232 VGMYKMDFI 1.500 38 LPVCHVALI 1.500 75 ASFYLRRVI 1.200 141 VASSNVTQI 1.000 233 GMYKMDFII 1.000 61 LFESLNFQN 0.900 161 PINSIIRGL 0.840 238 DFIISTSST 0.750 184 IMLFSSVYM 0.750 190 VYMMTLIQE 0.750 226 LPVSFSVGM 0.750 137 IFYTVASSN 0.700 186 LFSSVYMMT 0.700 151 LHVSKYCSL 0.600 70 DFKYEASFY 0.500 185 MLFSSVYMM 0.500 88 ICTTCLLGM 0.500 40 VCHVALIHM 0.500 118 IFTFHLFSW 0.500 15 LFSFFSASS 0.500 71 FKYEASFYL 0.480 182 KQIMLFSSV 0.432 184 RVLSICTTC 0.420 81 RVIRVLSIC 0.420 218 GKSHQHILL 0.400 222 QHILLPVSF 0.360 6 KLVLASQPT 0.360 59 PQLFESLNF 0.300 80 RRVIRVLSI 0.300 32 RPERTYLPV 0.300 104 PSISWLVRF 0.300 236 KMDFIISTS 0.280 95 GMLQVVNIS 0.252 V2-A24-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 3 ASQPTLCSF 3.600 4 SQPTLCSFF 3.600 7 TLCSFFSAS 0.120 8 LCSFFSASS 0.100 1 VLASQPTLC 0.100 5 QPTLCSFFS 0.100 2 LASQPTLCS 0.100 6 PTLCSFFSA 0.018 9 CSFFSASSP 0.010 V3-A24-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 2 YLRRVIRDL 5.600 9 DLSICTTCL 4.000 1 FYLRRVIRD 0.750 5 RVIRDLSIC 0.300 4 RRVIRDLSI 0.300 6 VIRDLSICT 0.144 8 RDLSICTIC 0.042 3 LRRVIRDLS 0.014 7 IRDLSICIT 0.010 V4-A24-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 3 CTTCLLDML 4.800 2 ICTTCLLDM 0.500 9 DMLQVVNIS 0.252 6 CLLDMLQVV 0.216 8 LDMLQVVNI 0.150 5 TCLLDMLQV 0.150 7 LLDMLQVVN 0.120 4 TTCLLDMLQ 0.012 1 SICTTCLLD 0.010 V12A-A24-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 4 SISWLIMLF 2.400 1 ISPSISWLI 2.100 3 PSISWLIML 0.600 2 SPSISWLIM 0.500 7 WLIMLFSSV 0.216 6 SWLIMLFSS 0.150 8 LIMLFSSVY 0.150 5 ISWLIMLFS 0.140 V12B-A24-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 579 RYGRTALIL 400.000 408 RYHVRREDL 400.000 282 SYPLVLRHI 105.000 364 GYGHSFLIM 30.000 872 GFSLRQLGL 20.000 832 GFSLRQLGL 20.000 528 KLMAKALLL 12.000 648 NSNPVITIL 10.080 218 RYRSGPSVS 10.000 946 RASPGTPSL 9.600 806 EIESVKEKL 9.240 523 IYNEDKLMA 9.000 86 GSLPAFADL 8.640 654 TILNIKLPL 8.400 461 NSEVVQLLL 8.400 460 GNSEVVQLL 8.064 447 KQKRTALHL 8.000 544 KNKCGLTPL 8.000 827 KTKVAGFSL 8.000 546 KCGLTPLLL 8.000 324 MQIKEFEEL 7,920 491 QCQEDECVL 7.200 883 HAQASVQQL 7.200 904 QAQEQGAAL 7.200 351 KNVDKWDDF 7.200 674 NPVGLPENL 7.200 277 CIPNLSYPL 7.200 843 HAQASVQQL 7.200 467 LLLDRRCQL 7.200 156 RAQGLTRAF 7.200 47 CNLEKGSWL 7.200 721 EQNDTQKQL 7.200 592 GSASIVNLL 6.720 521 YAIYNEDKL 6.600 566 FLIKKKANL 6.000 309 ELPATAARL 6.000 796 NQLRENKIL 6.000 735 TGISQDEIL 6.000 105 ATPAGAFLL 6.000 273 HLIQCIPNL 6.000 68 TTLTGHSAL 6.000 76 LSLSSSRAL 6.000 233 EPPAHQRLL 6.000 867 EQEVAGFSL 6.000 459 NGNSEVVQL 6.000 770 DLLRENSML 6.000 817 TIQLNEEAL 6.000 1042 QSPRHTKDL 6.000 197 GLEAASANL 6.000 286 VLRHIPEIL 5.600 593 SASIVNLLL 5.600 652 VITILNIKL 5.280 781 EIAKLRLEL 5.280 777 MLREEIAKL 5.280 302 GGGILGLEL 5.280 186 SYRLTHVRC 5.000 513 EYGNTALHY 5.000 916 IGDPGGVPL 4.800 80 SSRALPGSL 4.800 1051 GQDDRAGVL 4.800 152 AACLRAQGL 4.800 15 AATGLWAAL 4.800 104 SATPAGAFL 4.800 604 NVDVSSQDL 4.800 560 KQEVVKFLI 4.200 36 TWRKEPAVL 4.000 425 WGKVPRKDL 4.000 1106 AGGVGPTTL 4.000 958 ASGARAAAL 4.000 870 VAGFSLRQL 4.000 687 SAGNGDDGL 4.000 312 ATAARLSGL 4.000 266 SLSVFQLHL 4.000 147 HQRRDAACL 4.000 983 VCDSSGWIL 4.000 830 VAGFSLRQL 4.000 1060 APKCRPGTL 4.000 264 VGSLSVFQL 4.000 11 ATFAAATGL 4.000 763 LSHKKEEDL 4.000 591 CGSASIVNL 4.000 374 ETSTKISGL 4.000 300 ETGGGILGL 4.000 70 LTGHSALSL 4.000 5 ILLPTQATF 3.600 321 NSIMQIKEF 3.300 262 LGVGSLSVF 3.000 361 LSEGYGHSF 3.000 865 AQEQEVAGF 3.000 988 GWILPVPTF 3.000 83 ALPGSLPAF 3.000 1021 SNRETHQAF 2.880 660 LPLKVEEEI 2.310 558 EQKQEVVKF 2.200 270 FQLHLIQCI 2.160 825 LTKTKVAGF 2.000 396 WGDYDDSAF 2.000 1001 FLGRRCPMF 2.000
[1028] 27 TABLE XVII Pos 1234567890 Score V1-A-24-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 190 VYMMTLIQEL 475.200 77 FYLRRVIRVL 420.000 36 TYLPVCHVAL 360.000 238 DFIISTSSTL 30.000 17 SFFSASSPFL 20.000 186 LFSSVYMMTL 20.000 18 FFSASSPFLL 20.000 70 DFKYEASFYL 20.000 129 SFPVSSSLIF 15.000 84 RVLSICTTCL 12.00 6 KLVLASQPTL 12.00 155 KYCSLFPINS 10.00 202 ILVPSQPQPL 8.640 22 SSPFLLFLDL 8.640 164 SIIRGLFFTL 8.640 160 FPINSIIRGL 8.400 217 RGKSHQHILL 8.000 206 SQPSPLPKDL 7.200 194 TLIQELQEIL 7.200 52 LTMVFLSPQL 7.200 56 FLSPQLFESL 6.912 43 VALIHMVVLL 6.000 48 MVVLLTMVFL 6.000 167 RGLFFTLSLF 6.000 138 FYTVASSNVT 6.000 175 LFRDVFLKQI 6.000 100 VNISPSISWL 6.000 127 SLSFPVSSSL 5.600 10 ASQPTLFSFF 6.184 234 MYKMDFIIST 5.000 88 ICTTCLLGML 4.800 115 KSTIFTFHLF 4.800 85 VLSICTTCLL 4.000 20 SASSPFLLFL 4.000 172 TLSLFRDVFL 4.000 142 ASSNVTQINL 4.000 42 HVALIHMVVL 4.000 150 NLHVSKYCSL 4.000 119 FTFHLFSWSL 4.000 47 HMVVLLTMVF 3.600 53 TMVFLSPQLF 3.600 58 SPQLFESLNF 3.0001 230 FSVGMYKMDF 3.000 7 LVLASQPTLF 3.000 171 FTLSLFRDVF 3.000 19 FSASSPFLLF 2.400 221 HQHILLPVSF 2.400 97 LQVVNISPSI 2.100 157 CSLFPINSII 2.100 68 QNDFKYEASF 2.000 178 DVFLKQIMLF 2.000 9 LASQPTLFSF 2.000 162 INSIIRGLFF 2.000 103 SPSISWLVRF 2.000 16 FSFFSASSPF 2.000 193 MTLIQELQEI 1.980 37 YLPVCHVALI 1.500 72 KYEASFYLRR 1.500 232 VGMYKMDFII 1.500 177 RDVFLKQIML 1.200 156 YCSLFPINSI 1.200 215 LCRGKSHQHI 1.200 128 LSFPVSSSLI 1.200 74 EASFYLRRVI 1.200 179 VFLKQIMLFS 1.050 93 LLGMLQVVNI 1.000 153 VSKYCSLFPI 1.000 140 TVASSNVTQI 1.000 187 FSSVYMMTLI 1.000 231 SVGMYKMDFI 1.000 111 RFKWKSTIFT 1.000 109 LVRFKWKSTI 1.000 27 LFLDLRPERT 0.900 55 VFLSPQLFES 0.825 66 NFQNDFKYEA 0.825 184 IMLFSSVYMM 0.750 183 QIMLFSSVYM 0.750 225 LLPVSFSVGM 0.750 118 IFTFHLFSWS 0.720 170 FFTLSLFRDV 0.720 45 LIHMVVLLTM 0.700 123 LFSWSLSFPV 0.600 29 LDLRPERTYL 0.600 228 VSFSVGMYKM 0.550 137 IFYTVASSNV 0.500 120 TFHLFSWSLS 0.500 76 SFYLRRVIRV 0.500 87 SICTTCLLGM 0.500 161 PINSIIRGLF 0.420 166 IRGLFFTLSL 0.400 216 CRGKSHQHIL 0.400 114 WKSTIFTFHL 0.400 81 RVIRVLSICT 0.360 182 KQIMLFSSVY 0.300 32 RPRTYLPVC 0.300 151 LHVSKYCSLF 0.300 121 FHLFSWSLSF 0.300 219 KSHQHILLPV 0.280 35 RTYLPVCHVA 0.280 236 KMDFIISTSS 0.280 V2-A24-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine 4 ASQPTLCSFF 4.320 3 LASQPTLCSF 2.000 10 CSFFSASSPF 2.000 1 LVLASQPTLC 0.150 5 SQPTLCSFFS 0.150 6 QPTLCSFFSA 0.120 8 TLCSFFSASS 0.100 2 VLASQPTLCS 0.100 7 PTLCSFFSAS 0.018 9 LCSFFSASSP 0.010 V3-A24-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine 2 FYLRRVIRDL 420.000 10 DLSICTTCLL 4.000 9 RDLSICTTCL 1.200 6 RVIRDLSICT 0.360 3 YLRRVIRDLS 0.140 7 VIRDLSICTT 0.120 4 LRRVIRDLSI 0.100 1 SFYLRRVIRD 0.050 5 RRVIRDLSIC 0.030 8 IRDLSICTTC 0.014 V4-A24-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 3 ICTTCLLDML 4.800 8 LLDMLQVVNI 1.000 2 SICTTCLLDM 0.500 7 CLLDMLQVVN 0.216 6 TCLLDMLQVV 0.180 5 TTCLLDMLQV 0.100 9 LDMLQVVNIS 0.025 10 DMLQVVNISP 0.021 1 LSICTTCLLD 0.015 4 CTTCLLDMLQ 0.012 V12A-A24-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 3 SPSISWLIML 4.000 1 NISPSISWLI 1.680 9 LIMLFSSVYM 0.750 2 ISPSISWLIM 0.750 4 PSISWLIMLF 0.360 7 SWLIMLFSSV 0.216 8 WLIMLFSSVY 0.150 5 SISWLIMLFS 0.140 6 ISWLIMLFSS 0.100 V12B-A24-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 521 HYAIYNEDKL 220.000 636 DYKEKQMLKI 66.000 566 KFLIKKKANL 60.000 1009 MFDVSPAMRL 20.000 1001 SFLGRRCPMF 15.000 1029 AFRDKDDLPF 12.000 817 KTIQLNEEAL 12.000 270 VFQLHLIQCI 10.800 460 NGNSEVVQLL 10.080 219 RYRSGPSVSS 10.000 580 RYGRTALILA 10.000 545 KNKCGLTPLL 9.600 604 QNVDVSSQDL 8.640 277 QCIPNLSYPL 8.640 286 LVLRHIPEIL 8.400 115 GWERVVQRRL 8.400 654 ITILNIKLPL 8.400 830 KVAGFSLRQL 8.000 324 IMQIKEFEEL 7.920 537 LYGADIESKN 7.700 493 CQEDECVLML 7.200 83 RALPGSLPAF 7.200 674 SNPVGLPENL 7.200 197 QGLEAASANL 7.200 1051 LGQDDRAGVL 7.200 461 GNSEVVQLLL 6.720 592 CGSASIVNLL 6.720 777 SMLREEIAKL 6.600 753 VAEKEMNSEL 6.600 624 SSHHHVICEL 6.160 1082 TPPHRHTTTL 6.000 807 EIESVKEKLL 6.000 717 EYHSDEQNDT 6.000 541 DIESKNKCGL 6.000 1060 LAPKCRPGTL 6.000 362 LSEGYGHSFL 6.000 266 GSLSVFQLHL 6.000 796 QNQLRENKIL 6.000 279 IPNLSYPLVL 6.000 181 CPPSRNSYRL 6.000 467 QLLLDRRCQL 6.000 47 CCNLEKGSWL 6.000 559 EQKQEVVKFL 5.600 648 ENSNPVITIL 5.600 593 GSASIVNLLL 5.600 853 CYKWNHTEKT 5.500 893 CYKWGHTEKT 5.500 302 TGGGILGLEL 5.280 187 SYRLTHVRCA 5.000 514 EYGNTALHYA 5.000 620 EYAVSSHHHV 5.000 429 VPRKDLIVML 4.800 15 AAATGLWAAL 4.800 701 KSRKPENQQF 4.800 80 SSSRALPGSL 4.800 983 SVCDSSGWIL 4.800 916 QIGDPGGVPL 4.800 1042 QQSPRHTKDL 4.800 152 DAACLRAQGL 4.800 634 LSDYKEKQML 4.800 511 IQDEYGNTAL 4.800 105 SATPAGAFLL 4.800 190 LTHVRCAQGL 4.800 660 KLPLKVEEEI 4.620 411 HVRREDLDKL 4.400 904 QQAQEQGAAL 4.000 425 WWGKVPRKDL 4.000 687 ASAGNGDDGL 4.000 491 VQCQEDECVL 4.000 1091 LPHRDTTTSL 4.000 994 VPTFSSGSFL 4.000 932 AGDQGPGTHL 4.000 11 QATFAAATGL 4.000 459 ANGNSEVVQL 4.000 949 SPGTPSLVRL 4.000 763 SLSHKKEEDL 4.000 353 NVDKWDDFCL 4.000 41 EPAVLPCCNL 4.000 764 LSHKKEEDLL 4.000 62 AARKEFSTTL 4.000 161 LTRAFQVVHL 4.000 870 EVAGFSLRQL 4.000 209 APGRSSSCAL 4.000 478 VLDNKKRTAL 4.000 1027 HQAFRDKDDL 4.000 832 AGFSLRQLGL 4.000 1106 SAGGVGPTTL 4.000 70 TLTGHSALSL 4.000 68 STTLTGHSAL 4.000 735 NTGISQDEIL 4.000 958 LASGARAAAL 4.000 36 VTWRKEPAVL 4.000 872 AGFSLRQLGL 4.000 591 CCGSASIVNL 4.000 577 ALDRYGRTAL 4.000 76 ALSLSSSRAL 4.000 104 QSATPAGAFL 4.000 264 GVGSLSVFQL 4.000 865 QAQEQEVAGF 3.600 1021 DSNRETHQAF 3.600
[1029] 28 TABLE XVIII Pos 123456789 Score V1-B7-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 207 QPQPLPKDL 80.00 23 SPFLLFLDL 80.00 1 MPFISKLVL 80.00 30 DLRPERTYL 60.00 78 YLRRVIRVL 40.00 165 IIRGLFFTL 40.00 178 DVFLKQIML 20.00 203 LVPSQPQPL 20.00 7 LVLASQPTL 20.00 49 VVLLTMVFL 20.00 226 LPVSFSVGM 20.00 191 YMMTLIQEL 12.00 44 ALIHMVVLL 12.00 21 ASSPFLLFL 12.00 43 VALIHMVVL 12.00 38 LPVCHVALI 8.000 109 LVRFKWKST 5.000 195 LIQELQEIL 4.000 53 TMVFLSPQL 4.000 217 RGKSHQHIL 4.000 57 LSPQLFESL 4.000 86 LSICTTCLL 4.000 173 LSLFRDVFL 4.000 239 FIISTSSTL 4.000 115 KSTIFTFHL 4.000 85 VLSICTTCL 4.000 101 NISPSISWL 4.000 128 LSFPVSSSL 4.000 37 YLPVCHVAL 4.000 247 LPWAYDRGV 4.000 143 SSNVTQINL 4.000 19 FSASSPFLL 4.000 89 CTTCLLGML 4.000 167 RGLFFTLSL 4.000 187 FSSVYMMTL 4.000 98 QVVNISPSI 2.000 75 ASFYLRRVI 1.800 141 VASSNVTQI 1.200 232 VGMYKMDFI 1.200 32 RPERTYLPV 1.200 94 LGMLQVVNI 1.200 185 MLFSSVYMM 1.000 82 VIRVLSICT 1.000 88 ICTTCLLGM 1.000 40 VCHVALIHM 1.000 184 IMLFSSVYM 1.000 145 NVTQINLHV 1.000 42 HVALIHMVV 1.000 157 CSLFPINSI 0.600 74 EASFYLRRV 0.600 84 RVLSICTTC 0.500 81 RVIRVLSIC 0.500 12 QPTLFSFFS 0.400 151 LHVSKYCSL 0.400 18 FFSASSPFL 0.400 161 PINSIIRGL 0.400 58 SPQLFESLN 0.400 233 GMYKMDFII 0.400 120 TFHLFSWSL 0.400 194 TLIQELQEI 0.400 188 SSVYMMTLI 0.400 130 FPVSSSLIF 0.400 71 FKYEASFYL 0.400 158 SLFPINSII 0.400 218 GKSHQHILL 0.400 46 IHMVVLLTM 0.300 204 VPSQPQPLP 0.300 35 RTYLPVCHV 0.300 124 FSWSLSFPV 0.200 182 KQIMLFSSV 0.200 91 TCLLGMLQV 0.200 102 ISPSISWLV 0.200 171 FTLSLFRDV 0.200 133 SSSLIFYTV 0.200 103 SPSISWLVR 0.200 47 HMVVLLTMV 0.200 160 FPINSIIRG 0.200 209 QPLPKDLCR 0.200 211 LPKDLCRGK 0.200 224 ILLPVSFSV 0.200 92 CLLGMLQVV 0.200 231 SVGMYKMDF 0.100 6 KLVLASQPT 0.100 139 YTVASSNVT 0.100 229 SFSVGMYKM 0.100 45 LIHMVVLLT 0.100 99 VVNISPSIS 0.100 215 LCRGKSHQH 0.100 242 STSSTLPWA 0.100 149 INLHVSKYC 0.100 152 HVSKYCSLF 0.100 164 SIIRGLFFT 0.100 177 RDVFLKQIM 0.100 67 FQNDFKYEA 0.100 54 MVFLSPQLF 0.100 132 VSSSLIFYT 0.100 48 MVVLLTMVF 0.100 134 SSLIFYTVA 0.100 9 LASQPTLFS 0.090 20 SASSPFLLF 0.090 V2-B7-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 5 QPTLCSFFS 0.400 1 VLASQPTLC 0.100 2 LASQPTLCS 0.090 3 ASQPTLCSF 0.060 7 TLCSFFSAS 0.020 8 LCSFFSASS 0.020 4 SQPTLCSFF 0.020 6 PTLCSFFSA 0.010 9 CSFFSASSP 0.010 V3-B7-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 2 YLRRVIRDL 40.00 9 DLSICTTCL 4.000 6 VIRDLSICT 1.000 5 RVIRDLSIC 0.500 4 RRVIRDLSI 0.040 3 LRRVIRDLS 0.030 8 RDLSICTTC 0.010 7 IRDLSICTT 0.003 1 FYLRRVIRD 0.001 V4-B7-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. Pos 123456789 Score 3 CTTCLLDML 4.000 2 ICTTCLLDM 1.000 6 CLLDMLQVV 0.200 5 TCLLDMLQV 0.200 8 LDMLQVVNI 0.120 9 DMLQVVNIS 0.020 4 TTCLLDMLQ 0.010 1 SICTTCLLD 0.010 7 LLDMLQVVN 0.006 V12A-B7-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 2 SPSISWLIM 20.000 1 ISPSISWLI 0.400 3 PSISWLIML 0.400 7 WLIMLFSSV 0.200 8 LIMLFSSVY 0.060 4 SISWLIMLF 0.020 5 ISWLIMLFS 0.020 6 SWLIMLFSS 0.002 V12B-B7-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 1060 APKCRPGTL 240.000 428 VPRKDLIVM 200.000 233 EPPAHQRLL 80.000 674 NPVGLPENL 80.000 286 VLRHIPEIL 40.0 147 HQRRDAACL 40.000 777 MLREEIAKL 40.000 152 AACLRAQGL 36.000 15 AATGLWAAL 36.000 125 VPRPQAAPA 20.000 434 IVMLRDTDM 15.000 104 SATPAGAFL 12.000 830 VAGFSLRQL 12.000 958 ASGARAAAL 12.000 946 RASPGTPSL 12.000 687 SAGNGDDGL 12.000 312 ATAARLSGL 12.000 521 YAIYNEDKL 12.000 1106 AGGVGPTTL 12.000 883 HAQASVQQL 12.000 11 ATFAAATGL 12.000 870 VAGFSLRQL 12.000 843 HAQASVQQL 12.000 528 KLMAKALLL 12.000 904 QAQEQGAAL 12.000 593 SASIVNLLL 12.000 1027 QAFRDKDDL 12.00 105 ATPAGAFLL 12.000 61 AARKEFSTT 9.000 170 APTAPDGGA 9.000 425 WGKVPRKDL 9.000 1082 PPHRHTTTL 8.000 660 LPLKVEEEI 8.000 181 PPSRNSYRL 8.000 650 NPVITILNI 8.000 208 APGRSSSCA 6.000 621 AVSSHHHVI 6.000 604 NVDVSSQDL 6.000 781 EIAKLRLEL 6.000 1006 CPMFDVSPA 6.000 228 APSPAEPPA 6.000 467 LLLDRRCQL 6.000 588 AVCCGSASI 6.000 577 LDRYGRTAL 6.000 243 LPRAPQAVS 6.000 591 CGSASIVNL 4.000 76 LSLSSSRAL 4.000 817 TIQLNEEAL 4.000 309 ELPATAARL 4.000 544 KNKCGLTPL 4.000 491 QCQEDECVL 4.000 86 GSLPAFADL 4.000 735 TGISQDEIL 4.000 1042 QSPRHTKDL 4.000 592 GSASIVNLL 4.000 278 IPNLSYPLV 4.000 266 SLSVFQLHL 4.000 648 NSNPVITIL 4.000 36 TWRKEPAVL 4.000 460 GNSEVVQLL 4.000 256 QPSEEALGV 4.000 374 ETSTKISGL 4.000 566 FLIKKKANL 4.000 277 CIPNLSYPL 4.000 411 VRREDLDKL 4.000 302 GGGILGLEL 4.000 47 CNLEKGSWL 4.000 796 NQLRENKIL 4.000 264 VGSLSVFQL 4.000 447 KQKRTALHL 4.000 300 ETGGGILGL 4.000 324 MQIKEFEEL 4.000 827 KTKVAGFSL 4.000 68 TTLTGHSAL 4.000 654 TILNIKLPL 4.000 546 KCGLTPLLL 4.000 652 VITILNIKL 4.000 115 WERVVQRRL 4.000 763 LSHKKEEDL 4.000 721 EQNDTQKQL 4.000 770 DLLRENSML 4.000 459 NGNSEVVQL 4.000 209 PGRSSSCAL 4.000 273 HLIQCIPNL 4.000 470 DRRCQLNVL 4.000 70 LTGHSALSL 4.000 522 AIYNEDKLM 3.000 84 LPGSLPAFA 2.000 7 LPTQATFAA 2.000 285 LVLRHIPEI 2.000 944 EPRASPGTP 2.000 1073 TPPHRNADT 2.000 28 NPSRADPVT 2.000 1043 SPRHTKDLG 2.000 976 SPTKQKSVC 2.000 922 VPLSEGGTA 2.000 94 LPRSCPESE 2.000 1081 TPPHRHTTT 2.000 982 SVCDSSGWI 2.000
[1030] 29 TABLE XIX Pos 1234567890 Score V1-B7-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 160 FPINSIIRGL 80.000 48 MVVLLTMVFL 20.000 42 HVALIHMVVL 20.000 84 RVLSICTTCL 20.000 109 LVRFKWKSTI 20.000 142 ASSNVTQINL 12.000 20 SASSPFLLFL 12.000 43 VALIHMVVLL 12.000 52 LTMVFLSPQL 12.000 88 ICTTCLLGML 4.000 172 TLSLFRDVFL 4.000 119 FTFHLFSWSL 4.000 127 SLSFPVSSSL 4.000 85 VLSICTTCLL 4.000 22 SSPFLLFLDL 4.000 217 RGKSHQHILL 4.000 202 ILVPSQPQPL 4.000 100 VNISPSISWL 4.000 194 TLIQELQEIL 4.000 206 SQPQPLPKDL 4.000 6 KLVLASQPTL 4.000 150 NLHVSKYCSL 4.000 56 FLSPQLFESL 4.000 164 SIIRGLFFTL 4.000 215 LCRGKSHQHI 4.000 207 QPQPLPKDLC 3.000 183 QIMLFSSVYM 3.000 140 TVASSNVTQI 2.000 1 MPFISKLVLA 2.000 12 QPTLFSFFSA 2.000 231 SVGMYKMDFI 2.000 74 EASFYLRRVI 1.800 190 VYMMTLIQEL 1.200 232 VGMYKMDFII 1.200 184 IMLFSSVYMM 1.000 82 VIRVLSICTT 1.000 225 LLPVSFSVGM 1.000 87 SICTTCLLGM 1.000 45 LIHMVVLLTM 1.000 228 VSFSVGMYKM 1.000 29 LDLRPERTYL 0.600 156 YCSLFPINSI 0.600 32 RPERTYLPVC 0.600 211 LPKDLCRGKS 0.600 39 PVCHVALIHM 0.500 81 RVIRVLSICT 0.500 36 TYLPVCHVAL 0.400 70 DFKYEASFYL 0.400 18 FFSASSPFLL 0.400 97 LQVVNISPSI 0.400 186 LFSSVYMMTL 0.400 128 LSFPVSSSLI 0.400 77 FYLRRVIRVL 0.400 103 SPSISWLVRF 0.400 187 FSSVYMMTLI 0.400 193 MTLIQELQEI 0.400 238 DFIISTSSTL 0.400 175 LFRDVFLKQI 0.400 17 SFFSASSPFL 0.400 79 LRRVIRVLSI 0.400 216 CRGKSHQHIL 0.400 177 RDVFLKQIML 0.400 226 LPVSFSVGMY 0.400 166 IRGLFFTLSL 0.400 37 YLPVCHVALI 0.400 93 LLGMLQVVNI 0.400 153 VSKYCSLFPI 0.400 157 CSLFPINSII 0.400 114 WKSTIFTFHL 0.400 130 FPVSSSLIFY 0.400 58 SPQLFESLNF 0.400 44 ALIHMVVLLT 0.300 78 YLRRVIRVLS 0.300 91 TCLLGMLQVV 0.200 195 LIQELQEILV 0.200 90 TTCLLGMLQV 0.200 101 NISPSISWLV 0.200 219 KSHQHILLPV 0.200 40 VCHVALIHMV 0.200 209 QPLPKDLCRG 0.200 132 VSSSLIFYTV 0.200 144 SNVTQINLHV 0.200 165 IIRGLFFTLS 0.200 38 LPVCHVALIH 0.200 246 TLPWAYDRGV 0.200 204 VPSQPQPLPK 0.200 23 SPFLLFLDLR 0.200 223 HILLPVSFSV 0.200 99 VVNISPSISW 0.150 163 NSIIRGLFFT 0.100 98 QVVNISPSIS 0.100 241 ISTSSTLPWA 0.100 178 DVFLKQIMLF 0.100 35 RTYLPVCHVA 0.100 133 SSSLIFYTVA 0.100 49 VVLLTMVFLS 0.100 148 QINLHVSKYC 0.100 30 DLRPERTYLP 0.100 185 MLFSSVYMMT 0.100 7 LVLASQPTLF 0.100 V2-B7-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 6 QPTLCSFFSA 2.000 1 LVLASQPTLC 0.500 3 LASQPTLCSF 0.060 4 ASQPTLCSFF 0.060 2 VLASQPTLCS 0.030 10 CSFFSASSPF 0.020 8 TLCSFFSASS 0.020 5 SQPTLCSFFS 0.020 9 LCSFFSASSP 0.010 7 PTLCSFFSAS 0.002 V3-B7-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 10 DLSICTTCLL 4.000 7 VIRDLSICTT 1.000 6 RVIRDLSICT 0.500 2 FYLRRVIRDL 0.400 9 RDLSICTTCL 0.400 4 LRRVIRDLSI 0.400 3 YLRRVIRDLS 0.300 5 RRVIRDLSIC 0.010 8 IRDLSICTTC 0.003 1 SFYLRRVIRD 0.001 V4-B7-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 3 ICTTCLLDML 4.000 2 SICTTCLLDM 1.000 5 TTCLLDMLQV 0.200 6 TCLLDMLQVV 0.200 8 LLDMLQVVNI 0.120 7 CLLDMLQVVN 0.020 4 CTTCLLDMLQ 0.010 10 DMLQVVNISP 0.010 1 LSICTTCLLD 0.010 9 LDMLQVVNIS 0.006 V12A-B7-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 3 SPSISWLIML 80.000 9 LIMLFSSVYM 3.000 2 ISPSISWLIM 1.000 1 NISPSISWLI 0.400 6 ISWLIMLFSS 0.020 7 SWLIMLFSSV 0.020 5 SISWLIMLFS 0.020 8 WLIMLFSSVY 0.020 4 PSISWLIMLF 0.002 V12B-B7-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 429 VPRKDLIVML 800.000 62 AARKEFSTTL 360.000 209 APGRSSSCAL 240.000 411 HVRREDLDKL 200.000 41 EPAVLPCCNL 120.000 994 VPTFSSGSFL 80.000 279 IPNLSYPLVL 80.000 1091 LPHRDTTTSL 80.000 1082 TPPHRHTTTL 80.000 949 SPGTPSLVRL 80.000 181 CPPSRNSYRL 80.000 1007 CPMFDVSPAM 60.000 161 LTRAFQVVHL 40.000 315 AARLSGLNSI 36.000 15 AAATGLWAAL 36.000 983 SVCDSSGWIL 20.000 830 KVAGFSLRQL 20.000 126 VPRPQAAPAT 20.000 264 GVGSLSVFQL 20.000 286 LVLRHIPEIL 20.000 870 EVAGFSLRQL 20.000 1060 LAPKCRPGTL 12.000 958 LASGARAAAL 12.000 105 SATPAGAFLL 12.000 152 DAACLRAQGL 12.000 872 AGFSLRQLGL 12.000 76 ALSLSSSRAL 12.000 459 ANGNSEVVQL 12.000 1106 SAGGVGPTTL 12.000 832 AGFSLRQLGL 12.000 11 QATFAAATGL 12.000 687 ASAGNGDDGL 12.000 1061 APKCRPGTLC 9.000 235 PPAHQRLLFL 8.000 217 ALRYRSGPSV 6.000 945 EPRASPGTPS 6.000 353 NVDKWDDFCL 6.000 467 QLLLDRRCQL 6.000 577 ALDRYGRTAL 5.400 932 AGDQGPGTHL 5.400 955 LVRLASGARA 5.000 192 HVRCAQGLEA 5.000 428 KVPRKDLIVM 5.000 796 QNQLRENKIL 4.000 266 GSLSVFQLHL 4.000 559 EQKQEVVKFL 4.000 1027 HQAFRDKDDL 4.000 545 KNKCGLTPLL 4.000 916 QIGDPGGVPL 4.000 624 SSHHHVICEL 4.000 593 GSASIVNLLL 4.000 197 QGLEAASANL 4.000 654 ITILNIKLPL 4.000 70 TLTGHSALSL 4.000 68 STTLTGHSAL 4.000 1051 LGQDDRAGVL 4.000 460 NGNSEVVQLL 4.000 904 QQAQEQGAAL 4.000 777 SMLREEIAKL 4.000 104 QSATPAGAFL 4.000 763 SLSHKKEEDL 4.000 302 TGGGILGLEL 4.000 277 QCIPNLSYPL 4.000 591 CCGSASIVNL 4.000 190 LTHVRCAQGL 4.000 461 GNSEVVQLLL 4.000 470 LDRRCQLNVL 4.000 47 CCNLEKGSWL 4.000 604 QNVDVSSQDL 4.000 36 VTWRKEPAVL 4.000 817 KTIQLNEEAL 4.000 674 SNPVGLPENL 4.000 491 VQCQEDECVL 4.000 324 IMQIKEFEEL 4.000 592 CGSASIVNLL 4.000 80 SSSRALPGSL 4.000 1042 QQSPRHTKDL 4.000 648 ENSNPVITIL 4.000 735 NTGISQDEIL 4.000 764 LSHKKEEDLL 4.000 753 VAEKEMNSEL 3.600 253 GPQEQPSEEA 3.000 1003 LGRRCPMFDV 3.000 490 AVQCQEDECV 3.000 522 YAIYNEDKLM 3.000 675 NPVGLPENLT 3.000 95 LPRSCPESEQ 3.000 407 EPRYHVRRED 3.000 589 AVCCGSASIV 3.000 144 SPPCHQRRDA 3.000 128 RPQAAPATSA 3.000 138 TPSRDPSPPC 3.000 244 LPRAPQAVSG 2.000 8 LPTQATFAAA 2.000 652 PVITILNIKL 2.000 1065 RPGTLCHTDT 2.000 34 DPVTWRKEPA 2.000 923 VPLSEGGTAA 2.000 581 YGRTALILAV 2.000 1044 SPRHTKDLGQ 2.000
[1031] 30 TABLE XX Pos 123456789 Score V1-B35-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 226 LPVSFSVGM 40.000 1 MPFISKLVL 20.000 207 QPQPLPKDL 20.000 23 SPFLLFLDL 20.000 130 FPVSSSLIF 20.000 115 KSTIFTFHL 10.000 243 TSSTLPWAY 10.000 38 LPVCHVALI 8.000 217 RGKSHQHIL 6.000 187 FSSVYMMTL 5.000 21 ASSPFLLFL 5.000 19 FSASSPFLL 5.000 10 ASQPTLFSF 5.000 173 LSLFRDVFL 5.000 86 LSICTTCLL 5.000 128 LSFPVSSSL 5.000 143 SSNVTQINL 5.000 63 ESLNFQNDF 5.000 163 NSIIRGLFF 5.000 57 LSPQLFESL 5.000 30 DLRPERTYL 4.500 247 LPWAYDRGV 4.000 20 SASSPFLLF 3.000 165 IIRGLFFTL 3.000 43 VALIHMVVL 3.000 78 YLRRVIRVL 3.000 106 ISWLVRFKW 2.500 241 ISTSSTLPW 2.500 32 RPERTYLPV 2.400 184 IMLFSSVYM 2.000 12 QPTLFSFFS 2.000 157 CSLFPINSI 2.000 188 SSVYMMTLI 2.000 183 QIMLFSSVY 2.000 185 MLFSSVYMM 2.000 65 LNFQNDFKY 2.000 167 RGLFFTLSL 2.000 195 LIQELQEIL 2.000 58 SPQLFESLN 2.000 40 VCHVALIHM 2.000 75 ASFYLRRVI 2.000 148 QINLHVSKY 2.000 88 ICTTCLLGM 2.000 141 VASSNVTQI 1.200 211 LPKDLCRGK 1.200 116 STIFTFHLF 1.000 53 TMVFLSPQL 1.000 37 YLPVCHVAL 1.000 124 FSWSLSFPV 1.000 7 LVLASQPTL 1.000 49 VVLLTMVFL 1.000 85 VLSICTTCL 1.000 44 ALIHMVVLL 1.000 203 LVPSQPQPL 1.000 178 DVFLKQIML 1.000 239 FIISTSSTL 1.000 133 SSSLIFYTV 1.000 8 VLASQPTLF 1.000 101 NISPSISWL 1.000 162 INSIIRGLF 1.000 172 TLSLFRDVF 1.000 11 SQPTLFSFF 1.000 191 YMMTLIQEL 1.000 54 MVFLSPQLF 1.000 168 GLFFTLSLF 1.000 231 SVGMYKMDF 1.000 48 MVVLLTMVF 1.000 89 CTTCLLGML 1.000 102 ISPSISWLV 1.000 152 HVSKYCSLF 1.000 122 HLFSWSLSF 1.000 70 DFKYEASFY 0.900 194 TLIQELQEI 0.600 111 RFKWKSTIF 0.600 74 EASFYLRRV 0.600 113 KWKSTIFTF 0.600 142 ASSNVTQIN 0.500 134 SSLIFYTVA 0.500 132 VSSSLIFYT 0.500 100 VNISPSISW 0.500 104 PSISWLVRF 0.500 126 WSLSFPVSS 0.500 98 QVVNISPSI 0.400 233 GMYKMDFII 0.400 182 KQIMLFSSV 0.400 35 RTYLPVCHV 0.400 177 RDVFLKQIM 0.400 158 SLFPINSII 0.400 94 LGMLQVVNI 0.400 232 VGMYKMDFI 0.400 82 VIRVLSICT 0.300 9 LASQPTLFS 0.300 180 FLKQIMLFS 0.300 109 LVRFKWKST 0.300 145 NVTQINLHV 0.200 171 FTLSLFRDV 0.200 29 LDLRPERTY 0.200 131 PVSSSLIFY 0.200 160 FPINSIIRG 0.200 209 QPLPKDLCR 0.200 V2-B35-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 3 ASQPTLCSF 5.000 5 QPTLCSFFS 2.000 4 SQPTLCSFF 1.000 2 LASQPTLCS 0.300 8 LCSFFSASS 0.100 7 TLCSFFSAS 0.100 1 VLASQPTLC 0.100 9 CSFFSASSP 0.050 6 PTLCSFFSA 0.010 V3-B35-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 2 YLRRVIRDL 3.000 9 DLSICTTCL 1.000 6 VIRDLSICT 0.600 5 RVIRDLSIC 0.300 4 RRVIRDLSI 0.080 3 LRRVIRDLS 0.030 8 RDLSICTTC 0.020 7 IRDLSICTT 0.003 1 FYLRRVIRD 0.001 V4-B35-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 2 ICTTCLLDM 2.000 3 CTTCLLDML 1.000 6 CLLDMLQVV 0.400 5 TCLLDMLQV 0.300 9 DMLQVVNIS 0.100 8 LDMLQVVNI 0.040 7 LLDMLQVVN 0.030 4 TTCLLDMLQ 0.010 1 SICTTCLLD 0.010 V12A-B35-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 2 SPSISWLIM 40.000 8 LIMLFSSVY 2.000 1 ISPSISWLI 2.000 4 SISWLIMLF 1.000 3 PSISWLIML 0.500 5 ISWLIMLFS 0.500 7 WLIMLFSSV 0.200 6 SWLIMLFSS 0.010 V12B-B35-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 428 VPRKDLIVM 180.000 1060 APKCRPGTL 60.000 674 NPVGLPENL 20.000 993 VPTFSSGSF 20.000 233 EPPAHQRLL 20.000 211 RSSSCALRY 20.000 709 FPDTENEEY 18.000 80 SSRALPGSL 15.000 256 QPSEEALGV 12.000 612 LSGQTAREY 10.000 401 DSAFMEPRY 10.000 777 MLREEIAKL 9.000 650 NPVITILNI 8.000 660 LPLKVEEEI 8.000 981 KSVCDSSGW 7.500 946 RASPGTPSL 6.000 544 KNKCGLTPL 6.000 125 VPRPQAAPA 6.000 885 QASVQQLCY 6.000 447 KQKRTALHL 6.000 1021 SNRETHQAF 6.000 243 LPRAPQAVS 6.000 845 QASVQQLCY 6.000 156 RAQGLTRAF 6.000 827 KTKVAGFSL 6.000 904 QAQEQGAAL 6.000 1042 QSPRHTKDL 5.000 321 NSIMQIKEF 5.000 958 ASGARAAAL 5.000 103 QSATPAGAF 5.000 75 LSLSSSRAL 5.000 648 NSNPVITIL 5.000 86 GSLPAFADL 5.000 763 LSHKKEEDL 5.000 592 GSASIVNLL 5.000 1027 QAFRDKDDL 4.500 558 EQKQEVVKF 4.500 147 HQRRDAACL 4.500 628 VICELLSDY 4.000 316 RLSGLNSIM 4.000 278 IPNLSYPLV 4.000 351 KNVDKWDDF 4.000 127 RPQAAPATS 4.000 378 KISGLIQEM 4.000 687 SAGNGDDGL 3.000 491 QCQEDECVL 3.000 1006 CPMFDVSPA 3.000 522 AIYNEDKLM 3.000 870 VAGFSLRQL 3.000 1090 LPHRDTTTS 3.000 593 SASIVNLLL 3.000 830 VAGFSLRQL 3.000 521 YAIYNEDKL 3.000 633 LSDYKEKQM 3.000 922 VPLSEGGTA 3.000 825 LTKTKVAGF 3.000 15 AATGLWAAL 3.000 286 VLRHIPEIL 3.000 104 SATPAGAFL 3.000 940 LPPREPRAS 3.000 152 AACLRAQGL 3.000 883 HAQASVQQL 3.000 425 WGKVPRKDL 3.000 843 HAQASVQQL 3.000 371 IMKETSTKI 2.400 546 KCGLTPLLL 2.000 375 TSTKISGLI 2.000 228 APSPAEPPA 2.000 170 APTAPDGGA 2.000 275 IQCIPNLSY 2.000 208 APGRSSSCA 2.000 179 GCPPSRNSY 2.000 1035 LPFFKTQQS 2.000 391 SNVGTWGDY 2.000 1081 TPPHRHTTT 2.000 951 TPSLVRLAS 2.000 7 LPTQATFAA 2.000 84 LPGSLPAFA 2.000 234 PPAHQRLLF 2.000 516 NTALHYAIY 2.000 644 ISSENSNPV 2.000 434 IVMLRDTDM 2.000 40 EPAVLPCCN 2.000 267 LSVFQLHLI 2.000 1082 PPHRHTTTL 2.000 976 SPTKQKSVC 2.000 310 LPATAARLS 2.000 131 APATSATPS 2.000 28 NPSRADPVT 2.000 572 ALNLALDRY 2.000 460 GNSEVVQLL 2.000 467 LLLDRRCQL 2.000 1073 TPPHRNADT 2.000 181 PPSRNSYRL 2.000 205 LPGAPGRSS 2.000 529 LMAKALLLY 2.000 528 KLMAKALLL 2.000 721 EQNDTQKQL 2.000 47 CNLEKGSWL 2.000 222 GPSVSSAPS 2.000
[1032] 31 TABLE XXI Pos 1234567890 Score V1-B35-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 226 LPVSFSVGMY 40.000 130 FPVSSSLIFY 40.000 58 SPQLFESLNF 30.000 103 SPSISWLVRF 20.000 160 FPINSIIRGL 20.000 211 LPKDLCRGKS 12.000 228 VSFSVGMYKM 10.000 115 KSTIFTFHLF 10.000 217 RGKSHQHILL 6.000 153 VSKYCSLFPI 6.000 230 FSVGMYKMDF 5.000 19 FSASSPFLLF 5.000 16 FSFFSASSPF 5.000 22 SSPFLLFLDL 5.000 142 ASSNVTQINL 5.000 10 ASQPTLFSFF 5.000 182 KQIMLFSSVY 4.000 43 VALIHMVVLL 3.000 9 LASQPTLFSF 3.000 20 SASSPFLLFL 3.000 167 RGLFFTLSLF 2.000 184 IMLFSSVYMM 2.000 147 TQINLHVSKY 2.000 12 QPTLFSFFSA 2.000 128 LSFPVSSSLI 2.000 187 FSSVYMMTLI 2.000 87 SICTTCLLGM 2.000 84 RVLSICTTCL 2.000 207 QPQPLPKDLC 2.000 1 MPFISKLVLA 2.000 157 CSLFPINSII 2.000 6 KLVLASQPTL 2.000 242 STSSTLPWAY 2.000 183 QIMLFSSVYM 2.000 225 LLPVSFSVGM 2.000 219 KSHQHILLPV 2.000 45 LIHMVVLLTM 2.000 64 SLNFQNDFKY 2.000 32 RPERTYLPVC 1.200 109 LVRFKWKSTI 1.200 74 EASFYLRRVI 1.200 215 LCRGKSHQHI 1.200 171 FTLSLFRDVF 1.000 202 ILVPSQPQPL 1.000 221 HQHILLPVSF 1.000 132 VSSSLIFYTV 1.000 164 SIIRGLFFTL 1.000 178 DVFLKQIMLF 1.000 7 LVLASQPTLF 1.000 42 HVALIHMVVL 1.000 119 FTFHLFSWSL 1.000 150 NLHVSKYCSL 1.000 172 TLSLFRDVFL 1.000 88 ICTTCLLGML 1.000 58 MVVLLTMVFL 1.000 100 VNISPSISWL 1.000 47 HMVVLLTMVF 1.000 194 TLIQELQEIL 1.000 206 SQPQPLPKDL 1.000 162 INSIIRGLFF 1.000 127 SLSFPVSSSL 1.000 56 FLSPQLFESL 1.000 53 TMVFLSPQLF 1.000 85 VLSICTTCLL 1.000 52 LTMVFLSPQL 1.000 193 MTLIQELQEI 0.600 28 FLDLRPERTY 0.600 105 SISWLVRFKW 0.500 241 ISTSSTLPWA 0.500 124 FSWSLSFPVS 0.500 240 IISTSSTLPW 0.500 57 LSPQLFESLN 0.500 163 NSIIRGLFFT 0.500 99 VVNISPSISW 0.500 134 SSLIFYTVAS 0.500 117 TIFTFHLFSW 0.500 133 SSSLIFYTVA 0.500 126 WSLSFPVSSS 0.500 97 LQVVNISPSI 0.400 231 SVGMYKMDFI 0.400 93 LLGMLQVVNI 0.400 232 VGMYKMDFII 0.400 195 LIQELQEILV 0.400 140 TVASSNVTQI 0.400 37 YLPVCHVALI 0.400 156 YCSLFPINSI 0.400 69 NDFKYEASFY 0.300 68 QNDFKYEASF 0.300 180 FLKQIMLFSS 0.300 165 IIRGLFFTLS 0.300 82 VIRVLSICTT 0.300 78 YLRRVIRVLS 0.300 141 VASSNVTQIN 0.300 209 QPLPKDLCRG 0.300 70 DFKYEASFYL 0.300 175 LFRDVFLKQI 0.240 177 RDVFLKQIML 0.200 144 SNVTQINLHV 0.200 38 LPVCHVALIH 0.200 39 PVCHVALIHM 0.200 V2-B35-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 4 ASQPTLCSFF 5.000 10 CSFFSASSPF 5.000 3 LASQPTLCSF 3.000 6 QPTLCSFFSA 2.000 8 TLCSFFSASS 0.100 1 LVLASQPTLC 0.100 5 SQPTLCSFFS 0.100 2 VLASQPTLCS 0.100 7 PTLCSFFSAS 0.010 9 LCSFFSASSP 0.010 V3-B35-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 10 DLSICTTCLL 1.000 7 VIRDLSIGTT 0.600 3 YLRRVIRDLS 0.300 9 RDLSICTTCL 0.200 6 RVIRDLSICT 0.200 4 LRRVIRDLSI 0.120 2 FYLRRVIRDL 0.100 5 RRVIRDLSIC 0.030 8 IRDLSICTTC 0.003 1 SFYLRRVIRD 0.001 V4-B35-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 2 SICTTCLLDM 2.000 3 ICTTCLLDML 1.000 5 TTCLLDMLQV 0300 6 TCLLDMLQVV 0.200 7 CLLDMLQVVN 0.200 8 LLDMLQVVNI 0.120 1 LSICTTCLLD 0.050 4 CTTCLLDMLQ 0.010 10 DMLQVVNISP 0.010 9 LDMLQVVNIS 0.010 V12A-B35-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 3 SPSISWLIML 20.000 2 ISPSISWLIM 10.000 8 WLIMLFSSVY 2.000 9 LIMLFSSVYM 2.000 6 ISWLIMLFSS 0.500 4 PSISWLIMLF 0.500 1 NISPSISWLI 0.400 5 SISWLIMLFS 0.100 7 SWLIMLFSSV 0.020 V12B-B35-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 429 VPRKDLIVML 60.000 701 KSRKPENQQF 45.000 1007 CPMFDVSPAM 40.000 994 VPTFSSGSFL 20.000 234 EPPAHQRLLF 20.000 279 IPNLSYPLVL 20.000 181 CPPSRNSYRL 20.000 209 APGRSSSCAL 20.000 58 FPGTAARKEF 20.000 949 SPGTPSLVRL 20.000 1082 TPPHRHTTTL 20.000 391 KSNVGTWGDY 20.000 41 EPAVLPCCNL 20.000 1091 LPHRDTTTSL 20.000 29 NPSRADPVTW 15.000 572 KANLNALDRY 12.000 1000 GSFLGRRCPM 10.000 107 TPAGAFLLGW 10.000 62 AARKEFSTTL 9.000 522 YAIYNEDKLM 9.000 865 QAQEQEVAGF 9.000 749 KQIEVAEKEM 8.000 764 LSHKKEEDLL 7.500 428 KVPRKDLIVM 6.000 545 KNKCGLTPLL 6.000 945 EPRASPGTPS 6.000 492 QCQEDECVLM 6.000 83 RALPGSLPAF 6.000 1061 APKCRPGTLC 6.000 125 VPRPQAAPAT 6.000 1021 DSNRETHQAF 5.000 339 LSHKVIQCVF 5.000 624 SSHHHVICEL 5.000 266 GSLSVFQLHL 5.000 104 QSATPAGAFL 5.000 687 ASAGNGDDGL 5.000 80 SSSRALPGSL 5.000 593 GSASIVNLLL 5.000 348 FAKKKNVDKW 4.500 411 HVRREDLDKL 4.500 982 KSVCDSSGWI 4.000 128 RPQAAPATSA 4.000 1065 RPGTLCHTDT 4.000 633 LLSDYKEKQM 4.000 529 KLMAKALLLY 4.000 253 GPQEQPSEEA 4.000 645 ISSENSNPVI 4.000 315 AARLSGLNSI 3.600 11 QATFAAATGL 3.000 559 EQKQEVVKFL 3.000 1106 LAPKCRPGTL 3.000 914 RSQIGDPGGV 3.000 152 DAACLRAQGL 3.000 958 LASGARAAAL 3.000 89 LPAFADLPRS 3.000 15 AAATGLWAAL 3.000 105 SATPAGAFLL 3.000 847 ASVQQLCYKW 2.500 388 GSGKSNVGTW 2.500 887 ASVQQLCYKW 2.500 533 KALLLYGADI 2.400 810 SVKEKLLKTI 2.400 634 LSDYKEKQML 2.250 179 AGCPPSRNSY 2.000 275 LIQCIPNLSY 2.000 138 TPSRDPSPPC 2.000 817 KTIQLNEEAL 2.000 318 LSGLNSIMQI 2.000 364 EGYGHSFLIM 2.000 675 NPVGLPENLT 2.000 983 SVCDSSGWIL 2.000 1051 LGQDDRAGVL 2.000 144 SPPCHQRRDA 2.000 916 QIGDPGGVPL 2.000 361 CLSEGYGHSF 2.000 45 LPCCNLEKGS 2.000 34 DPVTWRKEPA 2.000 885 AWASVQQLCY 2.000 8 LPTQATFAAA 2.000 516 GNTALHYAIY 2.000 461 GNSEVVQLLL 2.000 923 VPLSEGGTAA 2.000 830 KVAGFSLRQL 2.000 612 DLSGQTAREY 2.000 628 HVICELLSDY 2.000 206 LPGAPGRSSS 2.000 282 LSYPLVLRHI 2.000 845 AQASVQQLCY 2.000 197 QGLEAASANL 2.000 604 QNVDVSSQDL 2.000 434 LIVMLRDTDM 2.000 235 PPAHQRLLFL 2.000 1013 SPAMRLKSDS 2.000 992 LPVPTFSSGS 2.000 688 SAGNGDDGLI 1.800 506 GADGNIQDEY 1.800 324 IMQIKEFEEL 1.500 777 SMLREEIAKL 1.500
[1033] Tables XXI-XLIX: 32 TABLE XXII Pos 123456789 Score V1A-A1-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 131 PVSSSLIFY 19 243 TSSTLPWAY 19 148 QINLHVSKY 18 196 IQELQEILV 18 65 LNFQNDFKY 17 227 PVSFSVGMY 17 29 LDLRPERTY 16 32 RPERTYLPV 16 183 QIMLFSSVY 16 205 PSQPQPLPK 16 21 ASSPFLLFL 15 70 DFKYEASFY 15 72 KYEASFYLR 15 20 SASSPFLLF 14 193 MTLIQELQE 14 116 STIFTFHLF 13 175 LFRDVFLKQ 13 212 PKDLCRGKS 13 28 FLDLRPFRT 12 51 LLTMVFLSP 12 219 KSHQHILLP 12 245 STLPWAYDR 12 23 SPFLLFLDL 11 45 LIHMVVLLT 11 61 LFESLNFQN 11 153 VSKYCSLFP 11 163 NSIIRGLFF 11 176 FRDVFLKQI 11 199 LQEILVESQ 11 68 QNDFKYEAS 10 87 SICTTCLLG 10 119 FTFHLFSWS 10 128 LSFPVSSSL 10 143 SSNVTQINL 10 171 FTLSLFRDV 10 209 QPLPKDLCR 10 236 KMDFIISTS 10 241 ISTSSTLPW 10 10 ASQPTLFSF 9 13 PTLFSFFSA 9 90 TTCLLGMLQ 9 103 SPSISWLVR 9 106 ISWLVRFKW 9 133 SSSLIFYTV 9 139 YTVASSNVT 9 V2A-HLA-A1-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 3 ASQPTLCSF 9 6 PTLCSFFSA 9 2 LASQPTLCS 7 1 VLASQPTLC 6 7 TLCSFFSAS 4 9 CSFFSASSP 4 V3A-HLA-A1-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 7 IRDLSICTT 10 1 FYLRRVIRD 6 4 RRVIRDLSI 6 2 YLRRVIRDL 5 6 VIRDLSICT 5 V4A-HLA-A1-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified,the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 7 LLDMLQVVN 12 1 SICTTCLLD 10 4 TTCLLDMLQ 8 3 CTTCLLDML 7 2 ICTTCLLDM 6 5 TCLLDMLQV 6 V12A-HLA-A1-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 8 LIMLFSSVY 16 5 ISWLIMLFS 10 3 PSISWLIML 9 2 SPSISWLIM 8
[1034] 33 TABLE XXIII Pos 123456789 Score V1A-HLA-A0201-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 44 ALIHMVVLL 30 92 CLLGMLQVV 28 191 YMMTLIQEL 28 224 8LLPVSFSV 28 78 YLRRVIRVL 26 194 TLIQELQEI 26 37 YLPVCHVAL 25 165 IIRGLFFTL 25 101 NISPSISWL 24 47 HMVVLLTMV 23 85 VLSICTTCL 23 158 SLFPINSII 23 195 LIQELQEIL 23 239 FIISTSSTL 23 164 SIIRGLFFT 22 7 LVLASQPTL 21 30 DLRPERTYL 21 161 PINSIIRGL 21 21 ASSPFLLFL 20 35 RTYLPVCHV 20 185 MLFSSVYMM 20 43 VALIHMVVL 19 45 LIHMVVLLT 19 49 VVLLTMVFL 19 53 TMVFLSPQL 19 56 FLSPQLFES 19 135 SLIFYTVAS 19 60 QLFESLNFQ 18 89 CTTCLLGML 18 127 SLSFPVSSS 18 136 LIFYTVASS 18 171 FTLSLFRDV 18 233 GMYKMDFII 18 26 LLFLDLRPE 17 50 VLLTMVFLS 17 77 FYLRRVIRV 17 94 LGMLQVVNI 17 128 LSFPVSSSL 17 141 VASSNVTQI 17 157 CSLFPINSI 17 167 RGLFFTLSL 17 174 SLFRDVFLK 17 184 IMLFSSVYM 17 220 SHQHILLPV 17 3 FISKLVLAS 16 38 LPVCHVALI 16 41 CHVALIHMV 16 42 HVALIHMVV 16 86 LSICTTCLL 16 95 GMLQVVNIS 16 182 KQIMLFSSV 16 203 LVPSQPQPL 16 6 KLVLASQPT 15 23 SPFLLFLDL 15 28 FLDLRPERT 15 57 LSPQLFESL 15 74 EASFYLRRV 15 80 RRVIRVLSI 15 91 TCLLGMLQV 15 93 EASFYLRRV 15 80 RRVIRVLSI 15 91 TCLLGMLQV 15 93 LLGMLQVVN 15 98 QVVNISPSI 15 105 SISWLVRFK 15 133 SSSLIFYTV 15 148 QINLHVSKY 15 151 LHVSKYCSL 15 154 SKYCSLFPI 15 168 GLFFTLSLF 15 173 LSLFRDVFL 15 178 DVFLKQIML 15 198 ELQEILVPS 15 202 ILVPSQPQP 15 223 HILLPVSFS 15 235 YKMDFIIST 15 242 STSSTLPWA 15 247 LPWAYDRGV 15 25 FLLFLDLRP 14 46 IHMVVLLTM 14 51 LLTMVFLSP 14 71 FKYEASFYL 14 82 VIRVLSICT 14 122 HLFSWSLSF 14 145 NVTQINLHV 14 187 FSSVYMMTL 14 236 KMDFIISTS 14 V2A-HLA-A0201-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 1 VLASQPTLC 13 7 TLCSFFSAS 12 2 LASQPTLCS 10 3 ASQPTLCSF 10 6 PTLCSFFSA 9 8 LCSFFSASS 6 V3A-HLA-A0201-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 2 YLRRVIRDL 26 9 DLSICTTCL 21 6 VIRDLSICT 15 7 IRDLSICTT 12 V4A-HLA-A0201-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 6 CLLDMLQVV 27 3 CTTCLLDML 18 8 LDMLQVVNI 17 7 LLDMLQVVN 15 9 DMLQVVNIS 14 5 TCLLDMLQV 13 V12A-HLA-A0201-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 7 WLIMLFSSV 25 4 SISWLIMLF 15 3 PSISWLIML 13 8 LIMLFSSVY 12 V12B-HLA-A0201-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 777 MLREEIAKL 30 548 GLTPLLLGV 29 802 KILEEIESV 29 18 GLWAALTTV 28 599 LLLEQNVDV 28 261 ALGVGSLSV 27 273 HLIQCIPNL 27 566 FLIKKKANL 26 111 FLLGWERVV 25 334 KLHSLSHKV 25 467 LLLDRRCQL 25 533 ALLLYGADI 25 266 SLSVFQLHL 24 528 KLMAKALLL 24 285 LVLRHIPEI 23 312 ATAARLSGL 23 946 RASPGTPSL 23 197 GLEAASANL 22 242 FLPRAPQAV 22 305 ILGLELPAT 22 327 KEFEELVKL 22 378 KISGLIQEM 22 15 AATGLWAAL 21 286 VLRHIPEIL 21 300 ETGGGILGL 21 315 ARLSGLNSI 21 371 IMKETSTKI 21 652 VITILNIKL 21 654 TILNIKLPL 21 770 DLLRENSML 21 781 EIAKLRLEL 21 784 KLRLELDET 21 1098 SLPHFHVSA 21 5 ILLPTQATF 20 83 ALPGSLPAF 20 159 GLTRAFQVV 20 277 CIPNLSYPL 20 325 QIKEFEELV 20 369 FLIMKETST 20 586 ILAVCCGSA 20 588 AVOCOSASI 20 840 LAQHAQASV 20 880 LAQHAQASV 20 14 AAATGLWAA 19 35 VTWRKEPAV 19 68 TTLTGHSAL 19 70 LTGHSALSL 19 104 SATPAGAFL 19 304 GILGLELPA 19 309 ELPATAARL 19 411 VRREDLDKL 19 432 DLIVMLRDT 19 457 SANGNSEVV 19 498 VLMLLEHGA 19 521 YAIYNEDKL 19 555 GVHEQKQEV 19 576 ALDRYGRTA 19 655 ILNIKLPLK 19 687 SAGNGDDGL 19 742 ILTNKQKQI 19 809 SVKEKLLKT 19 817 TIQLNEEAL 19 904 QAQEQGAAL 19 956 RLASGARAA 19 965 ALPPPTGKN 19 4 HILLPTQAT 18 11 ATFAAATGL 18 105 ATPAGAFLL 18 450 RTALHLASA 18 456 ASANGNSEV 18 477 VLDNKKRTA 18 485 ALIKAVQCQ 18 551 PLLLGVHEQ 18 584 ALILAVCCG 18 592 GSASIVNLL 18 595 SIVNLLLEQ 18 624 SHHHVICEL 18 656 LNIKLPLKV 18 659 KLPLKVEEE 18 830 VAGFSLRQL 18 843 HAQASVQQL 18 870 VAGFSLRQL 18 883 HAQASVQQL 18 916 IGDPGGVPL 18 953 SLVRLASGA 18 1112 TTLGSNREI 18 86 GSLPAFADL 17 117 RVVQRRLEV 17 152 AACLRAQGL 17 161 TRAFQVVHL 17 259 EEALGVGSL 17 316 RLSGLNSIM 17 330 EELVKLHSL 17 337 SLSHKVIQC 17 374 ETSTKISGL 17 385 EMGSGKSNV 17 403 AFMEPRYHV 17 427 KVPRKDLIV 17 459 NGNSEVVQL 17 460 GNSEVVQLL 17 493 QEDECVLML 17 529 LMAKALLLY 17 535 LLYGADIES 17 581 GRTALILAV 17 593 SASIVNLLL 17 620 YAVSSHHHV 17 648 NSNPVITIL 17 677 GLPENLTNG 17 813 KLLKTIQLN 17 914 SQIGDPGGV 17 939 HLPPREPRA 17 990 ILPVPTFSS 17 6 LLPTQATFA 16 62 ARKEFSTTL 16 75 ALSLSSSRA 16 110 AFLLGWERV 16 200 AASANLPGA 16 217 LRYRSGPSV 16 235 PAHQRLLFL 16 240 LLFLPRAPQ 16 270 FQLHLIQGI 16 280 NLSYPLVLR 16 307 GLELPATAA 16 318 SGLNSIMQI 16 338 LSHKVIQCV 16 500 MLLEHGADG 16 518 ALHYAIYNE 16 544 KNKCGLTPL 16 545 NKCGLTPLL 16 552 LLLGVHEQK 16 569 KKKANLNAL 16 591 CGSASIVNL 16 628 VICELLSDY 16 632 LLSDYKEKQ 16 694 GLIPQRKSR 16 806 EIESVKEKL 16 819 QLNEEALTK 16 827 KTKVAGFSL 16 911 ALRSQIGDP 16 923 PLSEGGTAA 16 985 DSSGWILPV 16 1050 LGQDDRAGV 16 1058 VLAPKCRPG 16 1089 TLPHRDTTT 16 1106 AGGVGPTTL 16 47 CNLEKGSWL 15 54 WLSFPGTAA 15 76 LSLSSSRAL 15 80 SSRALPGSL 15 82 RALPGSLPA 15 122 RLEVPRPQA 15 129 QAAPATSAT 15 168 HLAPTAPDG 15 264 VGSLSVFQL 15 293 ILKFSEKET 15 302 GGGILGLEL 15 306 LGLELRATA 15 345 CVFAKKKNV 15 370 LIMKETSTK 15 381 GLIQEMGSG 15 415 DLDKLHRPA 15 474 QLNVLDNKK 15 478 LDNKKRTAL 15 482 KRTALIKAV 15 531 AKALLLYGA 15 534 LLLYGADIE 15 559 QKQEVVKFL 15 600 LLEQNVDVS 15 611 DLSGQTARE 15 621 AVSSHHHVI 15 644 ISSENSNPV 15 728 QLSEEQNTG 15 753 AEKEMNSEL 15 755 KEMNSELSL 15 762 SLSHKKEED 15 799 RENKILEEI 15 834 SLRQLGLAQ 15 850 QLCYKWNHT 15 874 SLRQLGLAQ 15 957 LASGARAAA 15 958 ASGARAAAL 15 982 SVCDSSGWI 15 1027 QAFRDKDDL 15 1096 TTSLPHFHV 15 1104 VSAGGVGPT 15 1105 SAGGVGPTT 15 43 VLPCCNLEK 14 77 SLSSSRALP 14 112 LLGWERVVQ 14 155 LRAQGLTRA 14 162 RAFQVVHLA 14 232 AEPPAHQRL 14 256 QPSEEALGV 14 267 LSVFQLHLI 14 278 IPNLSYPLV 14 282 SYPLVLRHI 14 324 MQIKEFEEL 14 360 CLSEGYGHS 14 420 HRAAWWGKV 14 429 PRKDLIVML 14 452 ALHLASANG 14 470 DRRCQLNVL 14 501 LLEHGADGN 14 522 AIYNEDKLM 14 553 LLGVHEQKQ 14 577 LDRYGRTAL 14 578 DRYGRTALI 14 597 VNLLLEQNV 14 604 NVDVSSQDL 14 660 LPLKVEEEI 14 668 IKKHGSNPV 14 670 KHGSNPVGL 14 734 NTGISQDEI 14 771 LLRENSMLR 14 810 VKEKLLKTI 14 816 KTIQLNEEA 14 824 ALTKTKVAG 14 839 GLAQHAQAS 14 879 GLAQHAWAS 14 890 QLCYKWGHT 14 932 GDQGPGTHL 14 947 ASPGTPSLV 14 949 PGTPSLVRL 14 950 GTPSLVRLA 14 1088 TTLPHRDTT 14 1113 TLGSNREIT 14
[1035] 34 TABLE XXIV Pos 123456789 score V1A-HLA-A0202-9mers: 251P5G2 Noresultsfound. V2A-HLA-A0202-9mers: 251P5G2 Noresultsfound. V3A-HLA-A0202-9mers: 251P5G2 Noresultsfound. V4A-HLA-A0202-9mers: 251P5G2 Noresultsfound. V12A-HLA-A0202-9mers: 251P5G2 No results found. V12B-HLA-A0202-9mers: 251P5G2 Noresultsfound.
[1036] 35 TABLE XXV Pos 123456789 Score V1A-HLA-A0203-9mers: 251P5G2 Noresultsfound. V2A-HLA-A0203-9mers: 251P5G2 Noresultsfound. V3A-HLA-A0203-9mers: 251P5G2 Noresultsfound. V4A-HLA-A0203-9mers: 251P5G2 Noresultsfound. V12A-HLA-A0203-9mers: 251P5G2 Noresultsfound. V12B-HLA-A0203-9mers: 251P5G2 Noresultsfound.
[1037] 36 TABLE XXVI Pos 123456789 Score V1A-HLA-A3-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 81 RVIRVLSIC 24 174 SLFRDVFLK 24 183 QIMLFSSVY 24 39 PVCHVALIH 21 64 SLNFQNDFK 21 78 YLRRVIRVL 21 84 RVLSICTTC 21 44 ALIHMVVLL 20 48 MVVLLTMVF 20 147 TQINLHVSK 20 148 QINLHVSKY 20 172 TLSLFRDVF 20 224 ILLPVSFSV 20 105 SISWLVRFK 19 122 HLFSWSLSF 19 135 SLIFYTVAS 19 140 TVASSNVTQ 19 165 IIRGLFFTL 19 7 LVLASQPTL 18 30 DLRPERTYL 18 92 CLLGMLQVV 18 93 LLGMLQVVN 18 189 SVYMMTLIQ 18 202 ILVPSQPQP 18 214 DLCRGKSHQ 18 227 PVSFSVGMY 18 239 FIISTSSTL 18 8 VLASQPTLF 17 42 HVALIHMVV 17 49 VVLLTMVFL 17 76 SFYLRRVIR 17 152 HVSKYCSLF 17 158 SLFPINSII 17 164 SIIRGLFFT 17 168 GLFFTLSLF 17 205 PSQPQPLPK 17 222 QHILLPVSF 17 225 LLPVSFSVG 17 51 LLTMVFLSP 16 54 MVFLSPQLF 16 107 SWLVRFKWK 16 109 LVRFKWKST 16 127 SLSFPVSSS 16 131 PVSSSLIFY 16 163 NSIIRGLFF 16 213 KDLCRGKSH 16 231 SVGMYKMDF 16 25 FLLFLDLRP 15 29 LDLRPERTY 15 37 YLPVCHVAL 15 98 QVVNISPSI 15 99 VVNISPSIS 15 101 NISPSISWL 15 137 IFYTVASSN 15 198 ELQEILVPS 15 209 QPLPKDLCR 15 228 VSFSVGMYK 15 6 KLVLASQPT 14 14 TLFSFFSAS 14 28 FLDLRPERT 14 180 FLKQIMLFS 14 200 QEILVPSQP 14 223 HILLPVSFS 14 56 FLSPQLFES 13 60 QLFESLNFQ 13 70 DFKYEASFY 13 96 MLQVVNISP 13 103 SPSISWLVR 13 108 WLVRFKWKS 13 136 LIFYTVASS 13 145 NVTQINLHV 13 178 DVFLKQIML 13 194 TLIQELQEI 13 215 LCRGKSHQH 13 245 STLPWAYDR 13 3 FISKLVLAS 12 5 SKLVLASQP 12 10 ASQPTLFSF 12 45 LIHMVVLLT 12 50 VLLTMVFLS 12 80 RRVIRVLSI 12 85 VLSICTTCL 12 87 SICTTCLLG 12 113 KWKSTIFTF 12 211 LPKDLCRGK 12 26 LLFLDLRPE 11 43 VALIHMVVL 11 82 VIRVLSICT 11 91 TCLLGMLQV 11 104 PSISWLVRF 11 111 RFKWKSTIF 11 117 TIFTFHLFS 11 167 RGLFFTLSL 11 182 KQIMLFSSV 11 185 MLFSSVYMM 11 197 QELQEILVP 11 203 LVPSQPQPL 11 240 IISTSSTLP 11 V2A-HLA-A3-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 7 TLCSFFSAS 14 1 VLASQPTLC 13 3 ASQPTLCSF 12 4 SQPTLCSFF 8 9 CSFFSASSP 8 V3A-HLA-A3-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7, each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 5 RVIRDLSIC 23 2 YLRRVIRDL 17 4 RRVIRDLSI 12 6 VIRDLSICT 12 9 DLSICTTCL 12 8 RDLSICTTC 11 V4A-HLA-V3-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 7 LLDMLQVVN 18 6 CLLDMLQVV 17 1 SICTTCLLD 12 5 TCLLDMLQV 9 V12A-HLA-A3-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 9 LIMLFSSVY 22 8 WLIMLFSSV 18 5 SISWLIMLF 14 V12B-HLA-A3-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position each peptide is the start position plus eight. 819 QLNEEALTK 33 803 ILEEIESVK 30 154 CLRAQGLTR 29 410 HVRREDLDK 29 5 ILLPTQATF 28 552 LLLGVHEQK 28 341 KVIQCVFAK 27 370 LIMKETSTK 25 382 LIQEMGSGK 25 436 MLRDTDMNK 25 655 ILNIKLPLK 24 43 VLPCCNLEK 23 576 ALDRYGRTA 23 651 PVITILNIK 23 694 GLIPQRKSR 23 695 LIPQRKSRK 23 1010 DVSPAMRLK 23 342 VIQCVFAKK 22 474 QLNVLDNKK 22 528 KLMAKALLL 22 533 ALLLYGADI 22 631 ELLSDYKEK 22 332 LVKLHSLSH 21 563 VVKFLIKKK 21 661 PLKVEEEIK 21 834 SLRQLGLAQ 21 874 SLRQLGLAQ 21 956 RLASGARAA 21 1103 HVSAGGVGP 21 83 ALPGSLPAF 20 117 RVVQRRLEV 20 261 ALGVGSLSV 20 319 GLNSIMQIK 20 418 KLHRAAWWG 20 427 KVPRKDLIV 20 480 NKKRTALIK 20 562 EVVKFLIKK 20 588 AVCCGSASI 20 681 NLTNGASAG 20 786 RLELDETKH 20 953 SLVRLASGA 20 954 LVRLASGAR 20 964 AALPPPTGK 20 1001 FLGRRCPMF 20 56 SFPGTAARK 19 112 LLGWERVVQ 19 118 VVQRRLEVP 19 165 QVVHLAPTA 19 216 ALRYRSGPS 19 316 RLSGLNSIM 19 326 IKEFEELVK 19 457 LLLDRRCQL 19 500 MLLEHGADG 19 770 DLLRENSML 19 771 LLRENSMLR 19 829 KVAGFSLRQ 19 915 QIGDPGGVP 19 31 RADPVTWRK 18 42 AVLPCCNLE 18 111 FLLGWERVV 18 122 RLEVPRPQA 18 239 RLLFLPRAP 18 249 AVSGPQEQP 18 280 NLSYPLVLR 18 291 PEILKFSEK 18 309 ELPATAARL 18 557 HEQKQEVVK 18 618 REYAVSSHH 18 627 HVICELLSD 18 628 VICELLSDY 18 663 KVEEEIKKH 18 824 ALTKTKVAG 18 923 PLSEGGTAA 18 971 GKNGRSPTK 18 1062 KCRPGTLCH 18 18 GLWAALTTV 17 75 ALSLSSSRA 17 124 EVPRPQAAP 17 159 GLTRAFQVV 17 160 LTRAFQVVH 17 168 HLAPTAPDG 17 188 RLTHVRCAQ 17 203 ANLPGAPGR 17 218 RYRSGPSVS 17 288 RHIPEILKF 17 369 FLIMDETST 17 452 ALHLASANG 17 463 EVVQLLLDR 17 534 LLLYGADIE 17 566 FLIKKKANL 17 584 ALILAVCCG 17 598 NLLLEQNVD 17 599 LLLEQNVDV 17 621 AVSSHHHVI 17 728 QLSEEQNTG 17 740 DEILTNKQK 17 742 ILTNKQKQI 17 776 SMLREEIAK 17 777 MLREEIAKL 17 784 KLRLELDET 17 785 LRLELDETK 17 809 SVKEKLLKT 17 911 ALRSQIGDP 17 965 ALPPPTGKN 17 1054 DRAGVLAPK 17 1089 TLPHRDTTT 17 1098 SLPHFHVSA 17 87 SLPAFADLP 16 211 RSSSCALRY 16 307 GLELPATAA 16 347 FAKKKNVDK 16 381 GLIQEMGSG 16 423 AWWGKVPRK 16 434 IVMLRDTDM 16 466 QLLLDRRCQ 16 477 VLDNKKRTA 16 485 ALIKAVQCQ 16 522 AIYNEKDLM 16 535 LLYGADIES 16 548 GLTPLLLGV 16 561 QEVVKFLIK 16 586 ILAVCCGSA 16 596 IVNLLLEQN 16 747 QKQIEVAEK 16 797 QLRENKILE 16 802 KILEEIESV 16 821 NEEALTKTK 16 869 EVAGFSLRQ 16 973 NGRSPTKQK 16 989 WILPVPTFS 16 1108 GVGPTTLGS 16
[1038] 37 TABLE XXVII Pos 123456789 score V1A-HLA-A26-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 178 DVFLKQIML 28 101 NISPSISWL 26 116 STIFTFHLF 26 131 PVSSSLIFY 26 148 QINLHVSKY 26 227 PVSFSVGMY 26 165 IIRGLFFTL 25 168 GLFFTLSLF 25 185 MLFSSVYMM 25 30 DLRPERTYL 24 54 MVFLSPQLF 24 70 DFKYEASFY 24 89 CTTCLLGML 24 161 PINSIIRGL 24 44 ALIHMVVLL 23 49 VVLLTMVFL 23 179 VFLKQIMLF 23 48 MVVLLTMVF 22 122 HLFSWSLSF 22 152 HVSKYCSLF 22 203 LVPSQPQPL 22 239 FIISTSSTL 22 8 VLASQPTLF 21 183 QIMLFSSVY 21 195 LIQELQEIL 21 198 ELQEILVPS 21 231 SVGMYKMDF 21 7 LVLASQPTL 20 17 SFFSASSPF 20 78 YLRRVIRVL 20 37 YLPVCHVAL 19 104 PSISWLVRF 19 229 SFSVGMYKM 19 111 RFKWKSTIF 18 119 FTFHLFSWS 18 136 LIFYTVASS 18 172 TLSLFRDVF 18 3 FISKLVLAS 17 10 ASQPTLFSF 17 11 SWPTLFSFF 17 14 TLFSFFSAS 17 60 QLFESLNFQ 17 81 RVIRVLSIC 17 194 TLIQELEQI 17 201 EILVPSQPQ 17 238 DFIISTSST 17 13 PTLFSFFSA 16 18 FFSASSPFL 16 56 FLSPQLFES 16 57 LSPQLFESL 16 63 ESLNFQNDF 16 85 VLSICTTCL 16 113 KWKSTIFTF 16 164 SIIRGLFFT 16 171 FTLSLFRDV 16 214 DLCRGKSHQ 16 242 STSSTLPWA 16 2 PFISKLVLA 15 20 SASSPFLLF 15 23 SPFLLFLDL 15 35 TRYLPVCHV 15 52 LTMVFLSPQ 15 69 NDFKYEASF 15 92 CLLGMLQVV 15 105 SISWLVRFK 15 120 TFHLFSWSL 15 151 LHVSKYCSL 15 191 YMMTLIQEL 15 210 PLPKDLCRG 15 222 QHILLPVSF 15 225 LLPVSFSVG 15 21 ASSPFLLFL 14 26 LLFLDLRPE 14 45 LIHMVVLLT 14 65 LNFQNDFKY 14 127 SLSFPVSSS 14 128 LSFPVSSSL 14 140 TVASSNVTQ 14 146 VTQINLHVS 14 174 SLFRDVFLK 14 180 FLKQIMLFS 14 246 TLPWAYDRG 14 40 VCHVALIHM 13 43 VALIHMVVL 13 51 LLTMVFLSP 13 88 ICTTCLLGM 13 158 SLFPINSII 13 186 LFSSVYMMT 13 187 FSSVYMMTL 13 202 ILVPSQPQP 13 V2A-HLA-A26-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 3 ASQPTLCSF 17 6 PTLCSFFSA 16 7 TLCSFFSAS 15 4 SQPTLCSFF 13 1 VLASQPTLC 11 V3A-HLA-A269mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 9 DLSICTTCL 22 2 YLRRVIRDL 20 5 RVIRDLSIC 17 6 VIRDLSICT 12 V12A-HLA-A26-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 5 SISWLIMLF 26 9 LIMLFSSVY 21 8 WLIMLFSSV 17 4 PSISWLIML 16 2 ISPSISWLI 2 V12B-HLA-A26-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 1094 DTTTSLPHF 33 300 ETGGGILGL 31 374 ETSTKISGL 31 83 ALPGSLPAF 28 628 VICELLSDY 28 781 EIAKLRLEL 28 825 LTKTKVAGF 28 378 KISGLIQEM 27 516 NTALHYAIY 26 806 EIESVKEKL 26 273 HLIQCIPNL 25 309 ELPATAARL 25 312 ATAARLSGL 25 558 EQKQEVVKF 24 724 DTQKQLSEE 24 770 DLLRENSML 24 777 MLREEIAKL 24 994 PTFSSGSFL 23 463 EVVQLLLDR 22 562 EVVKFLIKK 22 566 FLIKKKANL 22 751 EVAEKEMNS 22 1010 DVSPAMRLK 22 5 ILLPTQATF 21 11 ATFAAATGL 21 604 NVDVSSQDL 21 1024 ETHQAFRDK 21 68 TTLTGHSAL 20 70 LTGHSALSL 20 259 EEALGVGSL 20 277 CIPNLSYPL 20 316 RLSGLNSIM 20 330 EELVKLHSL 20 357 DDFCLSEGY 20 432 DLIVMLRDT 20 631 ELLSDYKEK 20 652 VITILNIKL 20 667 EIKKHGSNP 20 827 KTKVAGFSL 20 869 EVAGFSLRQ 20 1001 FLGRRCPMF 20 1072 DTPPHRNAD 20 105 ATPAGAFLL 19 124 EVPRPQAAP 19 288 RHIPEILKF 19 328 EFEELVKLH 19 401 DSAFMEPRY 19 470 DRRCQLNVL 19 494 EDECVLMLL 19 606 DVSSQDLSG 19 611 DLSGQTARE 19 749 QIEVAEKEM 19 791 ETKHQNQLR 19 809 SVKEKLLKT 19 817 TIQLNEEAL 19 1080 DTPPHRHTT 19 93 DLPRSCPES 18 197 GLEAASANL 18 262 LGVGSLSVF 18 292 EILKFSEKE 18 321 NSIMQIKEF 18 327 KEFEELVKL 18 341 LVIQCVFAK 18 415 DLDKLHRAA 18 434 IVMLRDTDM 18 467 LLLDRRCQL 18 522 AIYNEDKLM 18 540 DIESKNKCG 18 654 TILNIKLPL 18 741 EILTNKQKQ 18 788 ELDETKHQN 18 65 EFSTTLTGH 17 266 SLSVFQLHL 17 289 HIPEILKFS 17 331 ELVKLHSLS 17 429 PRKDLIVML 17 439 DTDMNKRDK 17 450 RTALHLASA 17 513 EYGNTALHY 17 528 KLMAKALLL 17 563 VVKFLIKKK 17 627 HVICELLSD 17 663 KVEEEIKKH 17 711 DTENEEYHS 17 802 KILEEIESV 17 865 AQEQEVAGF 17 949 PGTPSLVRL 17 950 GTPSLVRLA 17 988 GWILPVPTF 17 1030 RDKDDLPFF 17 8 PTQATFAAA 16 161 TRAFQVVHL 16 285 LVLRHIPEI 16 286 VLRHIPEIL 16 324 MQIKEFEEL 16 342 VIQCVFAKK 16 381 GLIQEMGSG 16 394 GTWGDYDDS 16 464 VVQLLLDRR 16 485 ALIKAVQCQ 16 493 QEDECVLML 16 497 CVLMLLEHG 16 509 NIQDEYGNT 16 526 EDKLMAKAL 16 529 LMAKALLLY 16 548 GLTPLLLGV 16 549 LTPLLLGVH 16 582 RTALILAVC 16 595 SIVNLLLEQ 16 596 IVNLLLEQN 16 615 QTAREYAVS 16 651 PVITILNIK 16 653 ITILNIKLP 16 677 GLPENLTNG 16 721 EQNDTQKQL 16 760 ELSLSHKKE 16 790 DETKHQNQL 16 805 EEIESVKEK 16 812 EKLLKTIQL 16 1000 SFLGRRCPM 16 1034 DLPFFKTQQ 16 1049 DLGQDDRAG 16
[1039] 38 TABLE XXVIII Pos 123456789 score V1A-HLA-B0702-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 1 MPFISKLVL 23 23 SPFLLFLDL 23 207 QPQPLPKDL 21 32 RPERTYLPV 20 21 ASSPFLLFL 18 38 LPVCHVALI 18 130 FPVSSSLIF 18 226 LPVSFSVGM 18 247 LPWAYDRGV 17 165 IIRGLFFTL 16 204 VPSQPQPLP 16 18 FFSASSPFL 15 30 DLRPERTYL 15 44 ALIHMVVLL 15 103 SPSISWLVR 15 20 SASSPFLLF 14 85 VLSICTTCL 14 167 RGLFFTLSL 14 37 YLPVCHVAL 13 43 VALIHMVVL 13 49 VVLLTMVFL 13 78 YLRRVIRVL 13 101 NISPSISWL 13 173 LSLFRDVFL 13 209 QPLPKDLCR 13 7 LVLASQPTL 12 115 KSTIFTFHL 12 187 FSSVYMMTL 12 218 GKSHQHILL 12 12 QPTLFSFFS 11 19 FSASSPFLL 11 35 RTYLPVCHV 11 53 TMVFLSPQL 11 57 LSPQLFESL 11 86 LSICTTCLL 11 128 LSFPVSSSL 11 191 YMMTLIQEL 11 203 LVPSQPQPL 11 217 RGKSHQHIL 11 V2A-HLA-B0702-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 5 QPTLCSFFS 11 3 ASQPTLCSF 9 2 LASQPTLCS 8 4 SQPTLCSFF 7 6 PTLCSFFSA 7 V3A-HLA-B0702-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 9 DLSICTTCL 14 2 YLRRVIRDL 12 4 RRVIRDLSI 9 6 VIRDLSICT 8 7 IRDLSICTT 8 V4A-B0702-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 3 CTTCLLDML 10 8 LDMLQVVNI 10 2 ICTTCLLDM 9 5 TCLLDMLQV 8 6 CLLDMLQVV 7 7 LLDMLQVVN 4 V12A-B0702-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 3 SPSISWLIM 20 4 PSISWLIML 10 V12B-B0702-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 228 APSPAEPPA 23 170 APTAPDGGA 22 181 PPSRNSYRL 22 233 EPPAHQRLL 22 674 NPVGLPENL 22 1060 APKCRPGTL 22 1082 PPHRHTTTL 22 125 VPRPQAAPA 21 28 NPSRADPVTI 20 234 PPAHQRLLFJ 20 256 QPSEEALGV 20 428 VPRKDLIVM 20 84 LPGSLPAFA 19 208 APGRSSSCA 19 1006 CPMFDVSPA 19 7 LPTQATFAA 18 243 LPRAPQAVS 18 278 IPNLSYPLV 18 650 NPVITILNI 18 98 CPESEQSAT 17 144 PPCHQRRDA 17 660 LPLKVEEEI 17 678 LPENLTNGA 17 916 IGDPGGVPL 17 922 VPLSEGGTA 17 932 GDQGPGTHL 17 941 PPREPRASP 17 948 SPGTPSLVR 17 1073 TPPHRNADT 17 1081 TPPHRHTTT 17 15 AATGLWAAL 16 106 TPAGAFLLG 16 141 DPSPPCHQR 16 205 LPGAPGRSS 16 300 ETGGGILGL 16 781 EIAKLRLEL 16 946 RASPGTPSL 16 951 ASGARAAAL 16 958 ASGARAAAL 16 993 VPTFSSGSF 16 36 TWRKEPAVL 15 131 APATSATPS 15 173 APDGGAGCP 15 230 SPAEPPAHQ 15 528 KLMAKALLL 15 577 LDRYGRTAL 15 591 CGSASIVNL 15 918 DPGGVPLSE 15 11 ATFAAATGL 14 54 WLSFPGTAA 14 88 LPAFADLPR 14 104 SATPAGAFL 14 161 TRAFQVVHL 14 222 GSPVSSAPS 14 246 APQAVSGPQ 14 266 SLSVFQLHL 14 302 GGGILGLEL 14 312 ATAARLSGL 14 406 EPRYHVRRE 14 425 WGKVPRKDL 14 445 RDKQKRTAL 14 478 LDNKKRTAL 14 493 QEDECVLML 14 541 IESKNKCGL 14 545 NKCGLTPLL 14 546 KCGLTPLLL 14 579 RYGRTALIL 14 593 SASIVNLLL 14 670 KHGSNPVGL 14 755 KEMNSELSL 14 832 GFSLRQLGL 14 872 GFSLRQLGL 14 944 EPRASPGTP 14 1091 PHRDTTTSL 14 1106 AGGVGPTTL 14 41 PAVLPCCNL 13 62 ARKEFSTTL 13 70 LTGHSALSL 13 80 SSRALPGSL 13 86 GSLPAFADL 13 105 ATPAGAFLL 13 127 RPQAAPATS 13 137 TPSRDPSPP 13 147 HQRRDAACL 13 209 PGRSSSCAL 13 232 AEPPAHQRL 13 235 PAHQRLLFL 13 259 EEALGVGSL 13 264 VGSLSVFQL 13 279 PNLSYPLVL 13 309 ELPATAARL 13 327 KEFEELVKL 13 374 ETSTKISGL 13 403 AFMEPRYHV 13 447 KQKRTALHL 13 459 NGNSEVVQL 13 460 GNSEVVQLL 13 470 DRRCQLNVL 13 544 KNKCGLTPL 13 550 TPLLLGVHE 13 569 KKKANLNAL 13 647 ENSNPVITI 13 654 TILNIKLPL 13 777 MLREEIAKL 13 779 REEIAKLRL 13 807 IESVKEKLL 13 904 QAQEQGAAL 13 935 GPGTHLPPR 13 949 PGTPSLVRL 13 957 LASGARAAA 13 985 DSSGWILPV 13 1009 FDVSPAMRL 13 40 EPAVLPCCN 12 61 AARKEFSTT 12 83 ALPGSLPAF 12 94 LPRSCPESE 12 115 WERVVQRRL 12 122 RLEVPRPQA 12 128 PQAAPATSA 12 152 AACLRAQGL 12 197 GLEAASANL 12 200 AASANLPGA 12 254 QEQPSEEAL 12 286 VLRHIPEIL 12 298 EKETGGGIL 12 324 MQIKEFEEL 12 362 SEGYGHSFL 12 408 RYHVRREDL 12 411 VRREDLDKL 12 429 PRKDLIVML 12 461 NSEVVQLLL 12 511 QDEYGNTAL 12 526 EDKLMAKAL 12 559 QKQEVVKFL 12 566 FLIKKKANL 12 592 GSASIVNLL 12 648 NSNPVITIL 12 703 KPENQQFPD 12 753 AEKEMNSEL 12 812 EKLLKTIQL 12 827 KTKVAGFSL 12 843 HAQASVQQL 12 867 EQEVAGFSL 12 883 HAQASVQQL 12 966 LPPPTGKNG 12 968 PPTGKNGRS 12 976 SPTKQKSVC 12 983 VCDSSGWIL 12 991 LPVPTFSSG 12 994 PTFSSGSFL 12 1032 KDDLPFFKT 12 1043 SPRHTKDLG 12 1051 GQDDRAGVL 12 1064 RPGTLCHTD 12 1079 ADTPPHRHT 12 1090 LPHRDTTTS 12 1099 LPHFHVSAG 12 4 HILLPTQAT 11 13 FAAATGLWA 11 33 DPVTWRKEP 11 47 CNLEKGSWL 11 57 FPGTAARKE 11 68 TTLTGHSAL 11 76 LSLSSSRAL 11 82 RALPGSLPA 11 102 EQSATPAGA 11 103 QSATPAGAF 11 149 RRDAACLRA 11 156 RAQOLTRAF 11 261 ALGVGSLSV 11 273 HLIQCIPNL 11 277 CIPNLSYPL 11 283 YPLVLRHIP 11 290 IPEILKFSE 11 304 GILGLELPA 11 307 GLELPATAA 11 310 LPATAARLS 11 316 RLSGLNSIM 11 330 EELVKLHSL 11 378 KISGLIQEM 11 467 LLLDRRCQL 11 491 QCQEDECVL 11 494 EDEGVLMLL 11 527 DKLMAKALL 11 604 NVDVSSQDL 11 621 AVSSHHHVI 11 634 SDYKEKQML 11 687 SAGNGDDGL 11 696 IPQRKSRKP 11 709 FPDTENEEY 11 721 EQNDTQKQL 11 763 LSHKKEEDL 11 764 SHKKEEDLL 11 790 DETKHQNQL 11 806 EIESVKEKL 11 817 TIQLNEEAL 11 830 VAGFSLRQL 11 870 VAGFSLRQL 11 923 PLSEGGTAA 11 930 AAGDQGPGT 11 940 LPPREPRAS 11 962 RAAALPPPT 11 988 GWILPVPTF 11 1003 GRRCPMFDV 11 1012 SPAMRLKSD 11 1029 FRDKDDLPF 11 1035 LPFFKTQQS 11 1042 QSPRHTKDL 11 1096 TTSLPHFHV 11 1104 VSAGGVGPT 11 1105 SAGGVGPIT 11 1110 GPTTLGSNR 11
[1040] 39 TABLE XXIX Pos 123456789 score V1A-HLA-B08-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 111 RFKWKSTIF 26 78 YLRRVIRVL 23 30 DLRPERTYL 22 165 IIRGLFFTL 21 178 DVFLKQIML 21 151 LHVSKYCSL 20 217 RGKSHQHIL 20 23 SPFLLFLDL 19 37 YLPVCHVAL 19 173 LSLFRDVFL 19 44 ALIHMVVLL 18 232 VGMYKMDFI 18 85 VLSICTTCL 17 109 LVRFKWKST 17 113 KWKSTIFTF 17 180 FLKQIMLFS 17 207 QPQPLPKDL 17 1 MPFISKLVL 16 28 FLDLRPERT 16 43 VALIHMVVL 16 195 LIQELQEIL 16 209 QPLPKDLCR 16 211 LPKDLCRGK 16 215 LCRGKSHQH 16 80 RRVIRVLSI 15 101 NISPSISWL 15 158 SLFPINSII 15 161 PINSIIRGL 15 239 FIISTSSTL 15 163 NSIIRGLFF 14 8 VLASQPTLF 13 38 LPVCHVALI 13 49 VVLLTMVFL 13 71 FKYEASFYL 13 122 HLFSWSLSF 13 130 FPVSSSLIF 13 143 SSNVTQINL 13 168 GLFFTLSLF 13 172 TLSLFRDVF 13 194 TLIQELQEI 13 4 ISKLVLASQ 12 20 SASSPFLLF 12 76 SFYLRRVIR 12 82 VIRVLSICT 12 107 SWLVRFKWK 12 141 VASSNVTQI 12 153 VSDYCSLFP 12 187 FSSVYMMTL 12 191 YMMTLIQEL 12 V2A-HLA-B08-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 4 SQPTLCSFF 8 1 VLASQPTLC 7 5 QPTLCSFFS 7 7 TLCSFFSAS 7 3 ASQPTLCSF 6 2 LASQPTLCS 4 V3A-HLA-B08-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 2 YLRRVIRDL 23 9 DLSICTTCL 16 4 RRVIRDLSI 14 6 VIRDLSICT 11 V4A-HLA-B08-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 3 CTTCLLDML 10 8 LDMLQVVNI 9 7 LLDMLQVVN 7 1 SICTTCLLD 6 6 CLLDMLQVV 6 V12A-HLA-B08-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 5 SISWLIMLF 13 4 PSISWLIML 10 3 SPSISWLIM 8 2 ISPSISWLI 7 8 WLIMLFSSV 6 V12B-HLA-B08-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 1060 APKCRPGTL 34 445 RDKQKRTAL 31 347 FAKKKNVDK 27 566 FLIKKKANL 27 825 LTKTKVAGF 27 528 KLMAKALLL 26 526 EDKLMAKAL 25 777 MLREEIAKL 25 467 LLLDRRCQL 24 479 DNKKRTALI 24 567 LIKKKANLN 24 742 ILTNKQKQI 24 753 AEKEMNSEL 24 782 IAKLRLELD 24 809 SVKEKLLKT 24 152 AACLRAQGL 23 286 VLRHIPEIL 23 330 EELVKLHSL 23 374 ETSTKISGL 23 807 IESVKEKLL 23 812 EKLLKTIQL 23 47 CNLEKGSWL 22 62 ARKEFSTTL 22 235 PAHQRLLFL 22 425 WGKVPRKDL 22 429 PRKDLIVML 22 542 ESKNKCGLT 22 635 DYKEKQMLK 22 764 SHKKEEDLL 22 216 ALRYRSGPS 21 353 VDKWDDFCL 21 478 LDNKKRTAL 21 569 KKKANLNAL 21 744 TNKQKQIEV 21 827 KTKVAGFSL 21 977 PTKQKSVCD 21 1001 FLGRRCPMF 21 297 SEKETGGGI 20 447 KQKRTALHL 20 544 KNKCGLTPL 20 558 EQKQEVVKF 20 763 LSHKKEEDL 20 832 GFSLRQLGL 20 872 GFSLRQLGL 20 266 SLSVFQLHL 19 293 ILKFSEKET 19 337 SLSHKVIQC 19 339 SHKVIQCVF 19 371 IMKETSTKI 19 408 RYHVRREDL 19 411 VRREDLDKL 19 659 KLPLKVEEE 19 698 QRKSRKPEN 19 701 SRKPENQQF 19 762 SLSHKKEED 19 958 ASGARAAAL 19 1051 GQDDRAGVL 19 80 SSRALPGSL 18 233 EPPAHQRLL 18 273 HLIQCIPNL 18 309 ELPATAARL 18 312 ATAARLSGL 18 577 LDRYGRTAL 18 115 WERVVQRRL 17 147 HQRRDAACL 17 197 GLEAASANL 17 209 PGRSSSCAL 17 369 FLIMKETST 17 443 NKRDKQKRT 17 477 VLDNKKRTA 17 593 SASIVNLLL 17 655 ILNIKLPLK 17 661 PLKVEEEIK 17 696 IPQRKSRKP 17 770 DLLRENSML 17 781 EIAKLRLEL 17 823 EALTKTKVA 17 850 QLCYKWNHT 17 890 QLCYKWGHT 17 904 QAQEQGAAL 17 968 PPTGKNGRS 17 968 PPTGKNGRS 17 1012 SPAMRLKSD 17 1014 AMRLKSDSN 17 1021 SNRETHQAF 17 1058 VLAPKCRPG 17 36 TWRKEPAVL 16 49 LEKGSWLSF 16 104 SATPAGAFL 16 181 PPSRNSYRL 16 325 QIKEFEELV 16 351 KNVDKWDDF 16 406 EPRYHVRRE 16 470 DRRCQLNVL 16 540 DIESKNKCG 16 641 MLKISSENS 16 652 VITILNIKL 16 657 NIKLPLKVE 16 667 EIKKHGSNP 16 674 NPVGLPENL 16 687 SAGNGDDGL 16 806 EIESVKEKL 16 814 LLKTIQLNE 16 817 TIQLNEEAL 16 843 HAQASVQQL 16 883 HAQASVQQL 16 1016 RLKSDSNRE 16 1028 AFRDKDDLP 16 1030 RDKDDLPFF 16 1035 LPFFKTQQS 16 1082 PPHRHTTTL 16 1091 PHRDTTTSL 16
[1041] 40 TABLE XXX Pos 123456789 score V1A-HLA-B1510-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 46 IHMVVLLTM 20 151 LHVSKYCSL 20 222 QHILLPVSF 19 78 YLRRVIRVL 16 37 YLPVCHVAL 15 43 VALIHMVVL 15 101 NISPSISWL 14 165 IIRGLFFTL 14 187 FSSVYMMTL 14 1 MPFISKLVL 13 21 ASSPFLLFL 13 30 DLRPERTYL 13 44 ALIHMVVLL 13 49 VVLLTMVFL 13 161 PINSIIRGL 13 191 YMMTLIQEL 13 207 QPQPLPKDL 13 7 LVLASQPTL 12 18 FFSASSPFL 12 19 FSASSPFLL 12 53 TMVFLSPQL 12 128 LSFPVSSSL 12 173 LSLFRDVFL 12 218 GKSHQHILL 12 41 CHVALIHMV 11 57 LSPQLFESL 11 71 FKYEASFYL 11 85 VLSICTTCL 11 120 TFHLFSWSL 11 143 SSNVTQINL 11 172 TLSLFRDVF 11 195 LIQELQEIL 11 203 LVPSQPQPL 11 239 FIISTSSTL 11 23 SPFLLFLDL 10 86 LSICTTCLL 10 89 CTTCLLGML 10 104 PSISWLVRF 10 115 KSTIFTFHL 10 121 FHLFSWSLS 10 162 INSIIRGLF 10 167 RGLFFTLSL 10 178 DVFLKQIML 10 184 IMLFSSVYM 10 217 RGKSHQHIL 10 220 SHQHILLPV 10 229 SFSVGMYKM 10 V2A-B1510-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 3 ASQPTLCSF 8 4 SQPTLCSFF 6 V3A-HLA-B1510-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 2 YLRRVIRDL 14 9 DLSICTTCL 11 V4A-HLA-B1510-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 3 CTTCLLDML 10 2 ICTTCLLDM 8 7 LLDMLQVVN 4 V12A-HLA-B1510-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 1 ISPSISWL 16 4 PSISWLIML 10 5 SISWLIMLF 8 3 SPSISWLIM 7 V12B-HLA-B1510-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 670 KHGSNPVGL 24 624 SHHHVICEL 22 190 THVRCAQGL 21 625 HHHVICELL 21 764 SHKKEEDLL 21 1091 PHRDTTTSL 21 288 RHIPEILKF 19 339 SHKVIQCVF 19 916 IGDPGGVPL 17 541 IESKNKCGL 16 949 PGTPSLVRL 16 36 TWRKEPAVL 15 115 WERVVQRRL 15 161 TRAFQVVHL 15 781 EIAKLRLEL 15 938 THLPPREPR 15 946 RASPGTPSL 15 181 PPSRNSYRL 14 232 AEPPAHQRL 14 259 EEALGVGSL 14 300 ETGGGILGL 14 429 PRKDLIVML 14 460 GNSEVVQLL 14 556 VHEQKQEVV 14 591 CGSASIVNL 14 779 REEIAKLRL 14 807 IESVKEKLL 14 842 QHAQASVQQ 14 882 QHAQASVQQ 14 1051 GQDDRAGVL 14 1106 AGGVGPTTL 14 68 TTLTGHSAL 13 76 LSLSSSRAL 13 86 GSLPAFADL 13 233 EPPAHQRLL 13 254 QEQPSEEAL 13 273 HLIQCIPNL 13 279 PNLSYPLVL 13 298 EKETGGGIL 13 309 ELPATAARL 13 327 KEFEELVKL 13 335 LHSLSHKVI 13 366 GHSFLIMKE 13 374 ETSTKISGL 13 425 WGKVPRKDL 13 445 RDKQKRTAL 13 459 NGNSEVVQL 13 478 LDNKKRTAL 13 491 QCQEDECVL 13 511 QDEYGNTAL 13 577 LDRYGRTAL 13 592 GSASIVNLL 13 648 NSNPVITIL 13 717 YHSDEQNDT 13 793 KHQNQLREN 13 806 EIESVKEKL 13 817 TIQLNEEAL 13 867 EQEVAGFSL 13 932 GDQGPGTHL 13 1009 FDVSPAMRL 13 1069 CHTDTPPHR 13 15 AATGLWAAL 12 62 ARKEFSTTL 12 72 GHSALSLSS 12 104 SATPAGAFL 12 197 GLEAASANL 12 264 VGSLSVFQL 12 266 SLSVFQLHL 12 302 GGGILGLEL 12 324 MQIKEFEEL 12 330 EELVKLHSL 12 408 RYHVRREDL 12 461 NSEVVQLLL 12 467 LLLDRRCQL 12 470 DRRCQLNVL 12 493 QEDECVLML 12 494 EDECVLMLL 12 521 YAIYNEDKL 12 526 EDKLMAKAL 12 545 NKCGLTPLL 12 559 QKQEVVKFL 12 566 FLIKKKANL 12 569 KKKANLNAL 12 634 SDYKEKQML 12 654 TILNIKLPL 12 674 NPVGLPENL 12 721 EQNDTQKQL 12 753 AEKEMNSEL 12 777 MLREEIAKL 12 830 VAGFSLRQL 12 832 GFSLRQLGL 12 870 VAGFSLRQL 12 872 GFSLRQLGL 12 896 GHTEKTEQQ 12 904 QAQEQGAAL 12 1025 THQAFRDKD 12 1060 APKCRPGTL 12 1102 FHVSAGGVG 12 3 QHILLPTQA 11 5 ILLPTQATF 11 41 PAVLPCCNL 11 47 CNLEKGSWL 11 80 SSRALPGSL 11 105 ATPAGAFLL 11 146 CHQRRDAAC 11 167 VHLAPTAPD 11 209 PGRSSSCAL 11 235 PAHQRLLFL 11 286 VLRHIPEIL 11 312 ATAARLSGL 11 362 SEGYGHSFL 11 409 YHVRREDLD 11 411 VRREDLDKL 11 503 EHGADGNIQ 11 519 LHYAIYNED 11 527 DKLMAKALL 11 544 KNKCGLTPL 11 546 KCGLTPLLL 11 558 EQKQEVVKF 11 593 SASIVNLLL 11 604 NVDVSSQDL 11 687 SAGNGDDGL 11 735 TGISQDEIL 11 763 LSHKKEEDL 11 790 DETKHQNQL 11 812 EKLLKTIQL 11 827 KTKVAGFSL 11 843 HAQASVQQL 11 856 NHTEKTEQQ 11 883 HAQASVQQL 11 958 ASGARAAAL 11 988 GWILPVPTF 11 1027 QAFRDKDDL 11 1045 RHTKDLGQD 11 1082 PPHRHTTTL 11 1085 RHTTTLPHR 11
[1042] 41 TABLE XXXI Pos 123456789 score V1A-HLA-B2705-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 80 RRVIRVLSI 24 110 VRFKWKSTI 24 34 ERTYLPVCH 20 216 CRGKSHQHI 19 83 IRVLSICTT 18 111 RFKWKSTIF 18 128 LSFPVSSSL 18 167 RGLFFTLSL 18 168 GLFFTLSLF 18 176 FRDVFLKQI 18 178 DVFLKQIML 18 179 VFLKQIMLF 18 1 MPFISKLVL 17 7 LVLASQPTL 17 59 PQLFESLNF 17 69 NDFKYEASF 17 23 SPFLLFLDL 16 65 LNFQNDFKY 16 101 NISPSISWL 16 104 PSISWLVRF 16 113 KWKSTIFTF 16 122 HLFSWSLSF 16 169 LFFTLSLFR 16 209 QPLPKDLCR 16 217 RGKSHQHIL 16 222 QHILLPVSF 16 17 SFFSASSPF 15 27 LFLDLRPER 15 44 ALIHMVVLL 15 48 MVVLLTMVF 15 53 TMVFLSPQL 15 54 MVFLSPQLF 15 63 ESLNFQNDF 15 78 YLRRVIRVL 15 143 SSNVTQINL 15 147 TQINLHVSK 15 165 IIRGLFFTL 15 177 RDVFLKQIM 15 191 YMMTLIQEL 15 228 VSFSVGMYK 15 239 FIISTSSTL 15 245 STLPWAYDR 15 10 ASQPTLFSF 14 21 ASSPFLLFL 14 24 PFLLFLDLR 14 30 DLRPERTYL 14 43 VALIHMVVL 14 49 VVLLTMVFL 14 57 LSPQLFESL 14 71 FKYEASFYL 14 72 KYEASFYLR 14 76 SFYLRRVIR 14 120 TFHLFSWSL 14 130 FPVSSSLIF 14 157 CSLFPINSI 14 161 PINSIIRGL 14 166 IRGLFFTLS 14 173 LSLFRDVFL 14 174 SLFRDVFLK 14 184 IMLFSSVYM 14 185 MLFSSVYMM 14 195 LIQELQEIL 14 218 GKSHQHILL 14 233 GMYKMDFII 14 18 FFSASSPFL 13 46 IHMVVLLTM 13 73 YEASFYLRR 13 103 SPSISWLVR 13 144 SNVTQINLH 13 148 QINLHVSKY 13 151 LHVSKYCSL 13 152 HVSKYCSLF 13 158 SLFPINSII 13 163 NSIIRGLFF 13 194 TLIQELQEI 13 213 KDLCRGKSH 13 215 LCRGKSHQH 13 229 SFSVGMYKM 13 8 VLASQPTLF 12 11 SQPTLFSFF 12 20 SASSPFLLF 12 29 LDLRPERTY 12 31 LRPERTYLP 12 75 ASFYLRRVI 12 79 LRRVIRVLS 12 85 VLSICTTCL 12 86 LSICTTCLL 12 89 CTTCLLGML 12 105 SISWLVRFK 12 107 SWLVRFKWK 12 115 KSTIFTFHL 12 116 STIFTFHLF 12 159 LFPINSIIR 12 162 INSIIRGLF 12 172 TLSLFRDVF 12 183 QIMLFSSVY 12 187 FSSVYMMTL 12 205 PSQPQPLPK 12 207 QPQPLPKDL 12 231 SVGMYKMDF 12 19 FSASSPFLL 11 37 YLPVCHVAL 11 39 PVCHVALIH 11 64 SLNFQNDFK 11 70 DFKYEASFY 11 88 ICTTCLLGM 11 94 LGMLQVVNI 11 98 QVVNISPSI 11 114 WKSTIFTFH 11 131 PVSSSLIFY 11 211 LPKDLCRGK 11 226 LPVSFSVGM 11 V2A-B2705-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 3 ASQPTLCSF 14 4 SQPTLCSFF 12 9 CSFFSASSP 7 V3A-HLA-B2705-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 4 RRVIRDLSI 24 7 IRDLSICTT 17 2 YLRRVIRDL 14 9 DLSICTTCL 12 3 LRRVIRDLS 11 V4A-HLA-B2705-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 3 CTTCLLDML 12 2 ICTTCLLDM 11 8 LDMLQVVNI 11 9 DMLQVVNIS 7 5 TCLLDMLQV 5 7 LLDMLQVVN 5 V12A-HLA-B2705-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 5 SISWLIMLF 15 4 PSISWLIML 14 9 LIMLFSSVY 12 2 ISPSISWLI 10 3 SPSISWLIM 10 V12B-HLA-B2705-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 1015 MRLKSDSNR 29 62 ARKEFSTTL 26 785 LRLELDETK 26 210 GRSSSCALR 25 429 PRKDLIVML 25 1054 DRAGVLAPK 25 30 SRADPVTWR 24 288 RHIPEILKF 24 315 ARLSGLNSI 24 411 VRREDLDKL 24 412 RREDLDKLH 24 701 SRKPENQQF 24 835 LRQLGLAQH 24 875 LRQLGLAQH 24 1029 FRDKDDLPF 24 437 LRDTDMNKR 23 470 DRRCQLNVL 23 778 LREEIAKLR 23 1022 NRETHQAFR 23 161 TRAFQVVHL 22 287 LRHIPEILK 22 617 AREYAVSSH 22 139 SRDPSPPCH 21 148 QRRDAACLR 21 327 KEFEELVKL 21 578 DRYGRTALI 21 691 GDDGLIPQR 21 109 GAFLLGWER 20 183 SRNSYRLTH 20 273 HLIQCIPNL 20 988 GWILPVPTF 20 31 RADPVTWRK 19 155 LRAQGLTRA 19 445 RDKQKRTAL 19 471 RRCQLNVLD 19 566 FLIKKKANL 19 946 RASPGTPSL 19 1076 HRNADTPPH 19 1084 HRHTTTLPH 19 5 ILLPTQATF 18 121 RRLEVPRPQ 18 203 ANLPGAPGR 18 300 ETGGGILGL 18 423 AWWGKVPRK 18 430 RKDLIVMLR 18 581 GRTALILAV 18 610 QDLSGQTAR 18 777 MLREEIAKL 18 779 REEIAKLRL 18 932 GDQGPGTHL 18 971 GKNGRSPTK 18 11 ATFAAATGL 17 55 LSFPGTAAR 17 86 GSLPAFADL 17 114 GWERVVQRR 17 149 RRDAACLRA 17 156 RAQGLTRAF 17 176 GGAGCPPSR 17 197 GLEAASANL 17 238 QRLLFLPRA 17 262 LGVGSLSVF 17 316 RLSGLNSIM 17 341 KVIQCVFAK 17 378 KISGLIQEM 17 413 REDLDKLHR 17 538 GADIESKNK 17 544 KNKCGLTPL 17 558 EQKQEVVKF 17 562 EVVKFLIKK 17 591 CGSASIVNL 17 618 REYAVSSHH 17 648 NSNPVITIL 17 663 KVEEEIKKH 17 694 GLIPQRKSR 17 738 SQDEILTNK 17 786 RLELDETKH 17 832 GFSLRQLGL 17 872 GFSLRQLGL 17 905 AQEQGAALR 17 964 AALPPPTGK 17 974 GRSPTKQKS 17 1110 GPTTLGSNR 17 56 SFPGTAARK 16 74 SALSLSSSR 16 83 ALPGSLPAF 16 120 QRRLEVPRP 16 211 RSSSCALRY 16 219 YRSGPSVSS 16 281 LSYPLVLRH 16 291 PEILKFSEK 16 302 GGGILGLEL 16 321 NSIMQIKEF 16 324 MQIKEFEEL 16 444 KRDKQKRTA 16 460 GNSEVVQLL 16 463 EVVQLLLDR 16 473 CQLNVLDNK 16 482 KRTALIKAV 16 552 LLLGVHEQK 16 563 VVKFLIKKK 16 579 RYGRTALIL 16 592 GSASIVNLL 16 634 SDYKEKQML 16 662 LKVEEEIKK 16 674 NPVGLPENL 16 695 LIPQRKSRK 16 719 SDEQNDTQK 16 759 SELSLSHKK 16 790 DETKHQNQL 16 798 LRENKILEE 16 805 EEIESVKEK 16 808 ESVKEKLLK 16 812 EKLLKTIQL 16 865 AQEQEVAGF 16 949 PGTPSLVRL 16 955 VRLASGARA 16 1004 RRCPMFDVS 16 1009 FDVSPAMRL 16 1030 RDKDDLPFF 16 1085 RHTTTLPHR 16 1106 AGGVGPTTL 16 15 AATGLWAAL 15 36 TWRKEPAVL 15 41 PAVLPCCNL 15 47 CNLEKGSWL 15 49 LEKGSWLSF 15 68 TTLTGHSAL 15 115 WERVVQRRL 15 179 GCPPSRNSY 15 217 LRYRSGPSV 15 259 EEALGVGSL 15 330 EELVKLHSL 15 333 VKLHSLSHK 15 347 FAKKKNVDK 15 370 LIMKETSTK 15 374 ETSTKISGL 15 397 GDYDDSAFM 15 422 AAWWGKVPR 15 464 VVQLLLDRR 15 474 QLNVLDNKK 15 475 LNVLDNKKR 15 478 LDNKKRTAL 15 511 QDEYGNTAL 15 528 KLMAKALLL 15 571 KANLNALDR 15 624 SHHHVICEL 15 635 DYKEKQMLK 15 651 PVITILNIK 15 652 VITILNIKL 15 654 TILNIKLPL 15 740 DEILTNKQK 15 753 AEKEMNSEL 15 755 KEMNSELSL 15 770 DLLRENSML 15 781 EIAKLRLEL 15 794 HQNQLRENK 15 799 RENKILEEI 15 803 ILEEIESVK 15 819 QLNEEALTK 15 846 ASVQQLCYK 15 886 ASVQQLCYK 15 892 CYKWGHTEK 15 916 IGDPGGVPL 15 935 GPGTHLPPR 15 994 PTFSSGSFL 15 1047 TKDLGQDDR 15 1051 GQDDRAGVL 15 1077 RNADTPPHR 15 23 LTTVSNPSR 14 37 WRKEPAVLP 14 70 LTGHSALSL 14 76 LSLSSSRAL 14 105 ATPAGAFLL 14 113 LGWERVVQR 14 126 PRPQAAPAT 14 181 PPSRNSYRL 14 185 NSYRLTHVR 14 231 PAEPPAHQR 14 232 AEPPAHQRL 14 235 PAHQRLLFL 14 244 PRAPQAVSG 14 264 VGSLSVFQL 14 265 GSLSVFQLH 14 279 PNLSYPLVL 14 308 LELPATAAR 14 309 ELPATAARL 14 318 SGLNSIMQI 14 319 GLNSIMQIK 14 326 IKEFEELVK 14 339 SHKVIQCVF 14 342 VIQCVFAKK 14 343 IQCVFAKKK 14 351 KNVDKWDDF 14 407 PRYHVRRED 14 408 RYHVRREDL 14 436 MLRDTDMNK 14 441 DMNKRDKQK 14 447 KQKRTALHL 14 449 KRTALHLAS 14 459 NGNSEVVQL 14 461 NSEVVQLLL 14 521 YAIYNEDKL 14 527 DKLMAKALL 14 536 LYGADIESK 14 541 IESKNKCGL 14 545 NKCGLTPLL 14 546 KCGLTPLLL 14 557 HEQKQEVVK 14 569 KKKANLNAL 14 572 ANLNALDRY 14 577 LDRYGRTAL 14 629 ICELLSDYK 14 655 ILNIKLPLK 14 692 DDGLIPQRK 14 747 QKQIEVAEK 14 758 NSELSLSHK 14 765 HKKEEDLLR 14 772 LRENSMLRE 14 776 SMLREEIAK 14 796 NQLRENKIL 14 806 EIESVKEKL 14 828 TKVAGFSLR 14 849 QQLCYKWNH 14 868 QEVAGFSLR 14 931 AGDQGPGTH 14 938 THLPPREPR 14 948 SPGTPSLVR 14 967 PPPTGKNGR 14 996 FSSGSFLGR 14 1003 GRRCPMFDV 14 1007 PMFDVSPAM 14 1027 QAFRDKDDL 14 1038 FKTQQSPRH 14 1062 KCRPGTLCH 14 1093 RDTTTSLPH 14
[1043] 42 TABLE XXXII Pos 123456789 score V1A-HLA-B2709-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 80 RRVIRVLSI 25 110 VRFKWKSTI 20 176 FRDVFLKQI 19 216 CRGKSHQHI 18 167 RGLFFTLSL 17 35 RTYLPVCHV 15 217 RGKSHQHIL 15 44 ALIHMVVLL 14 1 MPFISKLVL 13 7 LVLASQPTL 13 21 ASSPFLLFL 13 23 SPFLLFLDL 13 32 RPERTYLPV 13 43 VALIHMVVL 13 49 VVLLTMVFL 13 53 TMVFLSPQL 13 115 KSTIFTFHL 13 128 LSFPVSSSL 13 168 GLFFTLSLF 13 173 LSLFRDVFL 13 177 RDVFLKQIM 13 185 MLFSSVYMM 13 218 GKSHQHILL 13 233 GMYKMDFII 13 34 ERTYLPVCH 12 59 PQLFESLNF 12 71 FKYEASFYL 12 77 FYLRRVIRV 12 79 LRRVIRVLS 12 83 IRVLSICTT 12 91 TCLLGMLQV 12 104 PSISWLVRF 12 111 RFKWKSTIF 12 122 HLFSWSLSF 12 151 LHVSKYCSL 12 158 SLFPINSII 12 161 PINSIIRGL 12 178 DVFLKQIML 12 184 IMLFSSVYM 12 224 ILLPVSFSV 12 239 FIISTSSTL 12 V2A-B2709-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 3 ASQPTLCSF 10 4 SQPTLCSFF 8 V3A-B2709-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 4 RRVIRDLSI 24 3 LRRVIRDLS 11 7 IRDLSICTT 11 V4A-HLA-B2709-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 5 TCLLDMLQV 13 2 ICTTCLLDM 11 3 CTTCLLDML 11 8 LDMLQVVNI 11 6 CLLDMLQVV 10 V12A-HLA-B2709-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 4 PSISWLIML 12 2 ISPSISWLI 11 3 SPSISWLIM 9 8 WLIMLFSSV 9 5 SISWLIMLF 8 V12B-B2709-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 581 GRTALILAV 23 161 TRAFQVVHL 22 315 ARLSGLNSI 22 62 ARKEFSTTL 21 217 LRYRSGPSV 21 411 VRREDLDKL 21 429 PRKDLIVML 21 482 KRTALIKAV 21 1003 GRRCPMFDV 21 470 DRRCQLNVL 20 578 DRYGRTALI 20 701 SRKPENQQF 20 1029 FRDKDDLPF 20 420 HRAAWWGKV 18 86 GSLPAFADL 16 121 RRLEVPRPQ 16 149 RRDAACLRA 16 579 RYGRTALIL 16 592 GSASIVNLL 16 779 REEIAKLRL 16 946 RASPGTPSL 16 288 RHIPEILKF 15 327 KEFEELVKL 15 471 RRCQLNVLD 15 1004 RRCPMFDVS 15 11 ATFAAATGL 14 117 RVVQRRLEV 14 197 GLEAASANL 14 210 GRSSSCALR 14 238 QRLLFLPRA 14 279 PNLSYPLVL 14 302 GGGILGLEL 14 397 GDYDDSAFM 14 408 RYHVRREDL 14 412 RREDLDKLH 14 445 RDKQKRTAL 14 447 KQKRTALHL 14 449 KRTALHLAS 14 459 NGNSEVVQL 14 460 GNSEVVQLL 14 528 KLMAKALLL 14 548 GLTPLLLGV 14 654 TILNIKLPL 14 670 KHGSNPVGL 14 832 GFSLRQLGL 14 872 GFSLRQLGL 14 949 PGTPSLVRL 14 974 GRSPTKQKS 14 988 GWILPVPTF 14 1009 FDVSPAMRL 14 1030 RDKDDLPFF 14 1051 GQDDRAGVL 14 76 LSLSSSRAL 13 120 QRRLEVPRP 13 187 YRLTHVRCA 13 232 AEPPAHQRL 13 244 PRAPQAVSG 13 273 HLIQCIPNL 13 426 GKVPRKDLI 13 467 LLLDRRCQL 13 515 GNTALHYAI 13 546 KCGLTPLLL 13 614 GQTAREYAV 13 755 KEMNSELSL 13 799 RENKILEEI 13 812 EKLLKTIQL 13 916 IGDPGGVPL 13 932 GDQGPGTHL 13 955 VRLASGARA 13 994 PTFSSGSFL 13 1015 MRLKSDSNR 13 1027 QAFRDKDDL 13 5 ILLPTQATF 12 15 AATGLWAAL 12 37 WRKEPAVLP 12 41 PAVLPCCNL 12 47 CNLEKGSWL 12 68 TTLTGHSAL 12 70 LTGHSALSL 12 104 SATPAGAFL 12 105 ATPAGAFLL 12 110 AFLLGWERV 12 126 PRPQAAPAT 12 139 SRDPSPPCH 12 147 HQRRDAACL 12 152 AACLRAQGL 12 156 RAQGLTRAF 12 158 QGLTRAFQV 12 159 GLTRAFQVV 12 181 PPSRNSYRL 12 183 SRNSYRLTH 12 184 RNSYRLTHV 12 190 THVRCAQGL 12 264 VGSLSVFQL 12 309 ELPATAARL 12 316 RLSGLNSIM 12 330 EELVKLHSL 12 364 GYGHSFLIM 12 407 PRYHVRRED 12 444 KRDKQKRTA 12 493 QEDECVLML 12 527 DKLMAKALL 12 544 KNKCGLTPL 12 566 FLIKKKANL 12 569 KKKANLNAL 12 591 CGSASIVNL 12 617 AREYAVSSH 12 634 SDYKEKQML 12 674 NPVGLPENL 12 698 QRKSRKPEN 12 735 TGISQDEIL 12 770 DLLRENSML 12 772 LRENSMLRE 12 778 LREEIAKLR 12 785 LRLELDETK 12 790 DETKHQNQL 12 796 NQLRENKIL 12 802 KILEEIESV 12 827 KTKVAGFSL 12 843 HAQASVQQL 12 883 HAQASVQQL 12 942 PREPRASPG 12 958 ASGARAAAL 12 961 ARAAALPPP 12 975 RSPTKQKSV 12 1076 HRNADTPPH 12 18 GLWAALTTV 11 30 SRADPVTWR 11 115 WERVVQRRL 11 116 ERVVQRRLE 11 148 QRRDAACLR 11 155 LRAQGLTRA 11 192 VRCAQGLEA 11 209 PGRSSSCAL 11 219 YRSGPSVSS 11 235 PAHQRLLFL 11 254 QEQPSEEAL 11 259 EEALGVGSL 11 266 SLSVFQLHL 11 277 CIPNLSYPL 11 285 LVLRHIPEI 11 286 VLRHIPEIL 11 300 ETGGGILGL 11 312 ATAARLSGL 11 318 SGLNSIMQI 11 324 MQIKEFEEL 11 334 KLHSLSHKV 11 345 CVFAKKKNV 11 351 KNVDKWDDF 11 353 VDKWDDFCL 11 427 KVPRKDLIV 11 437 LRDTDMNKR 11 461 NSEVVQLLL 11 491 QCQEDECVL 11 521 YAIYNEDKL 11 522 AIYNEDKLM 11 526 EDKLMAKAL 11 533 ALLLYGADI 11 541 IESKNKCGL 11 545 NKCGLTPLL 11 555 GVHEQKQEV 11 559 QKQEVVKFL 11 593 SASIVNLLL 11 597 VNLLLEQNV 11 599 LLLEQNVDV 11 625 HHHVICELL 11 648 NSNPVITIL 11 650 NPVITILNI 11 652 VITILNIKL 11 721 EQNDTQKQL 11 742 ILTNKQKQI 11 753 AEKEMNSEL 11 781 EIAKLRLEL 11 798 LRENKILEE 11 806 EIESVKEKL 11 807 IESVKEKLL 11 830 VAGFSLRQL 11 835 LRQLGLAQH 11 870 VAGFSLRQL 11 875 LRQLGLAQH 11 912 LRSQIGDPG 11 945 PRASPGTPS 11 983 VCDSSGWIL 11 1007 PMFDVSPAM 11 1044 PRHTKDLGQ 11 1060 APKCRPGTL 11 1084 HRHTTTLPH 11 1106 AGGVGPTTL 11
[1044] 43 TABLE XXXIII Pos 123456789 score V1A-HLA-B4402-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 21 ASSPFLLFL 19 44 ALIHMVVLL 19 101 NISPSISWL 18 10 ASQPTLFSF 17 116 STIFTFHLF 17 23 SPFLLFLDL 16 75 ASFYLRRVI 16 78 YLRRVIRVL 16 163 NSIIRGLFF 16 197 QELQEILVP 16 200 QEILVPSQP 16 207 QPQPLPKDL 16 222 QHILLPVSF 16 20 SASSPFLLF 15 54 MVFLSPQLF 15 63 ESLNFQNDF 15 86 LSICTTCLL 15 104 PSISWLVRF 15 113 KWKSTIFTF 15 128 LSFPVSSSL 15 158 SLFPINSII 15 161 PINSIIRGL 15 179 VFLKQIMLF 15 191 YMMTLIQEL 15 243 TSSTLPWAY 15 1 MPFISKLVL 14 11 SQPTLFSFF 14 29 LDLRPERTY 14 30 DLRPERTYL 14 37 YLPVCHVAL 14 62 FESLNFQND 14 65 LNFQNDFKY 14 100 VNISPSISW 14 162 INSIIRGLF 14 168 GLFFTLSLF 14 172 TLSLFRDVF 14 178 DVFLKQIML 14 239 FIISTSSTL 14 17 SFFSASSPF 13 33 PERTYLPVC 13 43 VALIHMVVL 13 48 MVVLLTMVF 13 49 VVLLTMVFL 13 69 NDFKYEASF 13 106 ISWLVRFKW 13 122 HLFSWSLSF 13 131 PVSSSLIFY 13 148 QINLHVSKY 13 157 CSLFPINSI 13 165 IIRGLFFTL 13 167 RGLFFTLSL 13 173 LSLFRDVFL 13 176 FRDVFLKQI 13 183 QIMLFSSVY 13 218 GKSHQHILL 13 7 LVLASQPTL 12 8 VLASQPTLF 12 19 FSASSPFLL 12 57 LSPQLFESL 12 59 PQLFESLNF 12 85 VLSICTTCL 12 94 LGMLQVVNI 12 115 KSTIFTFHL 12 141 VASSNVTQI 12 143 SSNVTQINL 12 152 HVSKYCSLF 12 187 FSSVYMMTL 12 194 TLIQELQEI 12 203 LVPSQPQPL 12 227 PVSFSVGMY 12 241 ISTSSTLPW 12 18 FFSASSPFL 11 53 TMVFLSPQL 11 70 DFKYEASFY 11 73 YEASFYLRR 11 80 RRVIRVLSI 11 89 CTTCLLGML 11 110 VRFKWKSTI 11 118 IFTFHLFSW 11 120 TFHLFSWSL 11 129 SFPVSSSLI 11 130 FPVSSSLIF 11 217 RGKSHQHIL 11 231 SVGMYKMDF 11 38 LPVCHVALI 10 71 FKYEASFYL 10 98 QVVNISPSI 10 111 RFKWKSTIF 10 151 LHVSKYCSL 10 154 SKYCSLFPI 10 188 SSVYMMTLI 10 195 LIQELQEIL 10 232 VGMYKMDFI 9 V2A-B4402-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 3 ASQPTLCSF 17 4 SQPTLCSFF 13 V3A-HLA-B4402-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 2 YLRRVIRDL 15 9 DLSICTTCL 12 4 RRVIRDLSI 10 V4A-HLA-B4402-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 8 LDMLQVVNI 12 3 CTTCLLDML 11 1 SICTTCLLD 4 5 TCLLDMLQV 4 6 CLLDMLQVV 4 V12A-HLA-B4402-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 1 ISPSISWL 16 5 SISWLIMLF 16 4 PSISWLIML 15 9 LIMLFSSVY 13 2 ISPSISWLI 11 V12B-HLA-B4402-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 232 AEPPAHQRL 27 327 KEFEELVKL 26 254 QEQPSEEAL 25 330 EELVKLHSL 25 259 EEALGVGSL 24 755 KEMNSELSL 24 807 IESVKEKLL 24 493 QEDECVLML 23 753 AEKEMNSEL 23 49 LEKGSWLSF 22 541 IESKNKCGL 22 790 DETKHQNQL 22 115 WERVVQRRL 21 288 RHIPEILKF 21 362 SEGYGHSFL 21 779 REEIAKLRL 21 799 RENKILEEI 20 297 SEKETGGGI 19 321 NSIMQIKEF 19 502 LEHGADGNI 19 83 ALPGSLPAF 18 526 EDKLMAKAL 18 780 EEIAKLRLE 18 805 EEIESVKEK 18 123 LEVPRPQAA 17 300 ETGGGILGL 17 308 LELPATAAR 17 374 ETSTKISGL 17 648 NSNPVITIL 17 740 DEILTNKQK 17 812 EKLLKTIQL 17 958 ASGARAAAL 17 11 ATFAAATGL 16 15 AATGLWAAL 16 64 KEFSTTLTG 16 105 ATPAGAFLL 16 152 AACLRAQGL 16 179 GCPPSRNSY 16 273 HLIQCIPNL 16 348 AKKKNVDKW 16 569 KKKANLNAL 16 572 ANLNALDRY 16 721 EQNDTQKQL 16 988 GWILPVPTF 16 1060 APKCRPGTL 16 5 ILLPTQATF 15 86 GSLPAFADL 15 156 RAQGLTRAF 15 233 EPPAHQRLL 15 291 PEILKFSEK 15 312 ATAARLSGL 15 318 SGLNSIMQI 15 429 PRKDLIVML 15 467 LLLDRRCQL 15 506 ADGNIQDEY 15 513 EYGNTALHY 15 521 YAIYNEDKL 15 528 KLMAKALLL 15 546 KCGLTPLLL 15 558 EQKQEVVKF 15 591 CGSASIVNL 15 647 ENSNPVITI 15 666 EEIKKHGSN 15 701 SRKPENQQF 15 768 EEDLLRENS 15 787 LELDETKHQ 15 796 NQLRENKIL 15 821 NEEALTKTK 15 916 IGDPGGVPL 15 946 RASPGTPSL 15 949 PGTPSLVRL 15 1042 QSPRHTKDL 15 1051 GQDDRAGVL 15 1106 AGGVGPTTL 15 62 ARKEFSTTL 14 68 TTLTGHSAL 14 76 LSLSSSRAL 14 101 SEQSATPAG 14 103 QSATPAGAF 14 104 SATPAGAFL 14 264 VGSLSVFQL 14 279 PNLSYPLVL 14 299 KETGGGILG 14 309 ELPATAARL 14 315 ARLSGLNSI 14 324 MQIKEFEEL 14 384 QEMGSGKSN 14 388 SGKSNVGTW 14 416 LDKLHRAAW 14 447 KQKRTALHL 14 459 NGNSEVVQL 14 462 SEVVQLLLD 14 494 EDECVLMLL 14 512 DEYGNTALH 14 545 NKCGLTPLL 14 592 GSASIVNLL 14 593 SASIVNLLL 14 621 AVSSHHHVI 14 624 SHHHVICEL 14 637 KEKQMLKIS 14 646 SENSNPVIT 14 654 TILNIKLPL 14 670 KHGSNPVGL 14 731 EEQNTGISQ 14 759 SELSLSHKK 14 767 KEEDLLREN 14 777 MLREEIAKL 14 781 EIAKLRLEL 14 817 TIQLNEEAL 14 822 EEALTKTKV 14 832 GFSLRQLGL 14 865 AQEQEVAGF 14 872 GFSLRQLGL 14 29 PSRADPVTW 13 39 KEPAVLPCC 13 58 PGTAARKEF 13 181 PPSRNSYRL 13 198 LEAASANLP 13 211 RSSSCALRY 13 234 PPAHQRLLF 13 235 PAHQRLLFL 13 262 LGVGSLSVF 13 266 SLSVFQLHL 13 282 SYPLVLRHI 13 285 LVLRHIPEI 13 298 EKETGGGIL 13 335 LHSLSHKVI 13 339 SHKVIQCVF 13 396 WGDYDDSAF 13 405 MEPRYHVRR 13 413 REDLDKLHR 13 417 DKLHRAAWW 13 425 WGKVPRKDL 13 426 GKVPRKDLI 13 445 RDKQKRTAL 13 460 GNSEVVQLL 13 461 NSEVVQLLL 13 470 DRRCQLNVL 13 478 LDNKKRTAL 13 525 NEDKLMAKA 13 529 LMAKALLLY 13 533 ALLLYGADI 13 559 QKQEVVKFL 13 566 FLIKKKANL 13 577 LDRYGRTAL 13 579 RYGRTALIL 13 630 CELLSDYKE 13 650 NPVITILNI 13 664 VEEEIKKHG 13 665 EEEIKKHGS 13 674 NPVGLPENL 13 679 PENLTNGAS 13 688 AGNGDDGLI 13 715 EEYHSDEQN 13 720 DEQNDTQKQ 13 730 SEEQNTGIS 13 735 TGISQDEIL 13 806 EIESVKEKL 13 830 VAGFSLRQL 13 858 TEKTEQQAQ 13 870 VAGFSLRQL 13 898 TEKTEQQAQ 13 906 QEQGAALRS 13 925 SEGGTAAGD 13 1021 SNRETHQAF 13 1027 QAFRDKDDL 13 1082 PPHRHTTTL 13
[1045] 44 TABLE XXXIV Pos 123456789 score V1A-HLA-B5101-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 38 LPVCHVALI 24 141 VASSNVTQI 24 247 LPWAYDRGV 24 1 MPFISKLVL 23 43 VALIHMVVL 23 74 EASFYLRRV 22 94 LGMLQVVNI 21 32 RPERTYLPV 20 23 SPFLLFLDL 19 207 QPQPLPKDL 18 232 VGMYKMDFI 18 92 CLLGMLQVV 16 154 SKYCSLFPI 16 233 GMYKMDFII 16 35 RTYLPVCHV 15 75 ASFYLRRVI 15 77 FYLRRVIRV 15 78 YLRRVIRVL 15 103 SPSISWLVR 15 110 VRFKWKSTI 15 160 FPINSIIRG 15 167 RGLFFTLSL 15 7 LVLASQPTL 14 157 CSLFPINSI 14 209 QPLPKDLCR 14 211 LPKDLCRGK 14 224 ILLPVSFSV 14 9 LASQPTLFS 13 71 FKYEASFYL 13 80 RRVIRVLSI 13 130 FPVSSSLIF 13 171 FTLSLFRDV 13 176 FRDVFLKQI 13 178 DVFLKQIML 13 216 CRGKSHQHI 13 226 LPVSFSVGM 13 20 SASSPFLLF 12 91 TCLLGMLQV 12 129 SFPVSSSLI 12 145 NVTQINLHV 12 158 SLFPINSII 12 165 IIRGLFFTL 12 188 SSVYMMTLI 12 194 TLIQELQEI 12 217 RGKSHQHIL 12 30 DLRPERTYL 11 42 HVALIHMVV 11 49 VVLLTMVFL 11 102 ISPSISWLV 11 128 LSFPVSSSL 11 133 SSSLIFYTV 11 173 LSLFRDVFL 11 196 IQELQEILV 11 204 VPSQPQPLP 11 V2A-B5101-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 2 LASQPTLCS 13 5 QPTLCSFFS 10 V3A-HLA-B5101-9mers Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 2 YLRRVIRDL 12 4 RRVIRDLSI 11 9 DLSICTTCL 11 1 FYLRRVIRD 7 7 IRDLSICTT 6 V4A-HLA-B5101-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 6 CLLDMLQVV 16 8 LDMLQVVNI 15 5 TCLLDMLQV 12 9 DMLQVVNIS 11 3 CTTCLLDML 7 V12A-HLA-B5101-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 2 ISPSISWLI 13 3 SPSISWLIM 12 8 WLIMLFSSV 9 4 PSISWLIML 8 9 LIMLFSSVY 5 6 ISWLIMLFS 4 7 SWLIMLFSS 4 5 SISWLIMLF 1 V12B-B5101-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 9 amino acids, and the end position for each peptide is the start position plus eight. 660 LPLKVEEEI 27 650 NPVITILNI 24 256 QPSEEALGV 22 457 SANGNSEVV 22 620 YAVSSHHHV 22 278 IPNLSYPLV 21 363 EGYGHSFLI 21 318 SGLNSIMQI 20 521 YAIYNEDKL 20 593 SASIVNLLL 20 830 VAGFSLRQL 20 840 LAQHAQASV 20 843 HAQASVQQL 20 870 VAGFSLRQL 20 880 LAQHAQASV 20 883 HAQASVQQL 20 233 EPPAHQRLL 19 578 DRYGRTALI 19 674 NPVGLPENL 19 918 DPGGVPLSE 19 1060 APKCRPGTL 19 1082 PPHRHTTTL 19 158 QGLTRAFQV 18 235 PAHQRLLFL 18 371 IMKETSTKI 18 823 EALTKTKVA 18 904 QAQEQGAAL 18 15 AATGLWAAL 17 152 AACLRAQGL 17 181 PPSRNSYRL 17 335 LHSLSHKVI 17 428 VPRKDLIVM 17 688 AGNGDDGLI 17 908 QGAALRSQI 17 1027 QAFRDKDDL 17 1050 LGQDDRAGV 17 1112 TTLGSNREI 17 21 AALTTVSNP 16 33 DPVTWRKEP 16 41 PAVLPCCNL 16 57 FPGTAARKE 16 104 SATPAGAFL 16 111 FLLGWERVV 16 243 LPRAPQAVS 16 264 VGSLSVFQL 16 282 SYPLVLRHI 16 459 NGNSEVVQL 16 484 TALIKAVQC 16 517 TALHYAIYN 16 591 CGSASIVNL 16 599 LLLEQNVDV 16 616 TAREYAVSS 16 636 YKEKQMLKI 16 687 SAGNGDDGL 16 696 IPQRKSRKP 16 729 LSEEQNTGI 16 810 VKEKLLKTI 16 946 RASPGTPSL 16 949 PGTPSLVRL 16 1106 AGGVGPTTL 16 44 LPCCNLEKG 15 130 AAPATSATP 15 162 RAFQVVHLA 15 169 LAPTAPDGG 15 217 LRYRSGPSV 15 260 EALGVGSLS 15 285 LVLRHIPEI 15 306 LGLELPATA 15 310 LPATAARLS 15 347 FAKKKNVDK 15 470 DRRCQLNVL 15 479 DNKKRTALI 15 550 TPLLLGVHE 15 575 NALDRYGRT 15 583 TALILAVCC 15 645 SSENSNPVI 15 647 ENSNPVITI 15 656 LNIKLPLKV 15 693 DGLIPQRKS 15 916 IGDPGGVPL 15 922 VPLSEGGTA 15 940 LPPREPRAS 15 944 EPRASPGTP 15 948 SPGTPSLVR 15 966 LPPPTGKNG 15 985 DSSGWILPV 15 1081 TPPHRHTTT 15 1090 LPHRDTTTS 15 1105 SAGGVGPTT 15 13 FAAATGLWA 14 18 GLWAALTTV 14 28 NPSRADPVT 14 61 AARKEFSTT 14 89 PAFADLPRS 14 106 TPAGAFLLG 14 113 LGWERVVQR 14 127 RPQAAPATS 14 131 APATSATPS 14 141 DPSPPCHQR 14 151 DAACLRAQG 14 205 LPGAPGRSS 14 230 SPAEPPAHQ 14 262 LGVGSLSVF 14 270 FQLHLIQCI 14 279 PNLSYPLVL 14 283 YPLVLRHIP 14 315 ARLSGLNSI 14 338 LSHKVIQCV 14 406 EPRYHVRRE 14 421 RAAWWGKVP 14 451 TALHLASAN 14 455 LASANGNSE 14 530 MAKALLLYG 14 742 ILTNKQKQI 14 910 AALRSQIGD 14 951 TPSLVRLAS 14 964 AALPPPTGK 14 968 PPTGKNGRS 14 976 SPTKQKSVC 14 991 LPVPTFSSG 14 1035 LPFFKTQQS 14 1059 LAPKCRPGT 14 1078 NADTPPHRH 14 1099 LPHFHVSAG 14 10 QATFAAATG 13 17 TGLWAALTT 13 31 RADPVTWRK 13 82 RALPGSLPA 13 84 LPGSLPAFA 13 88 LPAFADLPR 13 94 LPRSCPESE 13 107 PAGAFLLGW 13 143 SPPCHQRRD 13 172 TAPDGGAGC 13 209 PGRSSSCAL 13 215 CALRYRSGP 13 245 RAPQAVSGP 13 246 APQAVSGPQ 13 252 GPQEQPSEE 13 267 LSVFQLHLI 13 297 SEKETGGGI 13 302 GGGILGLEL 13 327 KEFEELVKL 13 386 MGSGKSNVG 13 402 SAFMEPRYH 13 425 WGKVPRKDL 13 502 LEHGADGNI 13 533 ALLLYGADI 13 556 VHEQKQEVV 13 559 QKQEVVKFL 13 621 AVSSHHHVI 13 644 ISSENSNPV 13 676 VGLPENLTN 13 678 LPENLTNGA 13 709 FPDTENEEY 13 735 TGISQDEIL 13 752 VAEKEMNSE 13 770 DLLRENSML 13 782 IAKLRLELD 13 795 QNQLRENKI 13 802 KILEEIESV 13 864 QAQEQEVAG 13 957 LASGARAAA 13 960 GARAAALPP 13 963 AAALPPPTG 13 967 PPPTGKNGR 13 1055 RAGVLAPKC 13 1064 RPGTLCHTD 13 1073 TPPHRNADT 13 1074 PPHRNADTP 13
[1046] 45 TABLE XXXV Pos 1234567890 score V1A-HLA-A0201-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is start position plus nine. 37 YLPVCHVALI 26 93 LLGMLQVVNI 26 56 FLSPQLFESL 25 6 KLVLASQPTL 24 164 SIIRGLFFTL 24 202 ILVPSQPQPL 24 43 VALIHMVVLL 23 44 ALIHMVVLLT 23 127 SLSFPVSSSL 23 150 NLHVSKYCSL 22 172 TLSLFRDVFL 22 194 TLIQELQEIL 22 195 LIQELQEILV 22 223 HILLPVSFSV 22 20 SASSPFLLFL 21 45 LIHMVVLLTM 21 85 VLSICTTCLL 21 135 SLIFYTVASS 21 246 TLPWAYDRGV 21 87 SICTTCLLGM 20 101 NISPSISWLV 20 184 IMLFSSVYMM 20 193 MTLIQELQEI 20 82 VIRVLSICTT 19 225 LLPVSFSVGM 19 42 HVALIHMVVL 18 52 LTMVFLSPQL 18 73 YEASFYLRRV 18 119 FTFHLFSWSL 18 140 TVASSNVTQI 18 160 FPINSIIRGL 18 190 VYMMTLIQEL 18 3 FISKLVLASQ 17 36 TYLPVCHVAL 17 46 IHMVVLLTMV 17 76 SFYLRRVIRV 17 77 FYLRRVIRVL 17 79 LRRVIRVLSI 17 100 VNISPSISWL 17 156 YCSLFPINSI 17 25 FLLFLDLRPE 16 26 LLFLDLRPER 16 40 VCHVALIHMV 16 48 MVVLLTMVFL 16 50 VLLTMVFLSP 16 84 RVLSICTTCL 16 91 TCLLGMLQVV 16 92 CLLGMLQVVN 16 174 SLFRDVFLKQ 16 180 FLKQIMLFSS 16 198 ELQEILVPSQ 16 219 KSHQHILLPV 16 224 ILLPVSFSVG 16 14 TLFSFFSASS 15 31 LRPERTYLPV 15 51 LLTMVFLSPQ 15 88 ICTTCLLGML 15 90 TTCLLGMLQV 15 108 WLVRFKWKST 15 109 LVRFKWKSTI 15 117 TIFTFHLFSW 15 122 HLFSWSLSFP 15 158 SLFPINSIIR 15 166 IRGLFFTLSL 15 183 QIMLFSSVYM 15 185 MLFSSVYMMT 15 8 VLASQPTLFS 14 17 SFFSASSPFL 14 29 LDLRPERTYL 14 64 SLNFQNDFKY 14 78 YLRRVIRVLS 14 96 MLQVVNISPS 14 132 VSSSLIFYTV 14 175 LFRDVFLKQI 14 181 LKQIMLFSSV 14 186 LFSSVYMMTL 14 231 SVGMYKMDFI 14 28 FLDLRPERTY 13 60 QLFESLNFQN 13 95 GMLQVVNISP 13 97 LQVVNISPSI 13 128 LSFPVSSSLI 13 137 IFYTVASSNV 13 144 SNVTQINLHV 13 238 DFIISTSSTL 13 9 LASQPTLFSF 12 22 SSPFLLFLDL 12 30 DLRPERTYLP 12 105 SISWLVRFKW 12 123 LFSWSLSFPV 12 136 LIFYTVASSN 12 142 ASSNVTQINL 12 165 IIRGLFFTLS 12 168 GLFFTLSLFR 12 215 LCRGKSHQHI 12 V2A-A0201-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 2 VLASQPTLCS 14 8 TLCSFFSASS 14 3 LASQPTLCSF 12 1 LVLASQPTLC 8 V3A-A0201-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 7 VIRDLSICTT 20 10 DLSICTTCLL 19 2 FYLRRVIRDL 17 4 LRRVIRDLSI 13 3 YLRRVIRDLS 12 9 RDLSICTTCL 12 6 RVIRDLSICT 10 V4A-A0201-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 8 LLDMLQVVNI 26 2 SICTTCLLDM 19 6 TCLLDMLQVV 16 3 ICTTCLLDML 15 5 TTCLLDMLQV 15 7 CLLDMLQVVN 15 V12A-HLA-A0201-9mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 2 NISPSISWLI 18 10 LIMLFSSVYM 17 4 SPSISWLIML 16 8 SWLIMLFSSV 16 9 WLIMLFSSVY 12 6 SISWLIMLFS 11 7 ISWLIMLFSS 9 V12B-HLA-A0201-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 777 SMLREEIAKL 30 840 GLAQHAQASV 27 880 GLAQHAQASV 27 338 SLSHKVIQCV 26 656 ILNIKLPLKV 26 267 SLSVFQLHLI 25 70 TLTGHSALSL 24 217 ALRYRSGPSV 24 599 NLLLEQNVDV 24 76 ALSLSSSRAL 23 305 GILGLELPAT 23 469 LLDRRCQLNV 23 577 ALDRYGRTAL 23 820 QLNEEALTKT 23 830 KVAGFSLRQL 23 161 LTRAFQVVHL 22 285 PLVLRHIPEI 22 324 IMQIKEFEEL 22 467 QLLLDRRCQL 22 478 VLDNKKRTAL 22 763 SLSHKKEEDL 22 1050 DLGQDDRAGV 22 15 AAATGLWAAL 21 315 AARLSGLNSI 21 371 LIMKETSTKI 21 536 LLYGADIESK 21 660 KLPLKVEEEI 21 729 QLSEEQNTGI 21 916 QIGDPGGVPL 21 958 LASGARAAAL 21 1059 VLAPKCRPGT 21 6 ILLPTQATFA 20 84 ALPGSLPAFA 20 306 ILGLELPATA 20 411 HVRREDLDKL 20 600 LLLEQNVDVS 20 644 KISSENSNPV 20 810 SVKEKLLKTI 20 1106 SAGGVGPTTL 20 264 GVGSLSVFQL 19 273 LHLIQCIPNL 19 278 CIPNLSYPLV 19 502 LLEHGADGNI 19 654 ITILNIKLPL 19 678 GLPENLTNGA 19 744 LTNKQKQIEV 19 798 QLRENKILEE 19 870 EVAGFSLRQL 19 957 RLASGARAAA 19 18 TGLWAALTTV 18 36 VTWRKEPAVL 18 62 AARKEFSTTL 18 113 LLGWERVVQR 18 155 CLRAQGLTRA 18 241 LLFLPRAPQA 18 261 EALGVGSLSV 18 286 LVLRHIPEIL 18 300 KETGGGILGL 18 309 LELPATAARL 18 327 IKEFEELVKL 18 335 KLHSLSHKVI 18 403 SAFMEPRYHV 18 456 LASANGNSEV 18 459 ANGNSEVVQL 18 548 CGLTPLLLGV 18 553 LLLGVHEQKQ 18 581 YGRTALILAV 18 586 LILAVCCGSA 18 588 LAVCCGSASI 18 624 SSHHHVICEL 18 802 NKILEEIESV 18 815 LLKTIQLNEE 18 817 KTIQLNEEAL 18 825 ALTKTKVAGF 18 835 SLRQLGLAQH 18 875 SLRQLGLAQH 18 949 SPGTPSLVRL 18 1060 LAPKCRPGTL 18 7 LLPTQATFAA 17 44 VLPCCNLEKG 17 105 SATPAGAFLL 17 112 FLLGWERVVQ 17 184 SRNSYRLTHV 17 190 LTHVRCAQGL 17 242 LFLPRAPQAV 17 259 SEEALGVGSL 17 282 LSYPLVLRHI 17 330 FEELVKLHSL 17 429 VPRKDLIVML 17 470 LDRRCQLNVL 17 529 KLMAKALLLY 17 530 LMAKALLLYG 17 533 KALLLYGADI 17 541 DIESKNKCGL 17 544 SKNKCGLTPL 17 556 GVHEQKQEVV 17 569 IKKKANLNAL 17 597 IVNLLLEQNV 17 647 SENSNPVITI 17 687 ASAGNGDDGL 17 804 ILEEIESVKE 17 983 SVCDSSGWIL 17 1099 SLPHFHVSAG 17 14 FAAATGLWAA 16 68 STTLTGHSAL 16 110 GAFLLGWERV 16 152 DAACLRAQGL 16 197 QGLEAASANL 16 262 ALGVGSLSVF 16 334 VKLHSLSHKV 16 490 AVQCQEDECV 16 494 QEDECVLMLL 16 510 NIQDEYGNTA 16 523 AIYNEDKLMA 16 549 GLTPLLLGVH 16 589 AVCCGSASIV 16 591 CCGSASIVNL 16 633 LLSDYKEKQM 16 670 KKHGSNPVGL 16 737 GISQDEILTN 16 753 VAEKEMNSEL 16 838 QLGLAQHAQA 16 843 QHAQASVQQL 16 878 QLGLAQHAQA 16 883 QHAQASVQQL 16 947 RASPGTPSLV 16 169 HLAPTAPDGG 15 277 QCIPNLSYPL 15 302 TGGGILGLEL 15 312 PATAARLSGL 15 361 CLSEGYGHSF 15 370 FLIMKETSTK 15 374 KETSTKISGL 15 455 HLASANGNSE 15 460 NGNSEVVQLL 15 468 LLLDRRCQLN 15 482 KKRTALIKAV 15 493 CQEDECVLML 15 501 MLLEHGADGN 15 511 IQDEYGNTAL 15 535 LLLYGADIES 15 566 KFLIKKKANL 15 585 ALILAVCCGS 15 587 ILAVCCGSAS 15 592 CGSASIVNLL 15 601 LLEQNVDVSS 15 621 YAVSSHHHVI 15 650 SNPVITILNI 15 652 PVITILNIKL 15 655 TILNIKLPLK 15 668 EIKKHGSNPV 15 695 GLIPQRKSRK 15 772 LLRENSMLRE 15 799 LRENKILEEI 15 832 AGFSLRQLGL 15 872 AGFSLRQLGL 15 904 QQAQEQGAAL 15 985 CDSSGWILPV 15 1017 RLKSDSNRET 15 5 HILLPTQATF 14 23 ALTTVSNPSR 14 27 VSNPSRADPV 14 80 SSSRALPGSL 14 83 RALPGSLPAF 14 104 QSATPAGAFL 14 111 AFLLGWERVV 14 123 RLEVPRPQAA 14 195 CAQGLEAASA 14 205 NLPGAPGRSS 14 220 YRSGPSVSSA 14 233 AEPPAHQRLL 14 235 PPAHQRLLFL 14 266 GSLSVFQLHL 14 279 IPNLSYPLVL 14 281 NLSYPLVLRH 14 318 LSGLNSIMQI 14 320 GLNSIMQIKE 14 353 NVDKWDDFCL 14 382 GLIQEMGSGK 14 383 LIQEMGSGKS 14 395 GTWGDYDDSA 14 420 LHRAAWWGKV 14 427 GKVPRKDLIV 14 434 LIVMLRDTDM 14 437 MLRDTDMNKR 14 453 ALHLASANGN 14 457 ASANGNSEVV 14 461 GNSEVVQLLL 14 486 ALIKAVQCQE 14 500 LMLLEHGADG 14 519 ALHYAIYNED 14 521 HYAIYNEDKL 14 534 ALLLYGADIE 14 555 LGVHEQKQEV 14 567 FLIKKKANLN 14 568 LIKKKANLNA 14 593 GSASIVNLLL 14 596 SIVNLLLEQN 14 614 SGQTAREYAV 14 688 SAGNGDDGLI 14 735 NTGISQDEIL 14 742 EILTNKQKQI 14 779 LREEIAKLRL 14 784 AKLRLELDET 14 814 KLLKTIQLNE 14 818 TIQLNEEALT 14 867 QEQEVAGFSL 14 930 TAAGDQGPGT 14 975 GRSPTKQKSV 14 990 WILPVPTFSS 14 991 ILPVPTFSSG 14 1089 TTLPHRDTTT 14 1091 LPHRDTTTSL 14
[1047] 46 TABLE XXXVI Pos 123456789 score V1A-HLA-A0202-10mers: 251P5G2 Noresultsfound. V2A-HLA-A0202-10mers: 251P5G2 Noresultsfound. V3A-HLA-A0202-10mers: 251P5G2 Noresultsfound. V4A-HLA-A0202-10mers: 251P5G2 Noresultsfound. V12A-HLA-A0202-10mers: 251P5G2 Noresultsfound. V12B-HLA-A0202-10mers: 251P5G2 Noresultsfound.
[1048] 47 TABLE XXXVII Pos 123456789 score V1A-HLA-A0203-10mers: 251P5G2 Noresultsfound. V2A-HLA-A0203-10mers: 251P5G2 Noresultsfound. V3A-HLA-A0203-10mers: 251P5G2 Noresultsfound. V4A-HLA-A0203-10mers: 251P5G2 Noresultsfound. V12A-HLA-A0203-10mers: 251P5G2 Noresultsfound. V12B-A0203-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 8 LPTQATFAAA 27 957 RLASGARAAA 27 7 LLPTQATFAA 19 14 FAAATGLWAA 19 54 SWLSFPGTAA 19 123 RLEVPRPQAA 19 145 PPCHQRRDAA 19 193 VRCAQGLEAA 19 307 LGLELPATAA 19 415 EDLDKLHRAA 19 903 EQQAQEQGAA 19 923 VPLSEGGTAA 19 956 VRLASGARAA 19 84 ALPGSLPAFA 18 102 SEQSATPAGA 18 125 EVPRPQAAPA 18 195 CAQGLEAASA 18 306 ILGLELPATA 18 450 KRTALHLASA 18 525 YNEDKLMAKA 18 680 PENLTNGASA 18 838 QLGLAQHAQA 18 878 QLGLAQHAQA 18 955 LVRLASGARA 18 9 PTQATFAAAT 17 15 AAATGLWAAL 17 55 WLSFPGTAAR 17 124 LEVPRPQAAP 17 146 PCHQRRDAAC 17 194 RCAQGLEAAS 17 308 GLELPATAAR 17 416 DLDKLHRAAW 17 904 QQAQEQGAAL 17 924 PLSEGGTAAG 17 958 LASGARAAAL 17
[1049] 48 TABLE XXXVIII Pos 1234567890 score V1A-HLA-A3-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 224 ILLPVSFSVG 24 227 PVSFSVGMYK 24 92 CLLGMLQVVN 22 28 FLDLRPERTY 21 78 YLRRVIRVLS 21 7 LVLASQPTLF 21 42 HVALIHMVVL 20 81 RVIRVLSICT 20 109 LVRFKWKSTI 20 210 PLPKDLCRGK 20 214 DLCRGKSHQH 20 44 ALIHMVVLLT 19 135 SLIFYTVASS 19 164 SIIRGLFFTL 19 182 KQIMLFSSVY 19 6 KLVLASQPTL 18 158 SLFPINSIIR 18 189 SVYMMTLIQE 18 50 VLLTMVFLSP 17 64 SLNFQNDFKY 17 84 RVLSICTTCL 17 98 QVVNISPSIS 17 146 VTQINLHVSK 17 168 GLFFTLSLFR 17 174 SLFRDVFLKQ 17 26 LLFLDLRPER 16 30 DLRPERTYLP 16 60 QLFESLNFQN 16 127 SLSFPVSSSL 16 136 LIFYTVASSN 16 165 IIRGLFFTLS 16 178 DVFLKQIMLF 16 202 ILVPSQPQPL 16 14 TLFSFFSASS 15 45 LIHMVVLLTM 15 48 MVVLLTMVFL 15 56 FLSPQLFESL 15 82 VIRVLSICTT 15 108 WINRFKWKST 15 140 TVASSNVTQI 15 172 TLSLFRDVFL 15 183 QIMLFSSVYM 15 8 VLASQPTLFS 14 54 MVFLSPQLFE 14 93 LLGMLQVVNI 14 106 ISWLVRFKWK 14 145 NVTQINLHVS 14 161 PINSIIRGLF 14 204 VPSQPQPLPK 14 225 LLPVSFSVGM 14 3 FISKLVLASQ 13 4 ISKLVASQP 13 10 ASQPTLFSFF 13 35 RTYLPVCHVA 13 37 YLPVCHVALI 13 49 VVLLTMVFLS 13 51 LLTMVFLSPQ 13 63 ESLNFQNDFK 13 96 MLQVVNISPS 13 99 VVNISPSISW 13 102 ISPSISWLVR 13 104 PSISWLVRFK 13 122 HLFSWSLSFP 13 147 TQINLHVSKY 13 152 HVSKYCSLFP 13 162 INSIIRGLFF 13 167 RGLFFTLSLF 13 180 FLKQIMLFSS 13 194 TLIQELQEIL 13 198 ELQEILVPSQ 13 201 EILVPSQPQP 13 223 HILLPVSFSV 13 240 IISTSSTLPW 13 25 FLLFLDLRPE 12 33 PERTYLPVCH 12 69 NDFKYEASFY 12 72 KYEASFYLRR 12 75 ASFYLRRVIR 12 87 SICTTCLLGM 12 150 NLHVSKYCSL 12 173 LSLFRDVFLK 12 185 MLFSSVYMMT 12 221 HQHILLPVSF 12 239 FIISTSSTLP 12 32 RPERTYLPVC 11 68 QNDFKYEASF 11 85 VLSICTTCLL 11 101 NISPSISWLV 11 121 FHLFSWSLSF 11 148 QINLHVSKYC 11 171 FTLSLFRDVF 11 231 SVGMYKMDFI 11 238 DFIISTSSTL 11 V2A-HLA-A3-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 1 LVLASQPTLC 16 8 TLCSFFSASS 15 2 VLASQPTLCS 14 4 ASQPTICSFF 13 9 LCSFFSASSP 8 10 CSFFSASSPF 7 V3A-HLA-A3-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 6 RVIRDLSICT 20 3 YLRRVIRDLS 17 7 VIRDLSICTT 16 10 DLSICTTCLL 11 4 LRRVIRDLSI 8 5 RRVIRDLSIC 8 8 IRDLSICTTC 8 V4A-HLA-A3-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 7 CLLDMLQVVN 21 8 LLDMLQVVNI 14 2 SICTTCLLDM 12 V12A-HLA-A3-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 9 WLIMLFSSVY 26 10 LIMLFSSVYM 13 6 SISWLIMLFS 12 V12B-HLA-A3-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 43 AVLPCCNLEK 31 785 KLRLELDETK 31 803 KILEEIESVK 31 370 FLIMKETSTK 30 326 QIKEFEELVK 29 342 KVIQCVFAKK 29 382 GLIQEMGSGK 29 695 GLIPQRKSRK 29 536 LLYGADIESK 28 419 KLHRAAWWGK 26 552 PLLLGVHEQK 26 160 GLTRAFQVVH 25 333 LVKLHSLSHK 25 819 IQLNEEALTK 25 262 ALGVGSLSVF 24 529 KLMAKALLLY 24 835 SLRQLGLAQH 24 875 SLRQLGLAQH 24 112 FLLGWERVVQ 23 287 VLRHIPEILK 23 5 HILLPTQATF 22 154 ACLRAQGLTR 22 549 GLTPLLLGVH 22 628 HVICELLSDY 22 6 ILLPTQATFA 21 113 LLGWERVVQR 21 119 VVQRRLEVPR 21 217 ALRYRSGPSV 21 306 ILGLELPATA 21 343 VIQCVFAKKK 21 612 DLSGQTAREY 21 655 TILNIKLPLK 21 662 PLKVEEEIKK 21 778 MLREEIAKLR 21 70 TLTGHSALSL 20 88 SLPAFADLPR 20 189 RLTHVRCAQG 20 240 RLLFLPRAPQ 20 243 FLPRAPQAVS 20 361 CLSEGYGHSF 20 428 KVPRKDLIVM 20 523 AIYNEDKLMA 20 563 EVVKFLIKKK 20 587 ILAVCCGSAS 20 771 DLLRENSMLR 20 825 ALTKTKVAGF 20 954 SLVRLASGAR 20 955 LVRLASGARA 20 957 RLASGARAAA 20 23 ALTTVSNPSR 19 49 NLEKGSWLSF 19 56 LSFPGTAARK 19 84 ALPGSLPAFA 19 125 EVPRPQAAPA 19 192 HVRCAQGLEA 19 275 LIQCIPNLSY 19 308 GLELPATAAR 19 332 ELVKLHSLSH 19 467 QLLLDRRCQL 19 486 ALIKAVQCQE 19 534 ALLLYGADIE 19 577 ALDRYGRTAL 19 589 AVCCGSASIV 19 629 VICELLSDYK 19 798 QLRENKILEE 19 830 KVAGFSLRQL 19 838 QLGLAQHAQA 19 878 QLGLAQHAQA 19 891 QLCYKWGHTE 19 912 ALRSQIGDPG 19 922 GVPLSEGGTA 19 966 ALPPPTGKNG 19 973 KNGRSPTKQK 19 55 WLSFPGTAAR 18 118 RVVQRRLEVP 18 433 DLIVMLRDTD 18 437 MLRDTDMNKR 18 513 DEYGNTALHY 18 557 VHEQKQEVVK 18 676 PVGLPENLTN 18 814 LKKLTIQLNE 18 916 QIGDPGGVPL 18 1040 KTQQSPRHTK 18 1054 DDRAGVLAPK 18 19 GLWAALTTVS 17 78 SLSSSRALPG 17 205 NLPGAPGRSS 17 241 LLFLPRAPQA 17 281 NLSYPLVLRH 17 294 ILKFSEKETG 17 317 RLSGLNSIMQ 17 335 KLHSLSHKVI 17 453 ALHLASANGN 17 477 NVLDNKKRTA 17 480 DNKKRTALIK 17 574 NLNALDRYGR 17 585 ALILAVCCGS 17 599 NLLLEQNVDV 17 600 LLLEQNVDVS 17 622 AVSSHHHVIC 17 656 ILNIKLPLKV 17 747 KQKQIEVAEK 17 772 LLRENSMLRE 17 804 ILEEIESVKE 17 810 SVKEKLLKTI 17 964 AAALPPPTGK 17 991 ILPVPTFSSG 17 993 PVPTFSSGSF 17 1011 DVSPAMRLKS 17 1031 RDKDDLPFFK 17 94 DLPRSCPESE 16 123 RLEVPRPQAA 16 155 CLRAQGLTRA 16 183 PSRNSYRLTH 16 286 LVLRHIPEIL 16 291 IPEILKFSEK 16 346 CVFAKKKNVD 16 410 YHVRREDLDK 16 411 HVRREDLDKL 16 436 VMLRDTDMNK 16 455 HLASANGNSE 16 469 LLDRRCQLNV 16 475 QLNVLDNKKR 16 484 RTALIKAVQC 16 501 MLLEHGADGN 16 510 NIQDEYGNTA 16 520 LHYAIYNEDK 16 561 KQEVVKFLIK 16 567 FLIKKKANLN 16 601 LLEQNVDVSS 16 635 SDYKEKQMLK 16 740 QDEILTNKQK 16 840 GLAQHAQASV 16 851 QLCYKWNHTE 16 870 EVAGFSLRQL 16 880 GLAQHAQASV 16 948 ASPGTPSLVR 16 971 TGKNGRSPTK 16 1109 GVGPTTLGSN 16 26 TVSNPSRADP 15 76 ALSLSSSRAL 15 166 QVVHLAPTAP 15 169 HLAPTAPDGG 15 179 AGCPPSRNSY 15 198 GLEAASANLP 15 218 LRYRSGPSVS 15 250 AVSGPQEQPS 15 310 ELPATAARLS 15 347 VFAKKKNVDK 15 416 DLDKLHRAAW 15 439 RDTDMNKRDK 15 473 RCQLNVLDNK 15 524 IYNEDKLMAK 15 556 GVHEQKQEVV 15 571 KKANLNALDR 15 586 LILAVCCGSA 15 616 QTAREYAVSS 15 719 HSDEQNDTQK 15 737 GISQDEILTN 15 787 RLELDETKHQ 15 794 KHQNQLRENK 15 808 IESVKEKLLK 15 821 LNEEALTKTK 15 852 LCYKWNHTEK 15 892 LCYKWGHTEK 15 924 PLSEGGTAAG 15 940 HLPPREPRAS 15 983 SVCDSSGWIL 15 990 WILPVPTFSS 15 1002 FLGRRCPMFD 15 1017 RLKSDSNRET 15 1059 VLAPKCRPGT 15 1090 TLPHRDTTTS 15 1099 SLPHFHVSAG 15
[1050] 49 TABLE XXXIX Pos 1234567890 score V1A-HLA-A26-10mers: 251P502 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 178 DVFLKQIMLF 33 42 HVALIHMVVL 24 164 SIIRGLFFTL 24 39 PVCHVALIHM 23 48 MVVLLTMVFL 23 56 FLSPQLFESL 23 87 SICTTCLLGM 23 238 DFIISTSSTL 23 45 LIHMVVLLTM 22 198 ELQEILVPSQ 22 242 STSSTLPWAY 22 70 DFKYEASFYL 21 119 FTFHLFSWSL 21 150 NLHVSKYCSL 21 161 PINSIIRGLF 21 171 FTLSLFRDVF 21 7 LVLASQPTLF 20 52 LTMVFLSPQL 20 185 LFSSVYMMTL 20 28 FLDLRPERTY 19 64 SLNFQNDFKY 19 84 RVLSICTTCL 19 183 QIMLFSSVYM 19 194 TLIQELQEIL 19 202 ILVPSQPQPL 19 225 LLPVSFSVGM 19 17 SFFSASSPFL 18 127 SLSFPVSSSL 18 147 TQINLHVSKY 18 201 EILVPSQPQP 18 3 FISKLVLASQ 17 6 KLVLASQPTL 17 10 ASQPTLFSFF 17 117 TIFTFHLFSW 17 129 SFPVSSSLIF 17 140 TVASSNVTQI 17 172 TLSLFRDVFL 17 185 MLFSSVYMMT 17 245 STLPWAYDRG 17 9 LASQPTLFSF 16 13 PTLFSFFSAS 16 19 FSASSPFLLF 16 30 DLRPERTYLP 16 85 VLSICTTCLL 16 100 VNISPSISWL 16 103 SPSISWLVRF 16 122 HLFSWSLSFP 16 135 SLIFYTVASS 16 165 IIRGLFFTLS 16 193 MTLIQELQQEI 16 226 LPVSFSVGMY 16 228 VSFSVGMYKM 16 V2A-A26-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5 each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 3 LASQPTLCSF 16 7 PTLCSFFSAS 16 4 ASQPTLCSFF 13 10 CSFFSASSPF 13 8 TLCSFFSASS 11 1 LVLASQPTLC 10 2 VLASQPTLCS 10 V3A-A26-HLA-10mers: 251P5G2 Each peptide is a portion OF SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 10 DLSICTTCLL 22 7 VIRDLSICTT 15 2 FYLRRVIRDL 12 6 RVIRDLSICT 12 1 SFYLRRVIRD 11 V4A-HLA-A26-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 2 SICTTCLLDM 22 5 TTCLLDMLQV 15 8 LLDMLQVVNI 15 3 ICTTCLLDML 13 4 CTTCLLDMLQ 11 7 CLLDMLQVVN 10 V12A-HLA-1A26-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 9 WLIMLFSSVY 21 10 LIMLFSSVYM 19 5 PSISWLIMLF 18 6 SISWLIMLFS 14 4 SPSISWLIML 13 2 NISPSISWLI 12 3 ISPSISWLIM 10 V12B-HLA-A26-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 870 EVAGFSLRQL 32 612 DLSGQTAREY 27 628 HVICELLSDY 27 541 DIESKNKCGL 26 830 KVAGFSLRQL 26 807 EIESVKEKLL 25 825 ALTKTKVAGF 25 161 LTRAFQVVHL 24 264 GVGSLSVFQL 24 752 EVAEKEMNSE 24 262 ALGVGSLSVF 23 411 HVRREDLDKL 23 428 KVPRKDLIVM 23 607 DVSSQDLSGQ 23 301 ETGGGILGLE 22 464 EVVQLLLDRR 22 529 KLMAKALLLY 22 563 EVVKFLIKKK 22 782 EIAKLRLELD 22 817 KTIQLNEEAL 22 993 PVPTFSSGSF 22 5 HILLPTQATF 21 275 LIQCIPNLSY 21 361 CLSEGYGHSF 21 652 PVITILNIKL 21 712 DTENEEYHSD 21 49 NLEKGSWLSF 20 68 STTLTGHSAL 20 190 LTHVRCAQGL 20 353 NVDKWDDFCL 20 364 EGYGHSFLIM 20 559 EQKQEVVKFL 20 654 ITILNIKLPL 20 916 QIGDPGGVPL 20 1001 SFLGRRCPMF 20 1073 DTPPHRNADT 20 36 VTWRKEPAVL 19 70 TLTGHSALSL 19 342 KVIQCVFAKK 19 513 DEYGNTALHY 19 709 QFPDTENEEY 19 789 ELDETKHQNQ 19 806 EEIESVKEKL 19 983 SVCDSSGWIL 19 1011 DVSPAMRLKS 19 1081 DTPPHRHTTT 19 83 RALPGSLPAF 18 125 EVPRPQAAPA 18 269 SVFQLHLIQC 18 286 LVLRHIPEIL 18 293 EILKFSEKET 18 305 GILGLELPAT 18 401 DDSAFMEPRY 18 416 DLDKLHRAAW 18 434 LIVMLRDTDM 18 440 DTDMNKRDKQ 18 478 VLDNKKRTAL 18 577 ALDRYGRTAL 18 633 LLSDYKEKQM 18 648 ENSNPVITIL 18 668 EIKKHGSNPV 18 725 DTQKQLSEEQ 18 735 NTGISQDEIL 18 792 ETKHQNQLRE 18 865 QAQEQEVAGF 18 1021 DSNRETHQAF 18 1025 ETHQAFRDKD 18 76 ALSLSSSRAL 17 103 EQSATPAGAF 17 152 DAACLRAQGL 17 234 EPPAHQRLLF 17 333 LVKLHSLSHK 17 378 TKISGLIQEM 17 467 QLLLDRRCQL 17 558 HEQKQEVVKF 17 742 EILTNKQKQI 17 781 EEIAKLRLEL 17 810 SVKEKLLKTI 17 1009 MFDVSPAMRL 17 1029 AFRDKDDLPF 17 1030 FRDKDDLPFF 17 1035 DLPFFKTQQS 17 1050 DLGQDDRAGV 17 1109 GVGPTTLGSN 17 41 EPAVLPCCNL 16 94 DLPRSCPESE 16 278 CIPNLSYPLV 16 332 ELVKLHSLSH 16 375 ETSTKISGLI 16 377 STKISGLIQE 16 433 DLIVMLRDTD 16 447 DKQKRTALHL 16 494 QEDECVLMLL 16 527 EDKLMAKALL 16 566 KFLIKKKANL 16 579 DRYGRTALIL 16 632 ELLSDYKEKQ 16 763 SLSHKKEEDL 16 769 EEDLLRENSM 16 770 EDLLRENSML 16 828 KTKVAGFSLR 16 843 QHAQASVQQL 16 883 QHAQASVQQL 16 978 PTKQKSVCDS 16 1094 RDTTTSLPHF 16 1095 DTTTSLPHFH 16 71 LTGHSALSLS 15 106 ATPAGAFLLG 15 118 RVVQRRLEVP 15 172 PTAPDGGAGC 15 288 LRHIPEILKF 15 300 KETGGGILGL 15 310 ELPATAARLS 15 321 LNSIMQIKEF 15 329 EFEELVKLHS 15 374 KETSTKISGL 15 429 VPRKDLIVML 15 516 GNTALHYAIY 15 536 LLYGADIESK 15 569 IKKKANLNAL 15 572 KANLNALDRY 15 591 CCGSASIVNL 15 596 SIVNLLLEQN 15 655 TILNIKLPLK 15 721 DEQNDTQKQL 15 737 GISQDEILTN 15 755 EKEMNSELSL 15 761 ELSLSHKKEE 15 771 DLLRENSMLR 15 772 LLRENSMLRE 15 798 QLRENKILEE 15 820 QLNEEALTKT 15 835 SLRQLGLAQH 15 848 SVQQLCYKWN 15 875 SLRQLGLAQH 15 888 SVQQLCYKWG 15 988 SGWILPVPTF 15 991 ILPVPTFSSG 15 1099 SLPHFHVSAG 15
[1051] 50 TABLE XL Pos 1234567890 score V1A-HLA-B0702-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 103 SPSISWLVRF 20 160 FPINSIIRGL 20 1 MPFISKLVLA 18 58 SPQLFESLNF 18 12 QPTLFSFFSA 17 20 SASSPFLLFL 15 166 IRGLFFTLSL 15 172 TLSLFRDVFL 15 204 VPSQPQPLPK 15 36 TYLPVGHVAL 14 42 HVALIHMVVL 14 56 FLSPQLFESL 14 142 ASSNVTQINL 14 18 FFSASSPFLL 13 22 SSPFLLFLDL 13 32 RPERTYLPVC 13 43 VALIHMVVLL 13 48 MVVLLTMVFL 13 84 RVLSIGTTCL 13 85 VLSICTTCLL 13 114 WKSTIFTFHL 13 202 ILVPSQPQPL 13 6 KLVLASQPTL 12 17 SFFSASSPFL 12 29 LDLRPERTYL 12 38 LPVCHVALIH 12 52 LTMVFLSPQL 12 127 SLSFPVSSSL 12 162 INSIIRGLFF 12 186 LFSSVYMMTL 12 216 CRGKSHQHIL 12 10 ASQPTLFSFF 11 33 ALIHMVVLLT 11 74 EASFYLRRVI 11 77 FYLRRVIRVL 11 79 LRRVIRVLSI 11 188 ICTTCLLGML 11 130 FPVSSSLIFY 11 164 SIIRGLFFTL 11 177 RDVFLKQIML 11 190 VYMMTLIQEL 11 207 QPQPLPKDLC 11 209 QPLPKDLCRG 11 211 LPKDLCRGKS 11 217 RGKSHQHILL 11 226 LPVSFSVGMY 11 19 FSASSPFLLF 10 23 SPFLLFLDLR 10 70 DFKYEASFYL 10 93 LLGMLQVVNI 10 100 VNISPSISWL 10 119 FTFHLFSWSL 10 133 SSSLIFYTVA 10 150 NLHVSKYCSL 10 194 TLIQELQEIL 10 206 SQPQPLPKDL 10 215 LCRGKSHQHI 10 219 KSHQHILLPV 10 238 DFIISTSSTL 10 31 LRPERTYLPV 9 46 IHMVVLLTMV 9 101 NISPSISWLV 9 111 RFKWKSTIFT 9 123 LFSWSLSFPV 9 132 VSSSLIFYTV 9 140 TVASSNVTQI 9 183 QIMLFSSVYM 9 187 FSSVYMMTLI 9 V2A-B0702-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 6 QPTLCSFFSA 17 4 ASQPTLCSFF 10 3 LASQPTLCSF 8 V3A-B0702-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 9 RDLSICTTGL 13 10 DLSICTTCLL 13 2 FYLRRVIRDL 10 4 LRRVIRDLSI 10 7 VIRDLSICTT 8 6 RVIRDLSICT 7 V4A-HLA-B0702-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 3 ICTTCLLDML 11 8 LLDMLQVVNI 10 2 SICTTCLLDM 8 5 TTGLLDMLQV 8 6 TCLLOMLQVV 7 V12A-HLA-B0702-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 4 SPSISWLIML 22 2 NISPSISWLI 9 3 ISPSISWLIM 9 10 LIMLFSSVYM 9 8 SWLIMLFSSV 7 5 PSISWLIMLF 6 6 SISWLIMLFS 5 7 ISWLIMLFSS 1 9 WLIMLFSSVY 1 V12B-HLA-B0702-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 429 VPRKDLIVML 25 209 APGRSSSCAL 24 235 PPAHQRLLFL 24 279 IPNLSYPLVL 24 949 SPGTPSLVRL 23 41 EPAVLPCCNL 22 1082 TPPHRHTTTL 22 1091 LPHRDTTTSL 22 126 VPRPQAAPAT 21 234 EPPAHQRLLF 21 994 VPTFSSGSFL 21 181 CPPSRNSYRL 20 8 LPTQATFAAA 19 128 RPQAAPATSA 19 704 KPENQQFPDT 19 1007 CPMFDVSPAM 19 1065 RPGTLCHTDT 19 58 FPGTAARKEF 18 145 PPCHQRRDAA 18 182 PPSRNSYRLT 18 675 NPVGLPENLT 18 923 VPLSEGGTAA 18 34 DPVTWRKEPA 17 144 SPPCHQRRDA 17 229 APSPAEPPAH 17 62 AARKEFSTTL 16 161 LTRAFQVVHL 16 174 APDGGAGCPP 16 253 GPQEQPSEEA 16 445 KRDKQKRTAL 16 459 ANGNSEVVQL 16 577 ALDRYGRTAL 16 945 EPRASPGTPS 16 958 LASGARAAAL 16 15 AAATGLWAAL 15 76 ALSLSSSRAL 15 107 TPAGAFLLGW 15 132 APATSATPSR 15 244 LPRAPQAVSG 15 300 KETGGGILGL 15 302 TGGGILGLEL 15 670 KKHGSNPVGL 15 687 ASAGNGDDGL 15 781 EEIAKLRLEL 15 29 NPSRADPVTW 14 70 TLTGHSALSL 14 86 PGSLPAFADL 14 138 TPSRDPSPPC 14 171 APTAPDGGAG 14 247 APQAVSGPQE 14 311 LPATAARLSG 14 407 EPRYHVRRED 14 461 GNSEVVQLLL 14 478 VLDNKKRTAL 14 511 IQDEYGNTAL 14 546 NKCGLTPLLL 14 579 DRYGRTALIL 14 592 CGSASIVNLL 14 648 ENSNPVITIL 14 654 ITILNIKLPL 14 830 KVAGFSLRQL 14 832 AGFSLRQLGL 14 870 EVAGFSLRQL 14 872 AGFSLRQLGL 14 916 QIGDPGGVPL 14 932 AGDQGPGTHL 14 1042 SPRHTKDLGQ 14 1106 SAGGVGPTTL 14 104 QSATPAGAFL 13 142 DPSPPCHQRR 13 206 LPGAPGRSSS 13 223 GPSVSSAPSP 13 233 AEPPAHQRLL 13 257 QPSEEALGVG 13 327 IKEFEELVKL 13 362 LSEGYGHSFL 13 425 WWGKVPRKDL 13 447 DKQKRTALHL 13 470 LDRRCQLNVL 13 493 CQEDECVLML 13 526 NEDKLMAKAL 13 544 SKNKCGLTPL 13 545 KNKCGLTPLL 13 559 EQKQEVVKFL 13 569 IKKKANLNAL 13 591 CCGSASIVNL 13 593 GSASIVNLLL 13 697 IPQRKSRKPE 13 755 EKEMNSELSL 13 904 QQAQEQGAAL 13 919 DPGGVPLSEG 13 942 PPREPRASPG 13 946 PRASPGTPSL 13 952 TPSLVRLASG 13 977 SPTKQKSVCD 13 1029 AFRDKDDLPF 13 1061 APKCRPGTLC 13 36 VTWRKEPAVL 12 80 SSSRALPGSL 12 84 ALPGSLPAFA 12 85 LPGSLPAFAD 12 89 LPAFADLPRS 12 95 LPRSCPESEQ 12 259 SEEALGVGSL 12 264 GVGSLSVFQL 12 266 GSLSVFQLHL 12 277 QCIPNLSYPL 12 291 IPEILKFSEK 12 309 LELPATAARL 12 324 IMQIKEFEEL 12 353 NVDKWDDFCL 12 364 EGYGHSFLIM 12 387 MGSGKSNVGT 12 411 HVRREDLDKL 12 527 EDKLMAKALL 12 528 DKLMAKALLL 12 566 KFLIKKKANL 12 763 SLSHKKEEDL 12 779 LREEIAKLRL 12 812 KEKLLKTIQL 12 817 KTIQLNEEAL 12 843 QHAQASVQQL 12 883 QHAQASVQQL 12 936 GPGTHLPPRE 12 941 LPPREPRASP 12 962 ARAAALPPPT 12 969 PPTGKNGRSP 12 1075 PPHRNADTPP 12 1083 PPHRHTTTLP 12
[1052] 51 TABLE XLI Pos 123456789 score V1A-HLA-B08-10mers: 251P5G2 Noresultsfound. V2A-HLA-B08-10mers: 251P5G2 Noresultsfound. V3A-HLA-B08-10mers: 251P5G2 Noresultsfound. V4A-HLA-B08-10mers: 251P5G2 Noresultsfound. V12A-HLA-B08-10mers: 251P5G2 Noresultsfound. V12B-HLA-B08-10mers: 251P5G2 Noresultsfound.
[1053] 52 TABLE XLII Pos 123456789 score V1A-HLA-B1510-10mers: 251P5G2 Noresultsfound. V2A-HLA-B1510-10mers: 251P5G2 Noresultsfound. V3A-HLA-B1510-10mers: 251P5G2 Noresultsfound. V4A-HLA-B1510-10mers: 251P5G2 Noresultsfound. V12A-HLA-B1510-10mers: 251P5G2 Noresultsfound. V12B-HLA-B1510-10mers: 251P5G2 Noresultsfound.
[1054] 53 TABLE XLIII Pos 123456789 score V1A-HLA-B2705-10mers: 251P5G2 Noresultsfound. V2A-HLA-B2705-10mers: 251P5G2 Noresultsfound. V3A-HLA-B2705-10mers: 251P5G2 Noresultsfound. V4A-HLA-B2705-10mers: 251P5G2 Noresultsfound. V12A-HLA-B2705-10mers: 251P5G2 Noresultsfound. V12B-HLA-B2705-10mers: 251P5G2 Noresultsfound.
[1055] 54 TABLE XLIV Pos 123456789 score V1A-HLA-B2709-10mers: 251P5G2 Noresultsfound. V2A-HLA-B2709-10mers: 251P5G2 Noresultsfound. V3A-HLA-B2709-10mers: 251P5G2 Noresultsfound. V4A-HLA-B2709-10mers: 251P5G2 Noresultsfound. V12A-HLA-B2709-10mers: 251P5G2 Noresultsfound. V12B-HLA-B2709-10mers: 251P5G2 Noresultsfound.
[1056] 55 TABLE XLV Pos 1234567890 score V1A-HLA-B4402-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 3; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 62 FESLNFQNDF 23 160 FPINSIIRGL 18 10 ASQPTLFSFF 17 36 TYLPVCHVAL 17 77 FYLRRVIRVL 17 164 SIIRGLFFTL 17 178 DVFLKQIMLF 17 100 VNISPSISWL 16 142 ASSNVTQINL 16 147 TQINLHVSKY 16 56 FLSPQLFESL 15 182 KQIMLFSSVY 15 206 SQPQPLPKDL 15 242 STSSTLPWAY 15 20 SASSPFLLFL 14 22 SSPFLLFLDL 14 28 FLDLRPERTY 14 43 VALIHMVVLL 14 69 NDFKYEASFY 14 74 EASFYLRRVI 14 103 SPSISWLVRF 14 105 SISWLVRFKW 14 112 FKWKSTIFTF 14 115 KSTIFTFHLF 14 117 TIFTFHLFSW 14 128 LSFPVSSSLI 14 156 YCSLFPINSI 14 190 VYMMTLIQEL 14 200 QEILVPSQPQ 14 202 ILVPSQPQPL 14 238 DFIISTSSTL 14 240 IISTSSTLPW 14 6 KLVLASQPTL 13 7 LVLASQPTLF 13 17 SFFSASSPFL 13 18 FFSASSPFLL 13 19 FSASSPFLLF 13 29 LDLRPERTYL 13 33 PERTYLPVCH 13 42 HVALIHMVVL 13 58 SPQLFESLNF 13 85 VLSICTTCLL 13 114 WKSTIFTFHL 13 129 SFPVSSSLIF 13 166 IRGLFFTLSL 13 167 RGLFFTLSLF 13 171 FTLSLFRDVF 13 172 TLSLFRDVFL 13 175 LFRDVFLKQI 13 194 TLIQELQEIL 13 197 QELQEILVPS 13 9 LASQPTLFSF 12 16 FSFFSASSPF 12 47 HMVVLLTMVF 12 48 MVVLLTMVFL 12 52 LTMVFLSPQL 12 53 TMVFLSPQLF 12 64 SLNFQNDFKY 12 73 YEASFYLRRV 12 84 RVLSICTTCL 12 88 ICTTCLLGML 12 110 VRFKWKSTIF 12 119 FTFHLFSWSL 12 121 FHLFSWSLSF 12 127 SLSFPVSSSL 12 130 FPVSSSLIFY 12 161 PINSIIRGLF 12 162 INSIIRGLFF 12 186 LFSSVYMMTL 12 217 RGKSHQHILL 12 221 HQHILLPVSF 12 37 YLPVCHVALI 11 68 QNDFKYEASF 11 99 VVNISPSISW 11 150 NLHVSKYCSL 11 151 LHVSKYCSLF 11 177 RDVFLKQIML 11 216 CRGKSHQHIL 11 226 LPVSFSVGMY 11 230 FSVGMYKMDF 11 V2A-B4402-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 5; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 4 ASQPTLCSFF 16 3 LASQPTLCSF 12 10 CSFFSASSPF 12 V3A-B4402-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 7; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 2 FYLRRVIRDL 16 10 DLSICTTCLL 13 9 RDLSICTTCL 12 4 LRRVIRDLSI 9 V4A-HLA-B4402-10mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 9; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 3 ICTTCLLDML 12 8 LLDMLQVVNI 11 1 LSICTTCLLD 5 V12A-HLA-B4402-10mers Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 5 PSISWLIMLF 17 2 NISPSISWLI 14 4 SPSISWLIML 14 9 WLIMLFSSVY 14 V12B-HLA-B4402-10mers Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 10 amino acids, and the end position for each peptide is the start position plus nine. 233 AEPPAHQRLL 29 526 NEDKLMAKAL 27 781 EEIAKLRLEL 27 300 KETGGGILGL 26 806 EEIESVKEKL 26 309 LELPATAARL 25 812 KEKLLKTIQL 25 374 KETSTKISGL 24 721 DEQNDTQKQL 24 330 FEELVKLHSL 23 494 QEDECVLMLL 23 513 DEYGNTALHY 23 558 HEQKQEWKF 23 259 SEEALGVGSL 22 647 SENSNPVITI 22 298 SEKETGGGIL 21 363 SEGYGHSFLI 20 867 QEQEVAGFSL 20 774 RENSMLREEI 19 76 ALSLSSSRAL 18 179 AGCPPSRNSY 18 577 ALDRYGRTAL 18 12 ATFAAATGLW 17 83 RALPGSLPAF 17 328 KEFEELVKLH 17 459 ANGNSEVVQL 17 648 ENSNPVITIL 17 732 EEQNTGISQD 17 777 SMLREEIAKL 17 823 EEALTKTKVA 17 1042 QQSPRHTKDL 17 15 AAATGLWAAL 16 29 NPSRADPVTW 16 103 EQSATPAGAF 16 105 SATPAGAFLL 16 209 APGRSSSCAL 16 234 EPPAHQRLLF 16 277 QCIPNLSYPL 16 321 LNSIMQIKEF 16 652 PVITILNIKL 16 817 KTIQLNEEAL 16 832 AGFSLRQLGL 16 870 EVAGFSLRQL 16 872 AGFSLRQLGL 16 124 LEVPRPQAAP 15 262 ALGVGSLSVF 15 288 LRHIPEILKF 15 292 PEILKFSEKE 15 445 KRDKQKRTAL 15 478 VLDNKKRTAL 15 529 KLMAKALLLY 15 546 NKCGLTPLLL 15 559 EQKQEVVKFL 15 654 ITILNIKLPL 15 667 EEIKKHGSNP 15 741 DEILTNKQKQ 15 742 EILTNKQKQI 15 756 KEMNSELSLS 15 760 SELSLSHKKE 15 769 EEDLLRENSM 15 807 EIESVKEKLL 15 830 KVAGFSLRQL 15 958 LASGARAAAL 15 1029 AFRDKDDLPF 15 58 FPGTAARKEF 14 62 AARKEFSTTL 14 65 KEFSTTLTGH 14 68 STTLTGHSAL 14 80 SSSRALPGSL 14 156 LRAQGLTRAF 14 255 QEQPSEEALG 14 260 EEALGVGSLS 14 273 LHLIQCIPNL 14 279 IPNLSYPLVL 14 315 AARLSGLNSI 14 331 EELVKLHSLS 14 406 MEPRYHVRRE 14 414 REDLDKLHRA 14 416 DLDKLHRAAW 14 429 VPRKDLIVML 14 467 QLLLDRRCQL 14 470 LDRRGQLNVL 14 527 EDKLMAKALL 14 528 DKLMAKALLL 14 569 IKKKANLNAL 14 591 CCGSASIVNL 14 592 CGSASIVNLL 14 612 DLSGQTAREY 14 624 SSHHHVICEL 14 628 HVICELLSDY 14 631 CELLSDYKEK 14 650 SNPVITILNI 14 670 KKHGSNPVGL 14 687 ASAGNGDDGL 14 754 AEKEMNSELS 14 770 EDLLRENSML 14 788 LELDETKHQN 14 825 ALTKTKVAGF 14 845 AQASVQQLCY 14 847 ASVQQLCYKW 14 885 AQASVQQLCY 14 887 ASVQQLCYKW 14 932 AGDQGPGTHL 14 944 REPRASPGTP 14 949 SPGTPSLVRL 14 1106 SAGGVGPTTL 14
[1057] 56 TABLE XLVI Pos 123456789 score V1A-HLA-B5101-10mers: 251P5G2 Noresultsfound. V2A-HLA-B5101-10mers: 251P5G2 Noresultsfound. V3A-HLA-B5101-10mers: 251P5G2 Noresultsfound. V4A-HLA-B5101-10mers: 251P5G2 Noresultsfound. V12A-HLA-B5101-10mers: 251P5G2 Noresultsfound. V12B-HLA-B5101-10mers: 251P5G2 Noresultsfound.
[1058] 57 TABLE XLVII Pos 123456789012345 score V12A-HLA-DRB10101-15mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 1 MLQVVNISPSISWL 33 2 MLQVVNISPSISWL 29 11 SISWLIMLFSSVYMM 27 9 SPSISWLIMLFSSVY 24 12 ISWLIMLFSSVYMMT 23 13 SWLIMLFSSVYMMTL 23 5 VVNISPSISWLIMLF 22 3 LQVVNISPSISWLIM 18 14 WLIMLFSSVYMMTLI 17 10 PSISWLIMLFSSVYM 15 6 VNISPSISWLIMLFS 14 V12B-HLA-DRB10101-15mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 169 AFQVVHLAPTAPDGG 32 40 PVTWRKEPAVLPCGN 30 386 SGLIQEMGSGKSNVG 30 644 EKQMLKISSENSNPV 30 656 NPVITILNIKLPLKV 29 10 HILLPTQATFAAATG 28 567 QEVVKFLIKKKANLN 28 993 SGWILPVPTFSSGSF 28 57 KGSWLSFPGTAARKE 27 428 AAWWGKVPRKDLIVM 27 437 KDLIVMLRDTDMNKR 27 501 DECVLMLLEHGADGN 27 529 IYNEDKLMAKALLLY 27 659 ITILNIKLPLKVEEE 27 958 PSLVRLASGARAAAL 27 6 KSHQHILLPTQATFA 26 16 QATFAAATGLWAALT 26 84 LSSSRALPGSLPAFA 26 94 LPAFADLPRSCPESE 26 155 RRDAACLRAQGLTRA 26 158 AACLRAQGLTRAFQV 26 195 LTHVRCAQGLEAASA 26 220 SCALRYRSGPSVSSA 26 222 ALRYRSGPSVSSAPS 26 332 IKEFEELVKLHSLSH 26 374 SFLIMKETSTKISGL 26 600 ASIVNLLLEQNVDVS 26 671 EEEIKKHGSNPVGLP 26 925 PGGVPLSEGGTAAGD 26 992 SSGWILPVPTFSSGS 26 1012 CPMFDVSPAMRLKSD 26 9 QHILLPTQATFAAAT 25 70 KEFSTTLTGHSALSL 25 73 STTLTGHSALSLSSS 25 118 LLGWERVVQRRLEVP 25 198 VRCAQGLEAASANLP 25 208 SANLPGAPGRSSSGA 25 244 QRLLFLPRAPQAVSG 25 286 NLSYPLVLRHIPEIL 25 310 GILGLELPATAARLS 25 313 GLELPATAARLSGLN 25 335 FEELVKLHSLSHKVI 25 338 LVKLHSLSHKVIQCV 25 536 MAKALLLYGADIESK 25 830 ALTKTKVAGFSLRQL 25 919 RSQIGDPGGVPLSEG 25 23 TGLWAALTFVSNPSR 24 69 RKEFSTTLTGHSALS 24 79 HSALSLSSSRALPGS 24 128 RLEVPRPQAAPATSA 24 201 AQGLEAASANLPGAP 24 245 RLLFLPRAPQAVSGP 24 267 ALGVGSLSVFQLHLI 24 281 IQCIPNLSYPLVLRH 24 317 PATAARLSGLNSIMQ 24 346 HKVIQCVFAKKKNVD 24 364 DFCLSEGYGHSFLIM 24 375 FLIMKETSTKISGLI 24 513 DGNIQDEYGNTALHY 24 590 ALILAVCGGSASIVN 24 592 ILAVCCGSASIVNLL 24 612 DVSSQDLSGQTAREY 24 681 PVGLPENLTNGASAG 24 699 DGLIPQRKSRKPENQ 24 815 SVKEKLLKTIQLNEE 24 915 GAALRSQIGDPGGVP 24 940 QGPGTHLPPREPRAS 24 943 GTHLPPREPRASPGT 24 986 QKSVCDSSGWILPVP 24 1104 SLPHFHVSAGGVGPT 24 22 ATGLWAALTTVSNPS 23 103 SCPESEQSATPAGAF 23 309 GGILGLELPATAARL 23 493 IKAVQCQEDECVLML 23 615 SQDLSGQTAREYAVS 23 655 SNPVITILNIKLPLK 23 810 LEEIESVKEKLLKTI 23 957 TPSLVRLASGARAAA 23 1038 KDDLPFFKTQQSPRH 23 1041 LPFFKIQQSPRHTKD 23 1093 TITLPHRDTTTSLPH 23 78 GHSALSLSSSRALPG 22 87 SRALPGSLPAFADLP 22 126 QRRLEVPRPQAAPAT 22 190 RNSYRLTHVRCAQGL 22 277 QLHLIQCIPNLSYPL 22 289 YPLVLRHIPEILKFS 22 305 KETGGGILGLELPAT 22 320 AARLSGLNSIMQIKE 22 362 WDDFCLSEGYGHSFL 22 524 ALHYAIYNEDKLMAK 22 549 SKNKCGLTPLLLGVH 22 559 LLGVHEQKQEVVKFL 22 589 TALILAVCCGSASIV 22 603 VNLLLEQNVDVSSQD 22 632 HHVICELLSDYKEKQ 22 647 MLKISSENSNPVITI 22 807 NKILEEIESVKEKLL 22 842 RQLGLAQHAQASVQQ 22 870 QAQEQEVAGFSLRQL 22 882 RQLGLAQHAQASVQQ 22 996 ILPVPTFSSGSFLGR 22 1109 HVSAGGVGPTTLGSN 22 14 PTQATFAAATGLWAA 21 264 SEEALGVGSLSVFQL 21 569 VVKFLIKKKANLNAL 21 607 LEQNVDVSSQDLSGQ 21 757 EVAEKEMNSELSLSH 21 804 LRENKILEEIESVKE 21 828 EEALTKTKVAGFSLR 21 60 WLSFPGTAARKEFST 20 114 AGAFLLGWERVVQRR 20 225 YRSGPSVSSAPSPAE 20 256 VSGPQEQPSEEALGV 20 300 LKFSEKETGGGILGL 20 306 ETGGGILGLELPATA 20 327 NSIMQIKEFEELVKL 20 372 GHSFLIMKETSTKIS 20 639 LSDYKEKQMLKISSE 20 733 KQLSEEQNTGISQDE 20 1011 RCPMFDVSPAMRLKS 20 1032 HQAFRDKDDLPFFKT 20 1106 PHFHVSAGGVGPTTL 20 65 GTAARKEFSTILTGH 19 123 RVVQRRLEVPRPQAA 19 241 PAHQRLLFLPRAPQA 19 259 PQEQPSEEALGVGSL 19 273 LSVFQLHLIQCIPNL 19 299 ILKFSEKETGGGILG 19 414 RYHVRREDLDKLHRA 19 456 RTALHLASANGNSEV 19 478 RCQLNVLDNKKRTAL 19 504 VLMLLEHGADGNIQD 19 534 KLMAKALLLYGADIE 19 571 KFLIKKKANLNALDR 19 577 KANLNALDRYGRTAL 19 588 RTALILAVCCGSASI 19 732 QKQLSEEQNTGISQD 19 781 NSMLREEIAKLRLEL 19 836 VAGFSLRQLGLAQHA 19 838 GFSLRQLGLAQHAQA 19 876 VAGFSLRQLGLAQHA 19 878 GFSLRQLGLAQHAQA 19 917 ALRSQIGDPGGVPLS 19 948 PREPRASPGTPSLVR 19 991 DSSGWILPVPTFSSG 19 1002 FSSGSFLGRRCPMFD 19 50 LPCCNLEKGSWLSFP 18 52 CGNLEKGSWLSFPGT 18 108 EQSATPAGAFLLGWE 18 113 PAGAFLLGWERVVQR 18 193 YRLTHVRCAQGLEAA 18 261 EQPSEEALGVGSLSV 18 288 SYPLVLRHIPEILKF 18 301 KFSEKETGGGILGLE 18 324 SGLNSIMQIKEFEEL 18 356 KKNVDKWDDFCLSEG 18 385 ISGLIQEMGSGKSNV 18 399 VGTWGDYDDSAFMEP 18 402 WGDYDDSAFMEPRYH 18 419 REDLDKLHRAAWWGK 18 427 RAAWWGKVPRKDLIV 18 450 KRDKQKRTALHLASA 18 468 SEVVQLLLDRRCQLN 18 502 ECVLMLLEHGADGNI 18 517 QDEYGNTALHYAIYN 18 537 AKALLLYGADIESKN 18 544 GADIESKNKCGLTPL 18 561 GVHEQKQEVVKFLIK 18 570 VKFLIKKKANLNALD 18 580 LNALDRYGRTALILA 18 583 LDRYGRTALILAVCC 18 623 AREYAVSSHHHVICE 18 670 VEEEIKKHGSNPVGL 18 683 GLPENLTNGASAGNG 18 780 ENSMLREEIAKLRLE 18 785 REEIAKLRLELDETK 18 798 TKHQNQLRENKILEE 18 813 IESVKEKLLKTIQLN 18 819 KLLKTIQLNEEALTK 18 825 QLNEEALTKTKVAGF 18 835 KVAGFSLRQLGLAQH 18 856 QLCYKWNHTEKTEQQ 18 875 EVAGFSLRQLGLAQH 18 896 QLCYKWGHTEKTEQQ 18 926 GGVPLSEGGTAAGDQ 18 956 GTPSLVRLASGARAA 18 959 SLVRLASGARAAALP 18 972 LPPPTGKNGRSPTKQ 18 997 LPVPTFSSGSFLGRR 18 1004 SGSFLGRRCPMFDVS 18 1053 TKDLGQDDRAGVLAP 18 1105 LPHFHVSAGGVGPTT 18 8 HQHILLPTQATFAAA 17 19 FAAATGLWAALTTVS 17 30 TTVSNPSRADPVTWR 17 38 ADPVTWRKEPAVLPC 17 112 TPAGAFLLGWERWQ 17 121 WERVVQRRLEVPRPQ 17 131 VPRPQAAPATSATPS 17 134 PQAAPATSATPSRDP 17 161 LRAQGLTRAFQVVHL 17 168 RAFQVVHLAPTAPDG 17 176 APTAPDGGAGCPPSR 17 243 HQRLLFLPRAPQAVS 17 262 QPSEEALGVGSLSVF 17 263 PSEEALGVGSLSVFQ 17 276 FQLHLIQCIPNLSYP 17 285 PNLSYPLVLRHIPEI 17 308 GGGILGLELPATAAR 17 379 KETSTKISGLIQEMG 17 383 TKISGLIQEMGSGKS 17 424 KLHRPAWWGKVPRKD 17 448 MNKRDKQKRTALHLA 17 453 KQKRTALHLASANGN 17 467 NSEVVQLLLDRRCQL 17 470 VVQLLLDRRCQLNVL 17 480 QLNVLDNKKRTALIK 17 481 LNVLDNKKRTALIKA 17 482 NVLDNKKRTALIKAV 17 503 CVLMLLEHGADGNIQ 17 531 NEDKLMAKALLLYGA 17 546 DIESKNKCGLTPLLL 17 552 KCGLTPLLLGVHEQK 17 553 CGLTPLLLGVHEQKQ 17 579 NLNALDRYGRTALIL 17 599 SASIVNLLLEQNVDV 17 602 IVNLLLEQNVDVSSQ 17 636 CELLSDYKEKQMLKI 17 640 SDYKEKQMLKISSEN 17 663 NIKLPLKVEEEIKKH 17 685 PENLTNGASAGNGDD 17 692 ASAGNGDDGLIPQRK 17 696 NGDDGLIPQRKSRKP 17 745 QDEILTNKQKQIEVA 17 747 EILTNKQKQIEVAEK 17 772 KKEEDLLRENSMLRE 17 778 LRENSMLREEIAKLR 17 790 KLRLELDETKHQNQL 17 829 EALTKTKVAGFSLRQ 17 833 KTKVAGFSLRQLGLA 17 839 FSLRQLGLAQHAQAS 17 841 LRQLGLAQHAQASVQ 17 851 QASVQQLCYKWNHTE 17 868 EQQAQEQEVAGFSLR 17 873 EQEVAGFSLRQLGLA 17 879 FSLRQLGLAQHAQAS 17 881 LRQLGLAQHAQASVQ 17 891 QASVQQLCYKWGHTE 17 935 TAAGDQGPGTHLPPR 17 969 AAALPPPTGKNGRSP 17 995 WILPVPTFSSGSFLG 17 1014 MFDVSPAMRLKSDSN 17 1099 RDTTTSLPHFHVSAG 17 1111 SAGGVGPTTLGSNRE 17 29 LTTVSNPSRADPVTW 16 44 RKEPAVLPCCNLEKG 16 58 GSWLSFPGTAARKEF 16 76 LTGHSALSLSSSRAL 16 81 ALSLSSSRALPGSLP 16 85 SSSRALPGSLPAFAD 16 88 RALPGSLPAFADLPR 16 90 LPGSLPAFADLPRSC 16 105 PESEQSATPAGAFLL 16 106 ESEQSATPAGAFLLG 16 107 SEQSATPAGAFLLGW 16 129 LEVPRPQAAPATSAT 16 150 PPCHQRRDAACLRAQ 16 167 TRAFQVVHLAPTAPD 16 172 VVHLAPTAPDGGAGC 16 177 PTAPDGGAGCPPSRN 16 205 EAASANLPGAPGRSS 16 226 RSGPSVSSAPSPAEP 16 230 SVSSAPSPAEPPAHQ 16 242 AHQRLLFLPRAPQAV 16 246 LLFLPRAPQAVSGPQ 16 248 FLPRAPQAVSGPQEQ 16 253 PQAVSGPQEQPSEEA 16 265 EEALGVGSLSVFQLH 16 270 VGSLSVFQLHLIQCI 16 272 SLSVFQLHLIQCIPN 16 274 SVFQLHLIQCIPNLS 16 282 QCIPNLSYPLVLRHI 16 290 PLVLRHIPEILKFSE 16 312 LGLELPATAARLSGL 16 323 LSGLNSIMQIKEFEE 16 341 LHSLSHKVIQCVFAK 16 342 HSLSHKVIQCVFAKK 16 373 HSFLIMKETSTKISG 16 382 STKISGLIQEMGSGK 16 431 WGKVPRKDLIVMLRD 16 458 ALHLASANGNSEVVQ 16 472 QLLLDRRCQLNVLDN 16 505 LMLLEHGADGNIQDE 16 526 HYAIYNEDKLMAKAL 16 530 YNEDKLMAKALLLYG 16 538 KALLLYGADIESKNK 16 574 IKKKANLNALDRYGR 16 581 NALDRYGRTALILAV 16 582 ALORYORTALILAVO 16 591 LILAVCCGSASIVNL 16 604 NLLLEQNVDVSSQDL 16 608 EQNVDVSSQDLSGQT 16 610 NVDVSSQDLSGQTAR 16 619 SGQTAREYAVSSHHH 16 668 LKVEEEIKKHGSNPV 16 673 EIKKHGSNPVGLPEN 16 682 VGLPENLTNGASAGN 16 766 ELSLSHKKEEDLLRE 16 771 HKKEEDLLRENSMLR 16 820 LLKTIQLNEEALTKT 16 826 LNEEALTKTKVAGFS 16 843 QLGLAQHAQASVQQL 16 869 QQAQEQEVAGFSLRQ 16 883 QLGLAQHAQASVQQL 16 894 VQQLCYKWGHTEKTE 16 907 TEQQAQEQGAALRSQ 16 909 QQAQEQGAALRSQIG 16 918 LRSQIGDPGGVPLSE 16 954 SPGTPSLVRLASGAR 16 960 LVRLASGARPAALPP 16 961 VRLASGARAAALPPP 16 962 RLASGARAAALPPPT 16 963 LASGARAAALPPPTG 16 1054 KDLGQDDRAGVLAPK 16 1055 DLGQDDRAGVLAPKC 16 1063 GVLAPKCRPGTLCHT 16 1069 CRPGTLCHTDTPPHR 16 1077 TDTPPHRNADTPPHR 16 1095 TLPHRDTTTSLPHFH 16 7 SHQHILLPTQATFAA 15 26 WAALTTVSNPSRADP 15 27 AALTTVSNPSRADPV 15 39 DPVTWRKEPAVLPCC 15 51 PCCNLEKGSWLSFPG 15 56 EKGSWLSFPGTAARK 15 99 DLPRSCPESEQSATP 15 120 GWERVVQRRLEVPRP 15 137 APATSATPSRDPSPP 15 159 ACLRAQGLTRAFQVV 15 165 GLTRAFQVVHLAPTA 15 170 FQVVHLAPTAPDGGA 15 181 DGGAGCPPSRNSYRL 15 192 SYRLTHVRGAQGLEA 15 196 THVRCAQGLEAASAN 15 221 CALRYRSGPSVSSAP 15 227 SGPSVSSAPSPAEPP 15 275 VFQLHLIQCIPNLSY 15 311 ILGLELPATAARLSG 15 333 KEFEELVKLHSLSHK 15 337 ELVKLHSLSHKVIQC 15 343 SLSHKVIQCVFAKKK 15 371 YGHSFLIMKETSTKI 15 393 GSGKSNVGTWGDYDD 15 405 YDDSAFMEPRYHVRR 15 438 DLIVMLRDTDMNKRD 15 487 KKRTALIKAVQCQED 15 490 TALIKAVQGQEDECV 15 548 ESKNKCGLTPLLLGV 15 556 TPLLLGVHEQKQEVV 15 586 YGRTALILAVCGGSA 15 641 DYKEKQMLKISSENS 15 737 EEQNTGISQDEILTN 15 752 KQKQIEVAEKEMNSE 15 760 EKEMNSELSLSHKKE 15 777 LLRENSMLREEIAKL 15 818 EKLLKTIQLNEEALT 15 863 HTEKTEQQAQEQEVA 15 866 KTEQQAQEQEVAGFS 15 903 HTEKTEQQAQEQGAA 15 906 KTEQQAQEQGAALRS 15 908 EQQAQEQGAALRSQI 15 927 GVPLSEGGTAAGDQG 15 936 AAGDQGPGTHLPPRE 15 949 REPRASPGTPSLVRL 15 980 GRSPTKQKSVCDSSG 15 985 KQKSVCDSSGWILPV 15 1010 RRCPMFDVSPAMRLK 15 1023 LKSDSNRETHQAFRD 15 1045 KTQQSPRHTKDLGQD 15 1058 QDDRAGVLAPKCRPG 15 1059 DDRAGVLAPKCRPGT 15 1085 ADTPPHRHTTTLPHR 15 1086 DTPPHRHTTTLPHRD 15 1094 TTLPHRDTTTSLPHF 15
[1059] 58 TABLE XLVIII Pos 123456789012345 score V12A-HLA-DRBI0301-15mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 1 MLQVVNISPSISWL 22 12 ISWLIMLFSSVYMMT 20 13 SWLIMLFSSVYMMTL 13 14 WLIMLFSSVYMMTLI 13 2 MLQVVNISPSISWLI 12 5 VVNISPSISWLIMLF 12 8 ISPSISWLIMLFSSV 12 3 LQVVNISPSISWLIM 11 4 QVVNISPSISWLIML 11 9 SPSISWLIMLFSSVY 11 15 LIMLFSSVYMMTLIQ 11 V12B-HLA-DRB1-15meres: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 470 VVQLLLDRRCQLNVL 37 635 ICELLSDYKEKQMLK 30 1020 AMRLKSDSNRETHQA 29 8 HQHILLPTQATFAAA 28 569 VVKFLIKKKANLNAL 27 655 SNPVITILNIKLPLK 27 810 LEEIESVKEKLLKTI 27 1053 TKDLGQDDRAGVLAP 27 278 LHLIQCIPNLSYPLV 26 481 LNVLDNKKRTALIKA 26 580 LNALDRYGRTALILA 26 544 GADIESKNKCGLTPL 25 766 ELSLSHKKEEDLLRE 25 724 HSDEQNDTQKQLSEE 24 439 LIVMLRDTDMNKRDK 23 712 NQQFPDTENEEYHSD 23 780 ENSMLREEIAKLRLE 23 740 NTGISQDEILTNKQK 22 790 KLRLELDETKHQNQL 22 73 STTLTGHSALSLSSS 21 79 HSALSLSSSRALPGS 21 432 GKVPRKDLIVMLRDT 21 828 EEALTKTKVAGFSLR 21 46 EPAVLPCCNLEKGSW 20 52 CCNLEKGSWLSFPGT 20 115 GAFLLGWERVVQRRL 20 243 HQRLLFLPRAPQAVS 20 267 ALGVGSLSVFQLHLI 20 289 YPLVLRHIPEILKFS 20 329 IMQIKEFEELVKLHS 20 414 RYHVRREDLDKLHRA 20 501 DECVLMLLEHGADGN 20 532 EDKLMAKALLLYGAD 20 556 TPLLLGVHEQKQEVV 20 600 ASIVNLLLEQNVDVS 20 602 IVNLLLEQNVDVSSQ 20 631 HHHVICELLSDYKEK 20 661 ILNIKLPLKVEEEIK 20 817 KEKLLKTIQLNEEAL 20 821 LKTIQLNEEALTKTK 20 919 RSQIGDPGGVPLSEG 20 38 ADPVTWRKEPAVLPC 19 265 EEALGVGSLSVFQLH 19 290 PLVLRHIPEILKFSE 19 296 IPEILKFSEKETGGG 19 327 NSIMQIKEFEELVKL 19 354 AKKKNVDKWDDFCLS 19 364 DFCLSEGYGHSFLIM 19 480 QLNVLDNKKRTALIK 19 571 KFLIKKKANLNALDR 19 645 KQMLKISSENSNPVI 19 665 KLPLKVEEEIKKHGS 19 679 SNPVGLPENLTNGAS 19 720 NEEYHSDEQNDTQKQ 19 745 QDEILTNKQKQIEVA 19 806 ENKILEEIESVKEKL 19 833 KTKVAGFSLRQLGLA 19 873 EQEVAGFSLRQLGLA 19 960 LVRLASGARAAALPP 19 986 QKSVCDSSGWILPVP 19 996 ILPVPTFSSGSFLGR 19 1014 MFDVSPAMRLKSDSN 19 26 WAALTTVSNPSRADP 18 29 LTTVSNPSRADPVTW 18 122 ERVVQRRLEVPRPQA 18 163 AQGLTRAFQVVHLAP 18 246 LLFLPRAPQAVSGPQ 18 269 GVGSLSVFQLHLIQC 18 293 LRHIPEILKFSEKET 18 297 PEILKFSEKETGGGI 18 323 LSGLNSIMQIKEFEE 18 349 IQCVFAKKKNVDKWD 18 356 KKNVDKWDDFCLSEG 18 419 REDLDKLHRAAWWGK 18 436 RKDLIVMLRDTDMNK 18 445 DTDMNKRDKQKRTAL 18 446 TDMNKRDKQKRTALH 18 472 QLLLDRRCQLNVLDN 18 479 CQLNVLDNKKRTALI 18 530 YNEDKLMAKALLLYG 18 559 LLGVHEQKQEVVKFL 18 567 QEVVKFLIKKKANLN 18 608 EQNVDVSSQDLSGQT 18 615 SQDLSGQTAREYAVS 18 698 DDGLIPQRKSRKPEN 18 711 ENQQFPDTENEEYHS 18 746 DEILTNKQKQIEVAE 18 773 KEEDLLRENSMLREE 18 1004 SGSFLGRRCPMFDVS 18 1032 HQAFRDKDDLPFFKT 18 1033 QAFRDKDDLPFFKTQ 18 1054 KDLGQDDRAGVLAPK 18 1094 TTLPHRDTTTSLPHF 18
[1060] 59 TABLE XLIX Pos 123456789012345 score V12A-HLA-DR1-0410-15mers: 251P5G2 Noresultsfound. V12B-DR-0401-15mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 23 TGLWAALTTVSNPSR 28 118 LLGWERVVQRRLEVP 28 428 AAWWGKVPRKDLIVM 28 720 NEEYHSDEQNDTQKQ 28 22 ATGLWAALTTVSNPS 26 243 HQRLLFLPRAPQAVS 26 246 LLFLPRAPQAVSGPQ 26 320 AARLSGLNSIMQIKE 26 338 LVKLHSLSHKVIQCV 26 374 SFLIMKETSTKISGL 26 470 VVQLLLDRRCQLNVL 26 602 IVNLLLEQNVDVSSQ 26 632 HHVICELLSDYKEKQ 26 644 EKQMLKISSENSNPV 26 647 MLKISSENSNPVITI 26 655 SNPVITILNIKLPLK 26 732 QKQLSEEQNTGISQD 26 790 KLRLELDETKHQNQL 26 841 LRQLGLAQHAQASVQ 26 881 LRQLGLAQHAQASVQ 26 993 SGWILPVPTFSSGSF 26 1014 MFDVSPAMRLKSDSN 26 1020 AMRLKSDSNRETHQA 26 1038 KDDLPFFKTQQSPRH 26 1053 TKDLGQDDRAGVLAP 26 16 QATFAAATGLWAALT 22 57 KGSWLSFPGTAARKE 22 69 RKEFSTTLTGHSALS 22 94 LPAFADLPRSCPESE 22 167 TRAFQVVHLAPTAPD 22 222 ALRYRSGPSVSSAPS 22 286 NLSYPLVLRHIPEIL 22 332 IKEFEELVKLHSLSH 22 527 YAIYNEDKLMAKALL 22 540 LLLYGADIESKNKCG 22 623 AREYAVSSHHHVICE 22 856 QLCYKWNHTEKTEQQ 22 896 QLCYKWGHTEKTEQQ 22 10 HILLPTQATFAAATG 20 26 WAALTTVSNPSRADP 20 46 EPAVLPCCNLEKGSW 20 79 HSALSLSSSRALPGS 20 115 GAFLLGWERVVQRRL 20 163 AQGLTRAFQVVHLAP 20 170 FQVVHLAPTAPDGGA 20 195 LTHVRCAQGLEAASA 20 208 SANLPGAPGRSSSCA 20 228 GPSVSSAPSPAEPPA 20 267 ALGVGSLSVFQLHLI 20 270 VGSLSVFQLHLIQCI 20 275 VFQLHLIQCIPNLSY 20 278 LHLIQCIPNLSYPLV 20 281 IQCIPNLSYPLVLRH 20 290 PLVLRHIPEILKFSE 20 296 IPEILKFSEKETGGG 20 308 GGGILGLELPATAAR 20 309 GGILGLELPATAARL 20 323 LSGLNSIMQIKEFEE 20 329 IMQIKEFEELVKLHS 20 335 FEELVKLHSLSHKVI 20 346 HKVIQCVFAKKKNVD 20 375 FLIMKETSTKISGLI 20 382 STKISGLIQEMGSGK 20 385 ISGLIQEMGSGKSNV 20 386 SGLIQEMGSGKSNVG 20 414 RYHVRREDLDKLHRA 20 419 REDLDKLHRAAWWGK 20 422 LDKLHRAAWWGKVPR 20 437 KDLIVMLRDTDMNKR 20 439 LIVMLRDTDMNKRDK 20 456 RTALHLASANGNSEV 20 478 RCQLNVLDNKKRTAL 20 489 RTALIKAVQCQEDEC 20 501 DECVLMLLEHGADGN 20 502 ECVLMLLEHGADGNI 20 513 DGNIQDEYGNTALHY 20 526 HYAIYNEDKLMAKAL 20 539 ALLLYGADIESKNKC 20 555 LTPLLLGVHEQKQEV 20 556 TPLLLGVHEQKQEVV 20 559 LLGVHEQKQEVVKFL 20 566 KQEVVKFLIKKKANL 20 567 QEVVKFLIKKKANLN 20 577 KANLNALDRYGRTAL 20 580 LNALDRYGRTALILA 20 588 RTALILAVCCGSASI 20 589 TALILAVCCGSASIV 20 599 SASIVNLLLEQNVDV 20 600 ASIVNLLLEQNVDVS 20 608 EQNVDVSSQDLSGQT 20 635 ICELLSDYKEKQMLK 20 658 VITILNIKLPLKVEE 20 665 KLPLKVEEEIKKHGS 20 671 EEEIKKHGSNPVGLP 20 679 SNPVGLPENLTNGAS 20 681 PVGLPENLTNGASAG 20 685 PENLTNGASAGNGDD 20 740 NTGISQDEILTNKQK 20 745 QDEILTNKQKQIEVA 20 753 QKQIEVAEKEMNSEL 20 760 EKEMNSELSLSHKKE 20 774 EEDLLRENSMLREEI 20 780 ENSMLREEIAKLRLE 20 788 IAKLRLELDETKHQN 20 792 RLELDETKHQNQLRE 20 806 ENKILEEIESVKEKL 20 807 NKILEEIESVKEKLL 20 810 LEEIESVKEKLLKTI 20 823 TIQLNEEALTKTKVA 20 833 KTKVAGFSLRQLGLA 20 843 QLGLAQHAQASVQQL 20 851 QASVQQLCYKWNHTE 20 873 EQEVAGFSLRQLGLA 20 883 QLGLAQHAQASVQQL 20 957 TPSLVRLASGARAAA 20 958 PSLVRLASGARAAAL 20 960 LVRLASGARAAALPP 20 996 ILPVPTFSSGSFLGR 20 7 SHQHILLPTQATFAA 18 65 GTAARKEFSTTLTGH 18 66 TAARKEFSTTLTGHS 18 75 TLTGHSALSLSSSRA 18 78 GHSALSLSSSRALPG 18 125 VQRRLEVPRPQAAPA 18 160 CLRAQGLTRAFQVVH 18 200 CAQGLEAASANLPGA 18 216 GRSSSCALRYRSGPS 18 225 YRSGPSVSSAPSPAE 18 236 SPAEPPAHQRLLFLP 18 249 LPRAPQAVSGPQEQP 18 264 SEEALGVGSLSVFQL 18 269 GVGSLSVFQLHLIQC 18 400 GTWGDYDDSAFMEPR 18 406 DDSAFMEPRYHVRRE 18 411 MEPRYHVRREDLDKL 18 446 TDMNKRDKQKRTALH 18 452 DKQKRTALHLASANG 18 464 ANGNSEVVQLLLDRR 18 469 EVVQLLLDRRCQLNV 18 518 DEYGNTALHYAIYNE 18 523 TALHYAIYNEDKLMA 18 541 LLYGADIESKNKCGL 18 596 CCGSASIVNLLLEQN 18 607 LEQNVDVSSQDLSGQ 18 611 VDVSSQDLSGQTARE 18 638 LLSDYKEKQMLKISS 18 652 SENSNPVITILNIKL 18 682 VGLPENLTNGASAGN 18 696 NGDDGLIPQRKSRKP 18 721 EEYHSDEQNDTQKQL 18 724 HSDEQNDTQKQLSEE 18 737 EEQNTGISQDEILTN 18 742 GISQDEILTNKQKQI 18 743 ISQDEILTNKQKQIE 18 756 IEVAEKEMNSELSLS 18 771 HKKEEDLLRENSMLR 18 815 SVKEKLLKTIQLNEE 18 824 IQLNEEALTKTKVAG 18 835 KVAGFSLRQLGLAQH 18 840 SLRQLGLAQHAQASV 18 859 YKWNHTEKTEQQAQE 18 875 EVAGFSLRQLGLAQH 18 880 SLRQLGLAQHAQASV 18 899 YKWGHTEKTEQQAQE 18 911 AQEQGAALRSQIGDP 18 954 SPGTPSLVRLASGAR 18 985 KQKSVCDSSGWILPV 18 1010 RRCPMFDVSPAMRLK 18 1017 VSPAMRLKSDSNRET 18 1068 KCRPGTLCHTDTPPH 18 1090 HRHTTTLPHRDTTTS 18 1094 TTLPHRDTTTSLPHF 18 1099 RDTTTSLPHFHVSAG 18 350 QCVFAKKKNVDKWDD 17 40 PVTWRKEPAVLPCCN 16 114 AGAFLLGWERVVQRR 16 190 RNSYRLTHVRCAQGL 16 273 LSVFQLHLIQCIPNL 16 359 VDKWDDFCLSEGYGH 16 368 SEGYGHSFLIMKETS 16 372 GHSFLIMKETSTKIS 16 517 QDEYGNTALHYAIYN 16 524 ALHYAIYNEDKLMAK 16 583 LDRYGRTALILAVCC 16 712 NQQFPDTENEEYHSD 16 858 CYKWNHTEKTEQQAQ 16 898 CYKWGHTEKTEQQAQ 16 992 SSGWILPVPTFSSGS 16 1012 CPMFDVSPAMRLKSD 16 1040 DLPFFKTQQSPRHTK 16 1041 LPFFKTQQSPRHTKD 16 373 HSFLIMKETSTKISG 15 438 DLIVMLRDTDMNKRD 15 472 QLLLDRRCQLNVLDN 15 481 LNVLDNKKRTALIKA 15 571 KFLIKKKANLNALDR 15 1062 AGVLAPKCRPGTLCH 15 1093 TTTLPHRDTTTSLPH 15 8 HQHILLPTQATFAAA 14 9 QHILLPTQATFAAAT 14 29 LTTVSNPSRADPVTW 14 58 GSWLSFPGTAARKEF 14 73 STTLTGHSALSLSSS 14 87 SRALPGSLPAFADLP 14 91 PGSLPAFADLPRSCP 14 116 AFLLGWERVVQRRLE 14 126 QRRLEVPRPQAAPAT 14 128 RLEVPRPQAAPATSA 14 158 AACLRAQGLTRAFQV 14 169 AFQVVHLAPTAPDGG 14 192 SYRLTHVRCAQGLEA 14 201 AQGLEAASANLPGAP 14 244 QRLLFLPRAPQAVSG 14 253 PQAVSGPQEQPSEEA 14 272 SLSVFQLHLIQCIPN 14 277 QLHLIQCIPNLSYPL 14 289 YPLVLRHIPEILKFS 14 293 LRHIPEILKFSEKET 14 311 ILGLEPATAARLSG 14 313 GLELPATAARLSGLN 14 326 LNSIMQIKEFEELVK 14 336 EELVKLHSLSHKVIQ 14 345 SHKVIQCVFAKKKNV 14 356 KKNVDKWDDFCLSEG 14 389 IQEMGSGKSNVGTWG 14 436 RKDLIVMLRDTDMNK 14 458 ALHLASANGNSEVVQ 14 467 NSEVVQLLLDRRCQL 14 468 SEVVQLLLDRRCQLN 14 480 QLNVLDNKKRTALIK 14 490 TALIKAVQCQEDECV 14 493 IKAVQCQEDECVLML 14 503 CVLMLLEHGADGNIQ 14 504 VLMLLEHGADGNIQD 14 505 LMLLEHGADGNIQDE 14 522 NTALHYAIYNEDKLM 14 533 DKLMAKALLLYGADI 14 538 KALLLYGADIESKNK 14 552 KCGLTPLLLGVHEQK 14 557 PLLLGVHEQKQEVVK 14 590 ALILAVCCGSASIVN 14 592 ILAVCCGSASIVNLL 14 603 VNLLLEQNVDVSSQD 14 604 NLLLEQNVDVSSQDL 14 610 NVDVSSQDLSGQTAR 14 625 EYAVSSHHHVICELL 14 631 HHHVICELLSDYKEK 14 636 CELLSDYKEKQMKLI 14 645 KQMLKISSENSNPVI 14 656 NPVITILNIKLPLKV 14 667 PLKVEEEIKKHGSNP 14 698 DDGLIPQRKSRKPEN 14 781 NSMLREEIAKLRLEL 14 785 REEIAKLRLELDETK 14 817 KEKLLKTIQLNEEAL 14 818 EKLLKTIQLNEEALT 14 821 LKTIQLNEEALTKTK 14 838 GFSLRQLGLAQHAQA 14 878 GFSLRQLGLAQHAQA 14 891 QASVQQLCYKWGHTE 14 919 RSQIGDPGGVPLSEG 14 925 PGGVPLSEGGTAAGD 14 927 GVPLSEGGTAAGDQG 14 986 QKSVCDSSGWILPVP 14 1011 RCPMFDVSPAMRLKS 14 1071 PGTLCHTDTPPHRNA 14 1102 TTSLPHFHVSAGGVG 14 1107 HFHVSAGGVGPTTLG 14 1112 AGGVGPTTLGSNREI 14 V12A-HLA-DRB1-15mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 1 MLQVVNISPSISWL 21 2 MLQVVNISPSISWLI 19 9 SPSISWLIMLFSSVY 18 11 SISWLIMLFSSVYMM 16 12 ISWLIMLFSSVYMMT 13 10 PSISWLIMLFSSVYM 12 15 LIMLFSSVYMMTLIQ 9 V12B-DRB1-1101-15mers: 251P5G2 Each peptide is a portion of SEQ ID NO: 25; each start position is specified, the length of peptide is 15 amino acids, and the end position for each peptide is the start position plus fourteen. 332 IKEFEELVKLHSLSH 32 286 NLSYPLVLRHIPEIL 31 94 LPAFADLPRSCPESE 30 567 QEVVKFLIKKKANLN 27 428 AAWWGKVPRKDLIVM 24 166 LTRAFQVVHLAPTAP 23 954 SPGTPSLVRLASGAR 22 118 LLGWERVVQRRLEVP 21 338 LVKLHSLSHKVIQCV 21 371 YGHSFLIMKETSTKI 21 436 RKDLIVMLRDTDMNK 21 480 QLNVLDNKKRTALIK 21 656 NPVITILNIKLPLKV 21 1002 FSSGSFLGRRCPMFD 21 1014 MFDVSPAMRLKSDSN 21 29 LTTVSNPSRADPVTW 20 125 VQRRLEVPRPQAAPA 20 192 SYRLTHVRCAQGLEA 20 243 HQRLLFLPRAPQAVS 20 293 LRHIPEILKFSEKET 20 297 PEILKFSEKETGGGI 20 419 REDLDKLHRAAWWGK 20 502 ECVLMLLEHGADGNI 20 526 HYAIYNEDKLMAKAL 20 577 KANLNALDRYGRTAL 20 641 DYKEKQMLKISSENS 20 667 PLKVEEEIKKHGSNP 20 668 LKVEEEIKKHGSNPV 20 771 HKKEEDLLRENSMLR 20 841 LRQLGLAQHAQASVQ 20 881 LRQLGLAQHAQASVQ 20 960 LVRLASGARAAALPP 20 386 SGLIQEMGSGKSNVG 19 623 AREYAVSSHHHVICE 19 169 AFQVVHLAPTAPDGG 18 382 STKISGLIQEMGSGK 18 501 DECVLMLLEHGADGN 18 569 VVKFLIKKKANLNAL 18 589 TALILAVCCGSASIV 18 644 EKQMLKISSENSNPV 18 858 CYKWNHTEKTEQQAQ 18 891 QASVQQLCYKWGHTE 18 898 CYKWGHTEKTEQQAQ 18 992 SSGWILPVPTFSSGS 18 993 SGWILPVPTFSSGSF 18 1105 LPHFHVSAGGVGPTT 18 40 PVTWRKEPAVLPCCN 17 119 LGWERWQRRLEVPR 17 167 TRAFQVVHLAPTAPD 17 190 RNSYRLTHVRCAQGL 17 442 MLRDTDMNKRDKQKR 17 486 NKKRTALIKAVQCQE 17 563 HEQKQEVVKFLIKKK 17 583 LDRYGRTALILAVCC 17 746 DEILTNKQKQIEVAE 17 836 VAGFSLRQLGLAQHA 17 876 VAGFSLRQLGLAQHA 17 1090 HRHTTTLPHRDTTTS 17 16 QATFAAATGLWAALT 16 23 TGLWAALTTVSNPSR 16 57 KGSWLSFPGTAARKE 16 62 SFPGTAARKEFSTTL 16 69 RKEFSTTLTGHSALS 16 189 SRNSYRLTHVRCAQG 16 216 GRSSSCALRYRSGPS 16 222 ALRYRSGPSVSSAPS 16 299 ILKFSEKETGGGILG 16 334 EFEELVKLHSLSHKV 16 349 IQCVFAKKKNVDKWD 16 359 VDKWDDFCLSEGYGH 16 372 GHSFLIMKETSTKIS 16 402 WGDYDDSAFMEPRYH 16 408 SAFMEPRYHVRREDL 16 517 QDEYGNTALHYAIYN 16 555 LTPLLLGVHEQKQEV 16 698 DDGLIPQRKSRKPEN 16 782 SMLREEIAKLRLELD 16 1060 DRAGVLAPKCRPGTL 16 1099 RDTTTSLPHFHVSAG 16 36 SRADPVTWRKEPAVL 15 80 SALSLSSSRALPGSL 15 115 GAFLLGWERVVQRRL 15 147 DPSPPCHQRRDAACL 15 287 LSYPLVLRHIPEILK 15 418 RREDLDKLHRAAWWG 15 445 DTDMNKRDKQKRTAL 15 447 DMNKRDKQKRTALHL 15 468 SEVVQLLLDRRCQLN 15 469 EVVQLLLDRRCQLNV 15 530 YNEDKLMAKALLLYG 15 552 KCGLTPLLLGVHEQK 15 568 EVVKFLIKKKANLNA 15 625 EYAVSSHHHVICELL 15 635 ICELLSDYKEKQMLK 15 725 SDEQNDTQKQLSEEQ 15 753 QKQIEVAEKEMNSEL 15 763 MNSELSLSHKKEEDL 15 814 ESVKEKLLKTIQLNE 15 825 QLNEEALTKTKVAGF 15 834 TKVAGFSLRQLGLAQ 15 874 QEVAGFSLRQLGLAQ 15 940 QGPGTHLPPREPRAS 15 977 GKNGRSPTKQKSVCD 15 1046 TQQSPRHTKDLGQDD 15 1074 LCHTDTPPHRNADTP 15
[1061] 60 TABLE L Protein Properties of 251P5G2 Bioinformatic Program URL Outcome ORF ORF finder 722-1489 Protein length 255aa Transmembrane TM Pred http://www.ch.embnet.org/ 6TM, N-terminal outside, aa 7-29, region 43-62, 82-100, 118-138, 158-176, 180-199 HMMTop http://www.enzim.hu/hmmtop/ 6TM, aa 7-29, 38-58, 85-109, 118-142, 169-193, 224-243 Sosui http://www.genome.ad.jp/SOSui/ 5TM, aa 6-28, 39-61, 86-108, 119-141, 166-188 TMHMM http://www.cbs.dtu.dk/services/TMHMM 5TM, N terminal inside, aa 7-29, 44-62, 83-102, 117-139, 159-181 Signal Peptide Signal P http://www.cbs.dtu.dk/services/SignalP/ cleavage between aa 129-130 pl pl/MW tool http://www.expasy.ch/tools/ pl 9.4 Molecular weight pl/MW tool http://www.expasy.ch/tools/ 29.3 kD Localization PSORT http://psort.nibb.ac.jp/ microbody (peroxisome) 74.8%, mitochondrial inner membrane 71.4%, plasma membrane 65.0%, mitochondrial intermembrane 30.4% PSORT II http://psort.nibb.ac.jp/ 44.4%: endoplasmic reticulum, 22.2%: mitochondrial, 22.2%: Golgi, 11.1%: nuclear Motifs Pfam http://www.sanger.ac.uk/Pfam/ Vomeronasal organ pheromone receptor family, V1R Prints http://www.biochem.ucl.ac.uk/ Rhodopsin-like GPCR superfamily signature Blocks http://www.blocks.fhcrc.org/ lodothyronine deiodinase aa 2-29
[1062] 61 TABLE LI Exon compositions of 251P5G2 v.1 Exon number Start End 1 1 2156
[1063] 62 TABLE LII(a). Nucleotide sequence of transcript variant 251P5G2 v.12 (SEQ ID NO: 70) gttttttttt tttttttttt tttttttttt tattttaagg gattcgttta ataggacttg 60 tggtaagtgg aataatgcca tgcaaaggtc cccatgtcta accaccagyt tctaggcatg 120 tattatggta tatgagaaat gggaattcag gctgcagatg aaatcaaggt tgataaccag 180 ctgactctaa aacaaaaaca ttaacttgaa ttacagattt gggcctaatg taattataag 240 cattcttaaa agtgaaagaa ataataagag aaactgagtg ctgtgatgtg agtcagttaa 300 actttttttt caactttttc tttaggtgat tattttccct taacataaaa tttactttag 360 ctcaactata caaacatgtg agttattgtt atgtaaccat cactcttcat taagaaatgc 420 tttgtaaaaa gtgagccagt ttttcatata cattcttcaa aatacattct caacattata 480 catcaaatta tatatacata catgcacaca tacactatat atatcaagga tttatatgag 540 aggattaatt aagaaaaaaa ttagtggaat aaaaataatg tttatgataa ttttggccat 600 agaatatata atacagatga tgtgaagtac aaaatgtttt ttatacttca tattttgatg 660 tacaaagtat gtttgtcttt gtaattcaga tgattacttt gcacttgtgt tcccatgaaa 720 aatgcctttc atttctaagc tggtattggc atctcagcca acacttttct ccttcttttc 780 tgcgtcttct ccttttctgc tttttctgga tctcaggcca gagcgcactt acctaccagt 840 ctgtcatgtg gccctcatcc acatggtggt ccttctcacc atggtgttct tgtctccaca 900 gctctttgaa tcactgaatt ttcagaatga cttcaaatat gaggcatctt tctacctgag 960 gagggtgatc agggtcctct ccatttgtac cacctgcctc ctggacatgc tgcaggtcgt 1020 caacatcagc cccagcattt cctggttgat aatgctgttc tcaagtgtct acatgatgac 1080 tctcattcag gaactacagg agatcctggt accttcacag ccccagcctc tacctaagga 1140 tctttgcaga ggcaagagcc atcagcacat cctgctgccg actcaagcaa cttttgctgc 1200 agcaactgga ctatgggctg cactaaccac cgtatcaaat ccaagcagag cagatcctgt 1260 gacctggaga aaggagccgg ctgtccttcc ctgctgtaac ctagagaaag gaagctggct 1320 gtccttccct ggcacagctg cacgcaagga attttccacc acgctcaccg ggcacagcgc 1380 gctgagcctc tccagttcgc gggccctccc cggctcgctc ccggctttcg cagacctccc 1440 ccgctcctgc cctgagtCCg agcagagcgC aacgccagcc ggcgcCttCC tcctgggctg 1500 ggagcgagtg gtgcagcggc ggctcgaagt cccccggcct caagcagccc ccgcgactag 1560 cgcgacaccc tcgcgggatc cgagtccacc ctgccaccag cgccgggacg ccgcgtgcct 1620 cagagcccaa gggctgaccc gggccttcca ggtggtccat ctcgctccta cggctcccga 1680 cggtggcgct gggtgtcccc catcccgcaa ttcctaccgg ctgacccatg tgcgctgcgc 1740 ccaggggctg gaggctgcca gcgccaacct tcccggcgct ccggggcgga gcagctcctg 1800 cgccctgcgc taccgcagcg gcccttcagt cagctccgcg ccgtcccccg cagagccccc 1860 ggcgcaccag cgcctgcttt tccttccccg agcgcctcaa gcagtctctg ggccgcagga 1920 acagccctct gaagaggcgc ttggtgtagg aagcctctca gttttccagt tacacctaat 1980 acagtgtatt ccaaatctaa gttacccact agtacttcgg cacattccag aaattctgaa 2040 attttctgaa aaggaaactg gtggtggaat tctaggctta gaattaccag cgacagctgc 2100 tcgcctctca ggattaaaca gcataatgca aatcaaagag tttgaagaat tggtaaaact 2160 tcacagcttg tcacacaaag tcattcagtg tgtgtttgca aagaaaaaaa atgtagacaa 2220 atgggatgac ttttgtctta gtgagggtta tggacattca ttcttaataa tgaaagaaac 2280 gtcgactaaa atatcaggtt taattcagga gatggggagc ggcaagagca acgtgggcac 2340 ttggggagac tacgacgaca gcgccttcat ggagccgagg taccacgtcc gtcgagaaga 2400 tctggacaag ctccacagag ctgcctggtg gggtaaagtc cccagaaagg atctcatcgt 2460 catgctcagg gacactgaca tgaacaagag ggacaagcaa aagaggactg ctctacattt 2520 ggcctctgcc aatggaaatt cagaagtagt acaactcctg ctggacagac gatgtcaact 2580 taacgtcctt gacaacaaaa aaaggacagc tctgataaag gccgtacaat gccaggaaga 2640 tgaatgtgtg ttaatgttgc tggaacatgg cgctgatgga aatattcaag atgagtatgg 2700 aaataccgct ctacactatg ctatctacaa tgaagataaa ttaatggcca aagcactgct 2760 cttatatggt gctgatattg aatcaaaaaa caagtgtggc ctcacaccac ttttgcttgg 2820 cgtacatgaa caaaaacagg aagtggtgaa atttttaatc aagaaaaaag ctaatttaaa 2880 tgcacttgat agatatggaa gaactgccct catacttgct gtatgttgtg gatcagcaag 2940 tatagtcaat cttctacttg agcaaaatgt tgatgtatct tctcaagatc tatctggaca 3000 gacggccaga gagtatgctg tttctagtca tcatcatgta atttgtgaat tactttctga 3060 ctataaagaa aaacagatgc taaaaatctc ttctgaaaac agcaatccag tgataaccat 3120 ccttaatatc aaacttccac tcaaggttga agaagaaata aagaagcatg gaagtaatcc 3180 tgtgggatta ccagaaaacc tgactaatgg tgccagtgct ggcaatggtg atgatggatt 3240 aattccacaa aggaagagca gaaaacctga aaatcagcaa tttcctgaca ctgagaatga 3300 agagtatcac agtgacgaac aaaatgatac ccagaaacaa ctttctgaag aacagaacac 3360 tggaatatca caagatgaga ttctgactaa taaacaaaag cagatagaag tggctgaaaa 3420 ggaaatgaat tctgagcttt ctcttagtca taagaaagaa gaagatctct tgcgtgaaaa 3480 cagcatgttg cgggaagaaa ttgccaagct aagactggaa ctagatgaaa caaaacatca 3540 gaaccagcta agggaaaata aaattttgga ggaaattgaa agtgtaaaag aaaaacttct 3600 aaagactata caactgaatg aagaagcatt aacgaaaacc aaggtggctg gtttctcttt 3660 gcgccagctt ggccttgccc agcatgcaca agcctcagtg caacagctgt gctacaaatg 3720 gaaccacaca gagaaaacag agcagcaggc tcaggagcag gaggtggctg gtttctcttt 3780 ycgccagctt ggccttgccc agcatgcaca agcctcagta caacaactgt gctacaaatg 3840 gggccacaca gagaaaacag agcagcaggc tcaggagcag ggagctgcgc tgaggtccca 3900 gataggcgac cctggcgggg tgcccctgag cgaagggggg acagcagcag gagaccaggg 3960 tccagggacc cacctcccac cgagggaacc tcgagcctcc cctggcaccc ctagcttggt 4020 ccgcctggcc tccggagccc gagctgctgc gcttccccca cccacaggga aaaacggccg 4080 atctccaacc aaacagaaat ctgtgtgtga ctcctctggt tggatactgc cagtccccac 4140 attttcttcc gggagttttc ttggcagaag gtgcccaatg tttgatgttt cgccagccat 4200 gaggctgaaa agtgacagca atagagaaac acatcagyct ttccgcgaca aagatgacct 4260 tcccttcttc aaaactcagc aatctccacg gcacacaaag gacttaggac aagatgaccg 4320 agctggagtg ctcgccccaa aatgcaggcc cggaacactc tgccacacgg acacaccacc 4380 acacagaaat geggacacac caccacacag acacaccacc acyctyccac acagagacac 4440 caccacatcg ttgccacact ttcatgtgtc agctggcggt gtgggcccca cgactctggg 4500 ctctaataga gaaattactt ag 4522
[1064] 63 TABLE LIII(a) Nucleotide seguence alignment of 251P5G2 v.1 (SEQ ID NO: 71) and 251P5G2 v.12 (SEQ ID NO: 72) Score = 2009 bits (1045), Expect = 0.0 Identites = 1047/1048(99%) Strand = Plus/Plus 251P5G2v.1: 1 gttttttttttttttttttttttttttttttattttaagggattcgtttaataggacttg 60 ||||||||||||||||||||||||||||||||||||||||||||||||| 251P5G2v.12: 1 gttttttttttttttttttttttttttttttattttaagggattcgtttaataggacttg 60 251P5G2v.1: 61 tggtaagtggaataatgccatgcaaaggtccccatgtctaaccaccaggttctaggcatg 120 ||||||||||||||||||||||||||||||||||||||||||||||||| 251P5G2v12: 61 tggtaagtggaataatyccatgcaaaggtccccatgtctaaccaccaggttctaggcatg 120 251P5G2v.1: 121 tattatggtatatgagaaatgggaattcaggctgcagatgaaatcaaggttgataaccag 180 ||||||||||||||||||||||||||||||||||||||||||||||||| 251P5G2v.12: 121 tattatggtatatgagaaatgggaattcaggctgcagatgaaatcaaggttgataaccag 180 251P5G2v.1: 181 ctgactctaaaacaaaaacattaacttgaattacagatttgggcctaatgtaattataag 240 ||||||||||||||||||||||||||||||||||||||||||||||||| 251P5G2v.12: 181 ctgactctaaaacaaaaacattaacttgaattacagatttgggcctaatgtaattataag 240 251P5G2v.1: 241 cattcttaaaagtgaaagaaataataagagaaactgagtgctgtgatgtgagtcagttaa 300 ||||||||||||||||||||||||||||||||||||||||||||||||| 251P5G2v.12: 241 cattcttaaaagtgaaagaaataataagagaaactgagtgctgtgatgtgagtcagttaa 300 251P5G2v.1: 301 acttttttttcaactttttctttaggtgattattttcccttaacataaaatttactttag 360 ||||||||||||||||||||||||||||||||||||||||||||||||| 251P5G2v.12: 301 acttttttttcaactttttctttaggtgattattttcccttaacataaaatttactttag 360 251P5G2v.1: 361 ctcaactatacaaacatgtgagttattgttatgtaaccatcactcttcattaagaaatgc 420 ||||||||||||||||||||||||||||||||||||||||||||||||| 251P5G2v.12: 361 ctcaactatacaaacatgtgagttattgttatgtaaccatcactcttcattaagaaatgc 420 251P5G2v.1: 421 tttgtaaaaagtgagccagtttttcatatacattcttcaaaatacattctcaacattata 480 ||||||||||||||||||||||||||||||||||||||||||||||||| 251P5G2v.12: 421 tttgtaaaaagtgagccagtttttcatatacattcttcaaaatacattctcaacattata 480 251P5G2v.1: 481 catcaaattatatatacatacatgcacacatacactatatatatcaaggatttatatgag 540 ||||||||||||||||||||||||||||||||||||||||||||||||| 251P5G2v.12: 481 catcaaattatatatacatacatgcacacatacactatatatatcaaggatttatatgag 540 251P5G2v.1: 541 aggattaattaagaaaaaaattagtggaataaaaataatgtttatgataattttggccat 600 ||||||||||||||||||||||||||||||||||||||||||||||||| 251P5G2v.12: 541 aggattaattaagaaaaaaattagtggaataaaaataatgtttatgataattttggccat 600 251P5G2v.1: 601 agaatatataatacagatgatgtgaagtacaaaatgttttttatacttcatattttgatg 660 ||||||||||||||||||||||||||||||||||||||||||||||||| 251P5G2v.12: 601 agaatatataatacagatgatgtgaagtacaaaatgttttttatacttcatattttgatg 660 251P5G2v.1: 661 tacaaagtatgtttgtctttgtaattcagatgattactttgcacttgtgttcccatgaaa 720 ||||||||||||||||||||||||||||||||||||||||||||||||| 251P5G2v.12: 661 tacaaagtatgtttgtctttgtaattcagatgattactttgcacttgtgttcccatgaaa 720 251P5G2v.1: 721 aatgcctttcatttctaagctggtattggcatctcagccaacacttttctccttcttttc 780 ||||||||||||||||||||||||||||||||||||||||||||||||| 251P5G2v.12: 721 aatgcctttcatttctaagctggtattggcatctcagccaacacttttctccttcttttc 780 251P5G2v.1: 781 tgcgtcttctccttttctgctttttctggatctcaggccagagcgcacttacctaccagt 840 ||||||||||||||||||||||||||||||||||||||||||||||||| 251P5G2v.12: 781 tgcgtcttctcottttctgctttttctggatctcaggccagagcgcacttacctaccagt 840 251P5G2v.1: 841 ctgtcatgtggccctcatccacatggtggtccttctcaccatggtgttcttgtctccaca 900 ||||||||||||||||||||||||||||||||||||||||||||||||| 251P5G2v.12: 841 ctgtcatgtggccctcatccacatggtggtccttctcaccatggtgttcttgtctccaca 900 251P5G2v.1: 901 gctctttgaatcactgaattttcagaatgacttcaaatatgaggcatctttctacctgag 960 ||||||||||||||||||||||||||||||||||||||||||||||||| 251P5G2v.12: 901 gctctttgaatcactgaattttcagaatgacttcaaatatgaggcatctttctacctgag 960 251P5G2v.1: 961 gagggtgatcagggtcctctccatttgtaccacctgcctcctgggcatgctgcaggtcgt 1020 ||||||||||||||||||||||||||||||||||||||||||||||||| 251P5G2v.12: 961 gagggtgatcagggtcctctccatttgtaccacctgcctcctggacatgctgcaggtcgt 1020 251P5G2v.1: 1021 caacatcagccccagcatttcctggttg 1048 |||||||||||||||||||||||||||| 251P5G2v.12: 1021 caacatcagccccagcatttcctggttg 1048 Score = 254 bits (132), Expect = 3e-64 Identities 132/132(100%) Strand = Plus/Plus 251P5G2v.1: 1271 ataatgctgttctcaagtgtctacatgatgactctcattcaggaactacaggagatcctg 1330 ||||||||||||||||||||||||||||||||||||||||||||||||| 251P5G2v.12: 1049 ataatgctgttctcaagtgtctacatgatgactctcattcaggaactacaggagatcctg 1108 251P5G2v.1: 1331 gtaccttcacagccccagcctctacctaaggatctttgcagaggcaagagccatcagcac 1390 ||||||||||||||||||||||||||||||||||||||||||||||||| 251P5G2v.12: 1109 gtaccttcacagccccagcctctacctaaggatctttgcagaggcaagagccatcagcac 1168 251P5G2v.1: 1391 atcctgctgccg 1402 |||||||||||| 251P5G2v.12: 1169 atcctgctgccg 1180
[1065] 64 TABLE LIV(a) Peptide sequences of protein coded by 251P5G2 v.12 (SEQ ID NO: 73) MPFISKLVLA SQPTLFSFFS ASSPFLLFLD LRPERTYLPV CHVALIHMVV LLTMVFLSPQ 60 LFESLNFQND FKYEASFYLR RVTRVLSICT TCLLDMLQVV NTSPSISWLI MLFSSVYMMT 120 LIQELQEILV PSQPQPLPKD LCRGKSHQHI LLPTQATFAA ATGLWAALTT VSNPSRADPV 180 TWRKEPAVLP CCNLEKGSWL SFPGTAARKE FSTTLTGHSA LSLSSSRALP GSLPAFADLP 240 RSCPESEQSA TPAGAFLLGW ERVVQRRLEV PRPQAAPATS ATPSRDPSPP CHQRRDAACL 300 RAQGLTRAFQ VVHLAPTAPD GGAGCPPSRN SYRLTHVRCA QGLEAASANL PGAPGRSSSC 360 ALRYRSGPSV SSAPSPAEPP AHQRLLFLPR APQAVSGPQE QPSEEALGVG SLSVFQLHLI 420 QCIPNLSYPL VLRHIPEILK FSEKETGGGI LGLELPATAA RLSGLNSIMQ IKEFEELVKL 480 HSLSHKVIQC VFAKKKNVDK WDDFCLSEGY GHSFLIMKET STKISGLIQE MGSGKSNVGT 540 WGDYDDSAFM EPRYHVRRED LDKLHRAAWW GKVPRKDLIV MLRDTDMNKR DKQKRTALHL 600 ASANGNSEVV QLLLDRRCQL NVLDNKKRTA LIKAVOCQED ECVLMLLEHG ADGNIQDEYG 660 NTALHYAIYN EDKLMAKALL LYGADIESKN KCGLTPLLLG VHEQKQEVVK FLIKKKANLN 720 ALDRYGRTAL ILAVCCGSAS IVNLLLEQNV DVSSQDLSGQ TAREYAVSSH HHVICELLSD 780 YKEKQMLKIS SENSNPVITI LNIKLPLKVE EEIKKHGSNP VGLPENLTNG ASAGNGDDGL 840 IPQRKSRKPE MQQFPDTENE EYHSDEQNDT QKQLSEEQNT GISQDEILTN KQKQIEVAEK 900 EMNSELSLSH KKEEDLLREH SMLREEIAKL RLELDETKHQ NQLRENKILE EIESVKEKLL 960 KTIQLNEEAL TKTKVAGFSL RQLGLAQHAQ ASVQQLCYKW NHTEKTEQQA QEQEVAGFSL 1020 RQLGLAQHAQ ASVQQLCYKW GHTEKTEQQA QEQGAALRSQ IGDPGGVPLS EGGTAAGDQG 1080 PGTHLPPREP RASPGTPSLV RLASGARAAA LPPPTGKNGR SPTKQKSVCD SSGWILPVPT 1140 FSSGSFLGRR CPMFDVSPAM RLKSDSNRET HQAFRDKDDL PFFKTQQSPR HTKDLGQDDR 1200 AGVLAPKCRP GTLCHTDTPP HRNADTPPHR HTTTLPHRDT TTSLPHFHVS AGGVGPTTLG 1260 SNREIT 1266
[1066] 65 TABLE LV(a) Amino acid seguence alignment of 121P1F1 v.1 (SEQ ID NO: 74) and 251P5G2 v.12 (SEQ ID NO: 75) Score = 269 bits (688), Expect = 2e−71 Identities = 152/227 (66%), Positives = 152/227 (66%), Gaps = 74/227 (32%) 251P5G2v.1: 1 MPFISKLVLASQPTLFSFFSASSPFLLFLDLRPERTYLPVCHVALIHMVVLLTMVFLSPQ 60 MPFISKLVLASQPTLFSFFSASSPFLLFLDLRPERTYLPVCHVALIHMVVLLTMVFLSPQ 251P5G2v.12: 1 MPFISKLVLASQPTLFSFFSASSPFLLFLDLRPERTYLPVCHVALIHMVVLLTMVFLSPQ 60 251P5G2v.1: 61 LFESLNFQNDFKYEASFYLRRVIRVLSICTTCLLCMLQVVNISPSISWLVRFKWKSTIFT 120 LFESLNFQNDFKYEASFYLRRVIRVLSICTTCLL MLQVVNISPSISWL 251P5G2v.12: 61 LFESLNFQNDFKYEASFYLRRVIRVLSICTTCLLDMLQVVNISPSISWL----------- 109 251P5G2v.1: 121 FHLFSWSLSFPVSSSLIFYTVASSNVTQINLHVSKYCSLFPINSIIRGLFFTLSLFRDVF 180 251P5G2v.12: 109 ------------------------------------------------------------ 109 251P5G2v.1: 181 LKQIMLFSSVYMMTLIQELQEILVPSQPQPLPKDLCRGKSHQHILLP 227 IMLFSSVYMMTLIQELQEILVPSQPQPLPKDLCRGKSHQHILLP 251P5G2v.12: 110 ---IMLFSSVYMMTLIQELQEILVPSQPQPLPKDLCRGKSHQHILLP 153
[1067] 66 TABLE LII(b) Nucleotide seguence of transcript variant 251P5G2v.13 (SEQ ID NO: 76) atgcctttca tttctaagct ggtattggca tctcagccaa cacttttctc cttcttttct 60 gcgtcttctc cttttctgct ttttctggat ctcaggccag agcgcactta cctaccagtc 120 tgtcatgtgg ccctcatcca catggtggtc cttctcacca tggtgttctt gtctccacag 180 ctctttgaat cactgaattt tcagaatgac ttcaaataty aggcatcttt ctacctgagg 240 agggtgatca gggtcctctc catttgtacc acctgcctcc tggacatgct ycaggtcgtc 300 aacatcagcc ccagcatttc ctggttgata atgctgttct caagtgtcta catgatgact 360 ctcattcagg aactacagga gatcctggta ccttcacagc cccagcctct acctaaggat 420 dtttgcagag gcaagagcca tcagcacatc ctgctgccga ctcaagcaac ttttgctgca 480 gcaactggac tatgggctgc actaaccacc gtatcaaatc caagcagagc agatcctgtg 540 acctggagaa aggagccggc tgtccttccc tgctgtaacc tagagaaagg aagctggctg 600 tccttccctg gcacagctgc acgcaaggaa ttttccacca cgctcaccgy gcacaycgcg 660 ctgagcctct ccagttcgcg ggccctcccc ggctcgctcc cggctttcgc agacctcccc 720 cgctcctgcc ctgagtccga gcagagcgca acyccagecy gcgccttcct cctyggctgg 780 yagcgagtgg tgcagcggcg gctcgaagtc ccccggcctc aagcagcccc cgcgactagc 840 gcgacaccct cgcgggatcc gagtccaccc tgccaccagc gccgggacgc cgcgtgcctc 900 agagcccaag ggctgacccg ggccttccag gtggtccatc tcgctcctac ggctcccgac 960 ggtggcgctg ggtgtccccc atcccgcaat tcctaccggc tgacccatgt gcgctgcgcc 1020 caggggctgg aggctgccag cgccaacctt cccggcgctc cggggcggag cagctcctgc 1080 gccctgcgct accgcagcgg cccttcagtc agctccgcgc cgtcccccgc agagcccccg 1140 gcgcaccagc gcctgctttt ccttccccga gcgcctcaag cagtctctgg gccgcaggaa 1200 cagccctctg aagaggcgct tggtgtagga agcctctcag ttttccagtt acacctaata 1260 cagtgtattc caaatctaag ttacccacta 4tacttcggc acattccaga aattctgaaa 1320 ttttctgaaa agyaaactgg tgytggaatt ctaggcttag aattaccagc gacagctgct 1380 cgcctctcag gattaaacag cataatgcaa atcaaagagt ttgaagaatt ggtaaaactt 1440 cacagcttgt cacacaaagt cattcagtgt gtgtttgcaa agaaaaaaaa tgtagacaaa 1500 tgggat9act tttgtcttag tgagggttat ggacattcat tcttaataat gaaagaaacg 1560 tcgactaaaa tatcaggttt aattcaggag atggggagcg gcaagagcaa cgtgggcact 1620 tggggagact acyacgacag cyccttcatg gagccgaggt accacgtccg tcgagaagat 1680 ctggacaagc tccacagagc tgcctggtgg ggtaaagtcc ccagaaagga tctcatcgtc 1740 atgctcaggg acactyacat gaacaagagg gacaagcaaa agaggactgc tctacatttg 1800 gcctctgcca atggaaattc agaagtagta caactcctgc tggacagacg atgtcaactt 1860 aacgtccttg acaacaaaaa aaggacagct ctgataaagg ccgtacaatg ccaggaagat 1920 gaatgtgtgt taatgttgct ggaacatggc gctyatggaa atattcaaga tgagtatgga 1980 aataccgctc tacactatgc tatctacaat gaagataaat taatggccaa agcactgctc 2040 ttatatggtg ctgatattga atcaaaaaac aagtytggcc toacaccact tttgcttggc 2100 gtacatgaac aaaaacagga agtggtgaaa tttttaatca agaaaaaagc taatttaaat 2160 gcacttgata gatatggaag aactgccctc atacttgctg tatgttgtgg atcagcaagt 2220 atagtcaatc ttctacttga gcaaaatgtt gatgtatctt ctcaagatct atctggacag 2280 acggccagag agtatgctgt ttctagtcat catcatgtaa tttgtgaatt actttctgac 2340 tataaagaaa aacagatgct aaaaatctct tctgaaaaca gcaatccagt gataaccatc 2400 cttaatatca aacttccact caaggttgaa gaagaaataa agaagcatgg aagtaatcct 2460 gtgggattac cagaaaacct gactaatggt gccagtgctg gcaatggtga tgatggatta 2520 attccacaaa ggaagagcag aaaacctgaa aatcagcaat ttcctgacac tgagaatgaa 2580 gagtatcaca gtgacgaaca aaatgatacc cagaaacaac tttctgaaga acagaacact 2640 ggaatatcac aagatgagat tctgactaat aaacaaaagc agatagaagt ggctgaaaag 2700 gaaatgaatt ctgagctttc tcttagtcat aagaaagaag aagatctctt gcgtgaaaac 2760 agcatgttgc gggaagaaat tgccaagcta agactggaac tagatgaaac aaaacatcag 2820 aaccagctaa gggaaaataa aattttggag gaaattgaaa gtgtaaaaga aaaacttcta 2880 aagactatac aactgaatga agaagcatta acgaaaacca aggtggctgg tttctctttg 2940 cgccagcttg gccttgccca gcatgcacaa gcctcagtgc aacagctgtg ctacaaatgg 3000 aaccacacag agaaaacaga gcagcaggct caggagcagg aggtggctgg tttctctttg 3060 cgccagcttg gccttgccca gcatgcacaa gcctcagtac aacaactgtg ctacaaatgg 3120 ggccacacag agaaaacaga gcagcaggct caggagcagg gagct9cgct gaggtcccag 3180 ataggcgacC ctggcggggt gcccctgagc gaagggggga cagcagcagg agaccagggt 3240 ccaggyacCC acctcccacC gagggaacct cgagcctccc ctggcacccc tagcttggtc 3300 cgcctggcct ccggagcccg agctgctgcg cttcccccac ccacagggaa aaacggccga 3360 tctccaacca aacagaaatc tgtgtgtgac tcctctggtt ggatactgcc agtccccaca 3420 ttttcttccg ggagttttct tggcagaayg tgcccaatgt ttgatgtttc gccagccatg 3480 aggctgaaaa gtgacagcaa tagagaaaca catcaggctt tccgcgacaa agatgacctt 3540 cccttcttca aaactcayca atctccacgg cacacaaagg acttaggaca agatgaccga 3600 gctggagtgc tcgccccaaa atgcaggccc ggaacactct gccacacgga cacaccacca 3660 cacagaaatg cggacacacc accacacaga cacaccacca cgctgccaca cagagacacc 3720 accacatcgt tgccacactt tcatgtgtca gctggcggtg tgggccccac gactctgggc 3780 tctaatagag aaattactta g 3801
[1068] 67 TABLE LIII(b) Nucleotide sequence alignment of 251P5G2 v.1 (SEQ ID NO: 77) and 251P5G2 v.13 (SEQ ID NO: 78) Score = 623 bits (324), Expect = e−175 Identities = 326/327 (99%) Strand = Plus/Plus Query: 722 atgcctttcatttctaagctggtattggcatctcagccaacacttttctccttcttttct 781 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 1 atgcctttcatttctaagctggtattggcatctcagccaacacttttctccttcttttct 60 Query: 782 gcgtcttctccttttctgctttttctggatctcaggccagagcgcacttacctaccagtc 841 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 61 ggtcttctccttttctgctttttctggatctcaggccagagcgcacttacctaccagtc 120 Query: 842 tgtcatgtggccctcatccacatggtggtccttctcaccatggtgttcttgtctccacag 901 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 121 tgtcatgtggccctcatccacatggtggtccttctcaccatggtgttcttgtctccacag 180 Query: 902 ctctttgaatcactgaattttcagaatgacttcaaatatgaggcatctttctacctgagg 961 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 181 ctctttgaatcactgaattttcagaatgacttcaaatatgaggcatctttctacctgagg 240 Query: 962 agggtgatcagggtcctctccatttgtaccacctycctcctgggcatgctgcaggtcgtc 1021 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 241 agggtgatcagggtcctctccatttgtaccacctgcctcctggacatgctgcaggtcgtc 300 Query: 1022 aacatcagccccagcatttcctggttg 1048 |||||||||||||||||||||||||| Sbjct: 301 aacatcagccccagcatttcctggttg 327 Score = 254 bits (132), Expect = 3e−64 Identities = 132/132(100%) Strand Plus/Plus Query: 1271 ataatgctgttctcaagtgtctacatgatgactctcattcaggaactacaggagatcctg 1330 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 328 ataatgctgttctcaagtgtctacatgatyactctcattcaggaactacaggagatcctg 387 Query: 1331 gtaccttcacagccccagcctctacctaaggatctttgcagaggcaagagccatcagcac 1390 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct: 388 gtaccttcacagccccagcctctacctaaggatctttgcagaggcaagagccatcagcac 447 Query: 1391 atcctgctgccg 1402 |||||||||||| Sbjct: 448 atcctgctgccg 459
[1069] 68 TABLE LIV(b) Peptide sequences of protein coded by 251P5G2 v.13 (SEQ ID NO: 79) MPFISKLVLA SQPTLFSFFS ASSPFLLFLD LRPERTYLPV CHVALIHMVV LLTMVFLSPQ 60 LFESLNFQND FKYEASFYLR RVIRVLSICT TCLLDMLQVV NISPSISWLI MLFSSVYMMT 120 LIQELQEILV PSQPQPLPKD LCRGKSHQHI LLPTQATFAA ATGLWAALTT VSNPSPADPV 180 TWRKEPAVLP CCNLEKGSWL SFPGTAARKE FSTTLTGHSA LSLSSSRALP GSLPAFADLP 240 RSCPESEQSA TPAGAFLLGW ERVVQRRLEV PRPQAAPATS ATPSRDPSPP CHQRRDAACL 300 RAQGLTRAFQ VVHLAPTAPD GGAGCPPSRN SYRLTHVRCA QGLEAASANL PGAPGRSSSC 360 ALRYRSGPSV SSAPSPAEPP AHQRLLFLPR APQAVSGPQE QPSEEALGVG SLSVFQLHLI 420 QCIPNLSYPL VLRHIPEILK FSEKETGGGI LGLELPATAA RLSGLNSIMQ IKEFEELVKL 480 HSLSHKVIQC VFAKKKNVDK WDDFCLSEGY GHSFLIMKET STKISGLIQE MGSGKSNVGT 540 WGDYDDSAFM EPRYHVRRED LDKLHRAAWW GKVPRKDLIV MLRDTDMNKR DKQKRTALHL 600 ASANGNSEVV QLLLDRRCQL NVLDNKKRTA LIKAVQCQED ECVLMLLEHG ADGNIQDEYG 660 NTALHYAIYN EDKLMAKALL LYGADIESKN KCGLTPLLLG VHEQKQEVVK FLIKKKANLN 720 ALDRYGRTAL ILAVCCGSAS IVNLLLEQNV DVSSQDLSGQ TAREYAVSSH HHVICELLSD 780 YKEKQMLKIS SEMSNPVITI LNIKLPLKVE EEIKKHGSNP VGLPENLTNG ASAGNGDDGL 840 IPQRKSRKPE NQQFPDTENE EYHSDEQNDT QKQLSEEQNT GISQDEILTN KQKQIEVAEK 900 EMNSELSLSH KKEEDLLREN SMLREEIAKL RLELDETKHQ NQLRENKILE EIESVKEKLL 960 KTIQLNEEAL TKTKVAGFSL RQLGLAQHAQ ASVQQLCYKW NHTEKTEQQA QEQEVAGFSL 1020 RQLGLAQHAQ ASVQQLCYKW GHTEKTEQQA QEQGAALRSQ IGDPGGVPLS EGGTAAGDQG 1080 PGTHLPPREP RASPGTPSLV RLASGARAAA LPPPTGKNGR SPTKQKSVCD SSGWILPVPT 1140 FSSGSFLGRR CPMFDVSPAM RLKSDSNRET HQAFRDKDDL PFFKTQQSPR HTKDLGQDDR 1200 AGVLAPKCRP GTLCHTDTPP HRNADTPPHR HTTTLPHRDT TTSLPHFHVS AGGVGPTTLG 1260 SNREIT 1266
[1070] 69 TABLE LV(b) Amino acid sequence alignment of 121P1F1 v.1 (SEQ ID NO: 80) and 251P5G2 v.13 (SEQ ID NO: 81) Score = 269 bits (688), Expect = 2e−71 Identities = 152/227 (66%), Positives = 152/227 (66%), Gaps = 74/227 (32%) 251P5G2v.1: 1 MPFISKLVLASQPTLFSFFSASSPFLLFLDLRPERTYLPVCHVALIHMVVLLTMVFLSPQ 60 MPFISKLVLASQPTLFSFFSASSPFLLFLDLRPERTYLPVCHVALIHMVVLLTMVFLSPQ 251P5G2v.13: 1 MPFISKLVLASQPTLFSFFSASSPFLLFLDLRPERTYLPVCHVALIHMVVLLTMVFLSPQ 60 251P5G2v.1: 61 LFESLNFQNDFKYEASFYLRRVIRVLSICTTCLLGMLQVVNISPSISWLVRFKWKSTIFT 120 LFESLNFQNDFKYEASFYLRRVIRVLSICTTCLL MLQVVNISPSISWL 251P5G2v.13: 61 LFESLNFQNDFKYEASFYLRRVIRVLSICTTCLLDMLQVVNISPSISWL----------- 109 251P5G2v. 1: 121 FHLFSWSLSFPVSSSLIFYTVASSNVTQINLHVSKYCSLFPINSIIRGLFFTLSLFRDVF 180 251P5G2v.13: 109 ------------------------------------------------------------ 109 251P5G2v.1: 181 LKQIMLFSSVYMMTLIQELQEILVPSQPQPLPKDLCRGKSHQHILLP 227 IMLFSSVYMMTLIQELQEILVPSQPQPLPKDLCRGKSHQHILLP 251P5G2v.13: 110 ---IMLFSSVYMMTLIQELQEILVPSQPQPLPKDLCRGKSHQHILLP 153
[1071]
Claims
1. A composition that comprises, consists essentially of, or consists of:
- a) a peptide of eight, nine, ten, or eleven contiguous amino acids of a protein of FIG. 2;
- b) a peptide of Tables VIII-XXI;
- c) a peptide of Tables XXII to XLV; or,
- d) a peptide of Tables XLVI to XLIX.
2. A composition of claim 1 that comprises a protein related to a protein of FIG. 2.
3. A protein of claim 2 that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to an entire amino acid sequence shown in FIG. 2.
4. A composition of claim 1 wherein the substance comprises a CTL polypeptide or an analog thereof, from the amino acid sequence of a protein of FIG. 2.
5. A composition of claim 4 further limited by a proviso that the epitope is not an entire amino acid sequence of FIG. 2.
6. A composition of claim 1 further limited by a proviso that the polypeptide is not an entire amino acid sequence of a protein of FIG. 2.
7. A composition of claim 1 that comprises an antibody polypeptide epitope from an amino acid sequence of FIG. 2.
8. A composition of claim 7 further limited by a proviso that the epitope is not an entire amino acid sequence of FIG. 2.
9. A composition of claim 7 wherein the antibody epitope comprises a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to the end of said peptide, wherein the epitope comprises an amino acid position selected from:
- a) an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5,
- b) an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
- c) an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
- d) an amino acid position having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
- e) an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIG. 9;
- f) a combination of at least two of a) through e);
- g) a combination of at least three of a) through e);
- h) a combination of at least four of a) through e); or
- i) a combination of five of a) through e).
10. A polynucleotide that encodes a protein of claim 1.
11. A polynucleotide of claim 10 that comprises a nucleic acid molecule set forth in FIG. 2.
12. A polynucleotide of claim 10 further limited by a proviso that the encoded protein is not an entire amino acid sequence of FIG. 2.
13. A composition of claim 11 wherein the substance comprises a polynucleotide that comprises a coding sequence of a nucleic acid sequence of FIG. 2.
14. A polynucleotide of claim 11 that further comprises an additional nucleotide sequence that encodes an additional peptide of claim 1.
15. A composition comprising a polynucleotide that is fully complementary to a polynucleotide of claim 10.
16. A method of generating a mammalian immune response directed to a protein of FIG. 2, the method comprising:
- exposing cells of the mammal's immune system to a portion of
- a) a 251P5G2-related protein and/or
- b) a nucleotide sequence that encodes said protein,
- whereby an immune response is generated to said protein.
17. A method of generating an immune response of claim 16, said method comprising:
- providing a 251P5G2-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 activated.
18. A method of claim 17 wherein the immune system cell is a B cell, whereby the induced B cell generates antibodies that specifically bind to the 251P5G2-related protein.
19. A method of claim 17 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 251P5G2-related protein.
20. A method of claim 17 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.
21. A method for detecting, in a sample, the presence of a 251P5G2-related protein or a 251P5G2-related polynucleotide, comprising steps of:
- contacting the sample with a substance that specifically binds to the 251P5G2-related protein or to the 251P5G2-related polynucleotide, respectively; and,
- determining that there is a complex of the substance with the 251P5G2-related protein or the substance with the 251P5G2-related polynucleotide, respectively.
22. A method of claim 21 for detecting the presence of a 251P5G2-related protein in a sample comprising steps of:
- contacting the sample with an antibody or fragment thereof either of which specifically bind to the 251P5G2-related protein; and,
- determining that there is a complex of the antibody or fragment thereof and the 251P5G2-related protein.
23. A method of claim 21 further comprising a step of:
- taking the sample from a patient who has or who is suspected of having cancer.
24. A method of claim 21 for detecting the presence of a protein of FIG. 2 mRNA in a sample comprising:
- producing cDNA from the sample by reverse transcription using at least one primer;
- amplifying the cDNA so produced using 251P5G2 polynucleotides as sense and antisense pimers, wherein the 251P5G2 polynucleotides used as the sense and antisense primers serve to amplify a 251P5G2 cDNA; and,
- detecting the presence of the amplified 251P5G2 cDNA.
25. A method of claim 21 for monitoring one or more 251P5G2 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 one or more 251P5G2 gene products expressed by cells in a tissue sample from an individual;
- comparing the status so determined to the status of one or more 251P5G2 gene products in a corresponding normal sample; and,
- identifying the presence of one or more aberrant gene products of 251P5G2 in the sample relative to the normal sample.
26. The method of claim 25 further comprising a step of determining if there are one or more elevated gene products of a 251P5G2 mRNA or a 251P5G2 protein, whereby the presence of one or more elevated gene products in the test sample relative to the normal tissue sample indicates the presence or status of a cancer.
27. A method of claim 26 wherein the cancer occurs in a tissue set forth in Table I.
28. A composition comprising:
- a substance that a) modulates the status of a protein of FIG. 2, or b) a molecule that is modulated by a protein of FIG. 2, whereby the status of a cell that expresses a protein of FIG. 2 is modulated.
29. A composition of claim 28, further comprising a physiologically acceptable carrier.
30. A pharmaceutical composition that comprises the composition of claim 28 in a human unit dose form.
31. A composition of claim 28 wherein the substance comprises an antibody or fragment thereof that specifically binds to a protein of FIG. 2.
32. An antibody or fragment thereof of claim 31, which is monoclonal.
33. An antibody of claim 31, which is a human antibody, a humanized antibody or a chimeric antibody.
34. A non-human transgenic animal that produces an antibody of claim 31.
35. A hybridoma that produces an antibody of claim 32.
36. A method of delivering a cytotoxic agent or a diagnostic agent to a cell that expresses a protein of FIG. 2, 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.
37. A composition of claim 28 wherein the substance comprises a polynucleotide that encodes an antibody or fragment thereof, either of which immunospecifically bind to a protein of FIG. 2.
38. A composition of claim 28 wherein the substance comprises a) a ribozyme that cleaves a polynucleotide having a 251P5G2 coding sequence, or b) a nucleic acid molecule that encodes the ribozyme; and, a physiologically acceptable carrier.
39. A composition of claim 28 wherein the substance comprises human T cells, wherein said T cells specifically recognize a 251P5G2 peptide subsequence in the context of a particular HLA molecule.
40. A method of inhibiting growth of cancer cells that express a protein of FIG. 2, the method comprising:
- administering to the cells the composition of claim 28.
41. A method of claim 40 of inhibiting growth of cancer cells that express a protein of FIG. 2, the method comprising steps of:
- administering to said cells an antibody or fragment thereof, either of which specifically bind to a 251P5G2-related protein.
42. A method of claim 40 of inhibiting growth of cancer cells that express a protein of FIG. 2, the method comprising steps of:
- administering to said cells a 251P5G2-related protein.
43. A method of claim 40 of inhibiting growth of cancer cells that express a protein of FIG. 2, the method comprising steps of:
- administering to said cells a polynucleotide comprising a coding sequence for a 251P5G2-related protein or comprising a polynucleotide complementary to a coding sequence for a 251P5G2-related protein.
44. A method of claim 40 of inhibiting growth of cancer cells that express a protein of FIG. 2, the method comprising steps of:
- administering to said cells a ribozyme that cleaves a polynucleotide that encodes a protein of FIG. 2.
45. A method of claim 40 of inhibiting growth of cancer cells that express a protein of FIG. 2 and a particular HLA molecule, the method comprising steps of:
- administering human T cells to said cancer cells, wherein said T cells specifically recognize a peptide subsequence of a protein of FIG. 2 while the subsequence is in the context of the particular HLA molecule.
46. A method of claim 40, the method comprising steps of:
- administering a vector that delivers a nucleotide that encodes a single chain monoclonal antibody, whereby the encoded single chain antibody is expressed intracellularly within cancer cells that express a protein of FIG. 2.
Type: Application
Filed: Apr 17, 2003
Publication Date: Apr 29, 2004
Inventors: Arthur B. Raitano (Los Angeles, CA), Pia M. Challita-Eid (Encino, CA), Aya Jakobovits (Beverly Hills, CA), Mary Faris (Los Angeles, CA), Wangmao Ge (Culver City, CA)
Application Number: 10418972
