Specific binding proteins and uses thereof

The invention relates to specific binding members, particularly antibodies and active fragments thereof, which recognize an aberrant post-translationally modified, particularly an aberrant glycosylated form of the EGFR. The binding members, particularly antibodies and fragments thereof, of the invention do not bind to EGFR on normal cells in the absence of amplification of the wild-type gene and are capable of binding the de2-7 EGFR at an epitope which is distinct from the junctional peptide. Antibodies of this type are exemplified by the novel antibody 806 whose VH and VL sequences are illustrated as SEQ ID NOs: 2 and 4 and chimeric antibodies thereof as exemplified by ch806.

Skip to: Description  ·  Claims  ·  References Cited  · Patent History  ·  Patent History
Description
CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 12/317,683, filed Dec. 23, 2008, now abandoned; which is a continuation of U.S. application Ser. No. 10/145,598, filed May 13, 2002, now U.S. Pat. No. 7,589,180, issued Sep. 15, 2009; which claims priority to U.S. Provisional Application No. 60/290,410, filed May 11, 2001, U.S. Provisional Application No. 60/326,019, filed Sep. 28, 2001, and U.S. Provisional Application No. 60/342,258, filed Dec. 21, 2001.

FIELD OF THE INVENTION

The present invention relates to specific binding members, particularly antibodies and fragments thereof, which bind to amplified epidermal growth factor receptor (EGFR) and to the de2-7 EGFR truncation of the EGFR. In particular, the epitope recognized by the specific binding members, particularly antibodies and fragments thereof, is enhanced or evident upon aberrant post-translational modification. These specific binding members are useful in the diagnosis and treatment of cancer. The binding members of the present invention may also be used in therapy in combination with chemotherapeutics or anti-cancer agents and/or with other antibodies or fragments thereof.

BACKGROUND OF THE INVENTION

The treatment of proliferative disease, particularly cancer, by chemotherapeutic means often relies upon exploiting differences in target proliferating cells and other normal cells in the human or animal body. For example, many chemical agents are designed to be taken up by rapidly replicating DNA so that the process of DNA replication and cell division is disrupted. Another approach is to identify antigens on the surface of tumor cells or other abnormal cells which are not normally expressed in developed human tissue, such as tumor antigens or embryonic antigens. Such antigens can be targeted with binding proteins such as antibodies which can block or neutralize the antigen. In addition, the binding proteins, including antibodies and fragments thereof, may deliver a toxic agent or other substance which is capable of directly or indirectly activating a toxic agent at the site of a tumor.

The EGFR is an attractive target for tumor-targeted antibody therapy because it is over-expressed in many types of epithelial tumors (27, 28). Moreover, expression of the EGFR is associated with poor prognosis in a number of tumor types including stomach, colon, urinary bladder, breast, prostate, endometrium, kidney and brain (e.g., glioma). Consequently, a number of EGFR antibodies have been reported in the literature with several undergoing clinical evaluation (18, 19, 29). Results from studies using EGFR mAbs in patients with head and neck cancer, squamous cell lung cancer, brain gliomas and malignant astrocytomas have been encouraging. The anti-tumor activity of most EGFR antibodies is enhanced by their ability to block ligand binding (30, 31). Such antibodies may mediate their efficacy through both modulation of cellular proliferation and antibody dependent immune functions (e.g. complement activation). The use of these antibodies, however, may be limited by uptake in organs that have high endogenous levels of EGFR such as the liver and skin (18, 19).

A significant proportion of tumors containing amplifications of the EGFR gene (i.e., multiple copies of the EGFR gene) also co-express a truncated version of the receptor (13) known as de2-7 EGFR, ΔEGFR, or Δ2-7 (terms used interchangeably herein) (2). The rearrangement seen in the de2-7 EGFR results in an in-frame mature mRNA lacking 801 nucleotides spanning exons 2-7 (6-9). The corresponding EGFR protein has a 267 amino acid deletion comprising residues 6-273 of the extracellular domain and a novel glycine residue at the fusion junction (9). This deletion, together with the insertion of a glycine residue, produces a unique junctional peptide at the deletion interface (9). The de2-7 EGFR (2) has been reported in a number of tumor types including glioma, breast, lung, ovarian and prostate (1-4). While this truncated receptor does not bind ligand, it possesses low constitutive activity and imparts a significant growth advantage to glioma cells grown as tumor xenografts in nude mice (10) and is able to transform NIH3T3 cells (11) and MCF-7 cells. The cellular mechanisms utilized by the de2-7 EGFR in glioma cells are not fully defined but are reported to include a decrease in apoptosis (12) and a small enhancement of proliferation (12).

As expression of this truncated receptor is restricted to tumor cells it represents a highly specific target for antibody therapy. Accordingly, a number of laboratories have reported the generation of both polyclonal (14) and monoclonal (3, 15, 16) antibodies specific to the unique peptide of de2-7 EGFR. A series of mouse mAbs, isolated following immunization with the unique de2-7 peptide, all showed selectivity and specificity for the truncated receptor and targeted de2-7 EGFR positive xenografts grown in nude mice (3, 25, 32).

However, one potential shortcoming of de2-7 EGFR antibodies is that only a proportion of tumors exhibiting amplification of the EGFR gene also express the de 2-7 EGFR (5). The exact percentage of tumors containing the de2-7 EGFR is not completely established, because the use of different techniques (i.e. PCR versus immunohistochemistry) and various antibodies, has produced a wide range of reported values for the frequency of its presence. Published data indicates that approximately 25-30% of gliomas express de2-7 EGFR with expression being lowest in anaplastic astrocytomas and highest in glioblastoma multiforme (6,13,17). The proportion of positive cells within de2-7 EGFR expressing gliomas has been reported to range from 37-86% (1). 27% of breast carcinomas and 17% of lung cancers were found to be positive for the de2-7 EGFR (1, 3, 13, 16). Thus, de2-7 EGFR specific antibodies would be expected to be useful in only a percentage of EGFR positive tumors.

Thus, while the extant evidence of activity of EGFR antibodies is encouraging, the observed limitations on range of applicability and efficacy reflected above remain.

Accordingly, it would be desirable to develop antibodies and like agents that demonstrate efficacy with a broad range of tumors, and it is toward the achievement of that objective that the present invention is directed.

The citation of references herein shall not be construed as an admission that such is prior art to the present invention.

SUMMARY OF THE INVENTION

In a broad aspect, the present invention provides an isolated specific binding member, particularly an antibody or fragment thereof, which recognizes an EGFR epitope which does not demonstrate any amino acid sequence alterations or substitutions from wild type EGFR and which is found in tumorigenic, hyperproliferative or abnormal cells and is not detectable in normal or wild type cells (the term “wild type cell” as used herein contemplates a cell that expresses endogenous EGFR but not the de 2-7 EGFR and the term specifically excludes a cell that overexpresses the EGFR gene; the term “wild type” refers to a genotype or phenotype or other characteristic present in a normal cell rather than in an abnormal or tumorigenic cell). In a further aspect, the present invention provides a specific binding member, particularly an antibody or fragment thereof, which recognizes an EGFR epitope which is found in tumorigenic, hyperproliferative or abnormal cells and is not detectable in normal or wild type cells, wherein the epitope is enhanced or evident upon aberrant post translational modification or aberrant expression. In a particular nonlimiting exemplification provided herein, the EGFR epitope is enhanced or evident wherein post-translational modification is not complete or full to the extent seen with normal expression of EGFR in wild type cells. In one aspect, the EGFR epitope is enhanced or evident upon initial or simple carbohydrate modification or early glycosylation, particularly high mannose modification, and is reduced or not evident in the presence of complex carbohydrate modification.

The specific binding member, which may be an antibody or a fragment thereof, such as an immunogenic fragment thereof, does not bind to or recognize normal or wild type cells containing normal or wild type EGFR epitope in the absence of aberrant expression and in the presence of normal EGFR post-translational modification. More particularly, the specific binding member of the invention, may be an antibody or fragment thereof, which recognizes an EGFR epitope which is present in cells overexpressing EGFR (e.g., EGFR gene is amplified) or expressing the de2-7 EGFR, particularly in the presence of aberrant post-translational modification, and that is not detectable in cells expressing EGFR under normal conditions, particularly in the presence of normal post-translational modification.

The present inventors have discovered novel monoclonal antibodies, exemplified herein by the antibody designated mAb 806, which specifically recognize aberrantly expressed EGFR. In particular, the antibodies of the present invention recognize an EGFR epitope which is found in tumorigenic, hyperproliferative or abnormal cells and is not detectable in normal or wild type cells, wherein the epitope is enhanced or evident upon aberrant post-translational modification. The antibodies of the present invention are further exemplified by the antibodies mAb 124 and mAb 1133 described herein. The novel antibodies of the invention also recognize amplified wild type EGFR and the de2-7 EGFR, yet bind to an epitope distinct from the unique junctional peptide of the de2-7 EGFR mutation. The antibodies of the present invention specifically recognize aberrantly expressed EGFR, including amplified EGFR and mutant EGFR (exemplified herein by the de2-7 mutation), particularly upon aberrant post-translational modification. Additionally, while mAb 806 does not recognize the EGFR when expressed on the cell surface of a glioma cell line expressing normal amounts of EGFR, it does bind to the extracellular domain of the EGFR (sEGFR) immobilized on the surface of ELISA plates, indicating the recognition of a conformational epitope. MAb 806 binds to the surface of A431 cells, which have an amplification of the EGFR gene but do not express the de2-7 EGFR. Importantly, mAb 806 did not bind significantly to normal tissues such as liver and skin, which express levels of endogenous, wild type (wt) EGFR that are higher than in most other normal tissues, but wherein EGFR is not aberrantly expressed or amplified.

The antibodies of the present invention can specifically categorize the nature of EGFR tumors or tumorigenic cells, by staining or otherwise recognizing those tumors or cells wherein aberrant EGFR expression, including EGFR amplification and/or EGFR mutation, particularly de2-7EGFR, is present. Further, the antibodies of the present invention, as exemplified by mAb 806, demonstrate significant in vivo anti-tumor activity against tumors containing amplified EGFR and against de2-7 EGFR positive xenografts.

The unique specificity of mAb 806, whereby mAb 806 binds to the de2-7 EGFR and amplified EGFR but not the normal, wild type EGFR, provides diagnostic and therapeutic uses to identify, characterize and target a number of tumor types, for example, head and neck, breast, or prostate tumors and glioma, without the problems associated with normal tissue uptake that may be seen with previously known EGFR antibodies.

Accordingly, the invention provides specific binding proteins, such as antibodies, which bind to the de2-7 EGFR at an epitope which is distinct from the junctional peptide but which do not bind to EGFR on normal cells in the absence of amplification of the EGFR gene. By amplification, it is meant to include that the cell comprises multiple copies of the EGFR gene.

Preferably the epitope recognized by mAb 806 is located within the region comprising residues 273-501 of the mature normal or wild type EGFR sequence. Therefore, also provided are specific binding proteins, such as antibodies, which bind to the de2-7 EGFR at an epitope located within the region comprising residues 273-501 of the EGFR sequence. The epitope may be determined by any conventional epitope mapping techniques known to the person skilled in the art. Alternatively, the DNA sequence encoding residues 273-501 could be digested, and the resultant fragments expressed in a suitable host. Antibody binding could be determined as mentioned above.

In a preferred aspect, the antibody is one which has the characteristics of the antibody which the inventors have identified and characterized, in particular recognizing aberrantly expressed EGFR, as found in amplified EGFR and de2-7EGFR. In a particularly preferred aspect the antibody is the mAb 806, or active fragments thereof. In a further preferred aspect the antibody of the present invention comprises the VH and VL amino acid sequences depicted in FIG. 14 (SEQ ID NO:2) and FIG. 15 (SEQ ID NO:4) respectively.

In another aspect, the invention provides an antibody capable of competing with the 806 antibody, under conditions in which at least 10% of an antibody having the VH and VL sequences of the 806 antibody is blocked from binding to de2-7EGFR by competition with such an antibody in an ELISA assay. In particular, anti-idiotype antibodies are contemplated and are exemplified herein. The anti-idiotype antibodies LMH-11, LMH-12 and LMH-13 are provided herein.

An isolated polypeptide consisting essentially of the epitope comprising residues 273-501 of the mature normal, wild type EGFR (residues 6-234 of mature de2-7 EGFR) forms another aspect of the present invention. The peptide of the invention is particularly useful in diagnostic assays or kits and therapeutically or prophylactically, including as a tumor or anti-cancer vaccine. Thus compositions of the peptide of the present invention include pharmaceutical compositions and immunogenic compositions.

The binding of an antibody to its target antigen is mediated through the complementarity-determining regions (CDRs) of its heavy and light chains, with the role of CDR3 being of particular importance. Accordingly, specific binding members based on the CDR3 regions of the heavy or light chain, and preferably both, of mAb806 will be useful specific binding members for in vivo therapy. The CDRs of the mAb 806 antibody are shown in FIGS. 16 and 17.

Accordingly, specific binding proteins such as antibodies which are based on the CDRs of the mAb 806 antibody identified, particularly the CDR 3 regions, will be useful for targeting tumors with amplified EGFR regardless of their de2-7 EGFR status. As mAb 806 does not bind significantly to normal, wild type receptor, there would be no significant uptake in normal tissue, a limitation of EGFR antibodies currently being developed (18, 19).

In the accompanying drawings, the nucleic acid sequence (SEQ ID NO:1) and translation (SEQ ID NO:2) thereof of the 806 VH gene is shown in FIG. 14. The VL gene of the 806 antibody is shown as FIG. 15 as nucleic acid sequence (SEQ ID NO:3) and predicted amino acid sequence (SEQ ID NO:4). In FIGS. 16 and 17, depicting the VH and VL polypeptide sequences of mAb 806, the CDRs are indicated in boxes.

In a further aspect, the present invention provides an isolated specific binding member capable of binding an antigen, wherein said specific binding member comprises a polypeptide binding domain comprising an amino acid sequence substantially as set out as residues 93-102 of SEQ ID NO:2. The invention further provides said isolated specific binding member which further comprises one or both of the polypeptide binding domains substantially as set out as residues 26-35A and 49-64 of SEQ ID NO:2, preferably both. One example of such an embodiment is the sequence substantially as shown in SEQ ID NO:2. In a preferred embodiment, the binding domains are carried by a human antibody framework.

In another aspect, the invention provides an isolated specific binding member capable of binding a tumor antigen, wherein said specific binding member comprises a polypeptide binding domain comprising a heavy chain sequence comprising at least the CDR3 sequence of SEQ ID NO:2, together with a light chain comprising CDRs whose amino acid sequences are substantially as found within SEQ ID NO:4. One example of such an embodiment is the sequence substantially as shown in SEQ ID NO:4. In a preferred embodiment, the CDRs are carried by a human antibody framework.

In further aspects, the invention provides an isolated nucleic acid which comprises a sequence encoding a specific binding member as defined above, and methods of preparing specific binding members of the invention which comprise expressing said nucleic acids under conditions to bring about expression of said binding member, and recovering the binding member.

Yet a further aspect of the invention are compositions of such binding proteins with additional binding proteins, such as binding proteins which bind to EGFR, preferably inhibiting ligand binding thereto. Such compositions can be “one pot” cocktails, kits, and so forth, preferably formulated for ease of administration.

Specific binding members according to the invention may be used in a method of treatment or diagnosis of the human or animal body, such as a method of treatment of a tumor in a human patient which comprises administering to said patient an effective amount of a specific binding member of the invention.

The present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes an antibody of the present invention; preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the antibody VH which has a nucleotide sequence or is complementary to a DNA sequence shown in FIG. 14 (SEQ ID NO:1). In another embodiment, the present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the antibody VL which has a nucleotide sequence or is complementary to a DNA sequence shown in FIG. 15 (SEQ ID NO:3).

The present invention also includes polypeptides or antibodies having the activities noted herein, and that display the amino acid sequences set forth and described above and in FIGS. 14 and 15 hereof and selected from SEQ ID NO:2 and 4.

In a further embodiment of the invention, the full DNA sequence of the recombinant DNA molecule or cloned gene provided herein may be operatively linked to an expression control sequence which may be introduced into an appropriate host. The invention accordingly extends to unicellular hosts transformed with the cloned gene or recombinant DNA molecule comprising a DNA sequence encoding the present VH and/or VL, or portions thereof, of the antibody, and more particularly, the VH and/or VL set forth above and in SEQ ID NO:1 and 3.

The present invention naturally contemplates several means for preparation of the antibodies and active fragments thereof, including as illustrated herein known recombinant techniques, and the invention is accordingly intended to cover such synthetic or chimeric antibody preparations within its scope. The isolation of the cDNA and amino acid sequences disclosed herein facilitates the reproduction of the antibody of the present invention by such recombinant techniques, and accordingly, the invention extends to expression vectors prepared from the disclosed DNA sequences for expression in host systems by recombinant DNA techniques, and to the resulting transformed hosts.

The present invention provides drugs or other entities, including antibodies such as anti-idiotype antibodies, that are capable of binding to the antibody thereby modulating, inhibiting or potentiating the antibody activity. Thus, anti-idiotype antibodies to mAb806 are provided and exemplified herein. Such anti-idiotype antibodies would be useful in the development of drugs that would specifically bind the antibodies such as mAb806 or its epitope or that would potentiate its activity.

The diagnostic utility of the present invention extends to the use of the antibodies of the present invention in assays to characterize tumors or cellular samples or to screen for tumors or cancer, including in vitro and in vivo diagnostic assays.

In an immunoassay, a control quantity of the antibodies, or the like may be prepared and labeled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a cellular sample. After the labeled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached.

Specific binding members of the invention may carry a detectable or functional label. The specific binding members may carry a radioactive label, such as the isotopes 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 121I, 124I, 125I, 131I, 111In, 211At, 198Au, 67Cu, 225Ac, 213Bi, 99Tc and 186Re. When radioactive labels are used, known currently available counting procedures may be utilized to identify and quantitate the specific binding members. In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.

The radiolabelled specific binding members, particularly antibodies and fragments thereof, are useful in in vitro diagnostics techniques and in in vivo radioimaging techniques. In a further aspect of the invention, radiolabelled specific binding members, particularly antibodies and fragments thereof, particularly radioimmunoconjugates, are useful in radioimmunotherapy, particularly as radiolabelled antibodies for cancer therapy. In a still further aspect, the radiolabelled specific binding members, particularly antibodies and fragments thereof, are useful in radioimmuno-guided surgery techniques, wherein they can identify and indicate the presence and/or location of cancer cells, precancerous cells, tumor cells, and hyperproliferative cells, prior to, during or following surgery to remove such cells.

Immunoconjugates or antibody fusion proteins of the present invention, wherein the specific binding members, particularly antibodies and fragments thereof, of the present invention are conjugated or attached to other molecules or agents further include, but are not limited to binding members conjugated to a chemical ablation agent, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent or drug.

The present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of, for instance, amplified EGFR or de2-7EGFR. The system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the antibody, and one or more additional immunochemical reagents, at least one of which is a free or immobilized components to be determined or their binding partner(s).

In a further embodiment, the present invention relates to certain therapeutic methods which would be based upon the activity of the binding member, antibody, or active fragments thereof, or upon agents or other drugs determined to possess the same activity. A first therapeutic method is associated with the prevention or treatment of cancer, including but not limited to head and neck, breast, prostate and glioma.

In particular, the binding members and antibodies of the present invention, and in a particular embodiment the 806 antibody whose sequences are presented in SEQ ID NOS: 2 and 4 herein, or active fragments thereof, and chimeric (bispecific) or synthetic antibodies derived therefrom can be prepared in pharmaceutical compositions, including a suitable vehicle, carrier or diluent, for administration in instances wherein therapy is appropriate, such as to treat cancer. Such pharmaceutical compositions may also include methods of modulating the half-life of the binding members, antibodies or fragments by methods known in the art such as pegylation. Such pharmaceutical compositions may further comprise additional antibodies or therapeutic agents.

Thus, a composition of the present invention may be administered alone or in combination with other treatments, therapeutics or agents, either simultaneously or sequentially dependent upon the condition to be treated. In addition, the present invention contemplates and includes compositions comprising the binding member, particularly antibody or fragment thereof, herein described and other agents or therapeutics such as anti-cancer agents or therapeutics, anti-EGFR agents or antibodies, or immune modulators. More generally these anti-cancer agents may be tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post-translational modulators, cell growth or division inhibitors (e.g. anti-mitotics), PDGFR inhibitors or signal transduction inhibitors. Other treatments or therapeutics may include the administration of suitable doses of pain relief drugs such as non-steroidal anti-inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such as morphine, or anti-emetics. Thus, these agents may be anti-EGFR specific agents, such as AG1478, or may be more general anti-cancer and anti-neoplastic agents, non limiting examples including doxorubicin, carboplatin and cisplatin. In addition, the composition may be administered with immune modulators, such as interleukins, tumor necrosis factor (TNF) or other growth factors, cytokines or hormones such as dexamethasone which stimulate the immune response and reduction or elimination of cancer cells or tumors. The composition may also be administered with, or may include combinations along with other anti-EGFR antibodies, including but not limited to the anti-EGFR antibodies 528; 225; SC-03; 108 (ATCC HB9764) U.S. Pat. No. 6,217,866; 14E1 (U.S. Pat. No. 5,942,602); DH8.3; L8A4; Y10; HuMAX-EGFr (Genmab/Medarex); ICR62; and ABX-EGF (Abgenix).

The present invention also includes binding members, including antibodies and fragments thereof, which are covalently attached to or otherwise associated with other molecules or agents. These other molecules or agents include, but are not limited to, molecules (including antibodies or antibody fragments) with distinct recognition characteristics, toxins, ligands, and chemotherapeutic agents.

Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing detailed description, which proceeds with reference to the following illustrative drawings, and the attendant claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-F present the results of flow cytometric analysis of glioma cell lines. U87MG (light gray histograms) and U87MG.Δ2-7 (dark gray histograms) cells were stained with either an irrelevant IgG2b antibody (open histograms), DH8.3 (specific for de2-7 EGFR), MAb 806 or 528 (binds both wild type and de2-7 EGFR) as indicated.

FIGS. 2A-C present the results of ELISA of MAb 806, DH8.3 and 528 antibodies. (A) binding of increasing concentrations of MAb 806 (▴) DH8.3 (●) or 528 (▪) antibody to sEGFR coated ELISA plates. (B) inhibition of MAb 806 and 528 binding to sEGFR coated ELISA plates by increasing concentrations of sEGFR in solution. (C) binding of increasing concentrations of DH8.3 to the de2-7 junctional peptide illustrates binding curves for mAb 806 and 528 antibodies to immobilized wild-type sEGFR.

FIGS. 2D-E graphically present the results of BIAcore™ binding studies using C-terminal biotinylated peptide and including a monoclonal antibody of the invention, along with other known antibodies, among them the L8A4 antibody which recognizes the junction peptide of the de2-7 EGFR mutant, and controls.

FIG. 3 depicts the internalization of MAb 806 and the DH8.3 antibody. U87MG.Δ2-7 cells were pre-incubated with MAb 806 (▴) or DH8.3 (●) at 4° C., transferred to 37° C. and internalization determined by FACS. Data represents mean internalization at each time point±SE of 3 (DH8.3) or 4 (MAb 806) separate experiments.

FIGS. 4A-B illustrate biodistribution (% ID/g tumor) of radiolabeled (a) 125I-MAb 806 and (b) 131I-DH8.3 in nude mice bearing U87MG and U87MG.Δ2-7 xenografts. Each point represents the mean of 5 mice±SE except for 1 hr where n=4.

FIGS. 5A-B illustrate biodistribution of radiolabeled 125I-MAb 806 (open bar) and 131I-DH8.3 (filled bar) antibodies expressed as (a) tumor:blood or (b) tumor:liver ratios in nude mice bearing U87MG.Δ2-7 xenografts. Each bar represents the mean of 5 mice±SE except for 1 hr where n=4

FIGS. 6A-C illustrate flow cytometric analysis of cell lines containing amplification of the EGFR gene. A431 cells were stained with either MAb 806, DH8.3 or 528 (black histograms) and compared to an irrelevant IgG2b antibody (open histogram)

FIGS. 7A-B illustrate biodistribution (% ID/g tumor) of radiolabeled (a) 125I-MAb 806 and (b) 131I-528 in nude mice bearing U87MG.Δ2-7 and A431 xenografts.

FIGS. 8A-D illustrate biodistribution of radiolabeled 125I-MAb 806 (open bar) and 131I-528 (filled bar) antibodies expressed as (a,b) tumor:blood or (c,d) tumor:liver ratios in nude mice bearing (a,c) U87MGA2-7 and (b,d) A431 xenografts.

FIGS. 9A-B illustrate anti-tumor effect of mAb 806 on A) U87MG and B) U87MG.Δ2-7 xenograft growth rates in a preventative model. 3×106 U87MG or U87MG.Δ2-7 cells were injected s.c. into both flanks of 4-6 week old BALB/c nude mice, (n=5) at day 0. Mice were injected i.p. with either 1 mg of mAb 806 (●); 0.1 mg of mAb 806 (▴); or vehicle (∘) starting one day prior to tumor cell inoculation. Injections were given three times per week for two weeks as indicated by the arrows. Data are expressed as mean tumor volume±S.E.

FIGS. 10A-C illustrate the anti-tumor effect of mAb 806 on A) U87MG, B) U87MG.Δ2-7 and C) U87MG.wtEGFR xenografts in an established model. 3×106 U87MG, U87MG.Δ2-7, or U87MG.wtEGFR cells, were injected s.c. into both flanks of 4-6 week old BALB/c nude mice, (n=5). Mice were injected i.p. with either 1 mg doses of mAb 806 (●); 0.1 mg doses of mAb 806 (▴); or vehicle (∘) starting when tumors had reached a mean tumor volume of 65-80 mm3. Injections were given three times per week for two weeks as indicated by the arrows. Data are expressed as mean tumor volume±S.E.

FIGS. 11A-B illustrate anti-tumor effect of mAb 806 on A431 xenografts in A) preventative and B) established models. 3×106 A431 cells were injected s.c. into both flanks of 4-6 week old BALB/c nude mice (n=5). Mice were injected i.p. with either 1 mg doses of mAb 806 (●); or vehicle (∘), starting one day prior to tumor cell inoculation in the preventative model, or when tumors had reached a mean tumor volume of 200 mm3. Injections were given three times per week for two weeks as indicated by the arrows. Data are expressed as mean tumor volume±S.E.

FIG. 12 illustrates the anti-tumor effect of treatment with mAb 806 combined with treatment with AG1478 on A431 xenografts in a preventative model. Data are expressed as mean tumor volume±S.E.

FIG. 13 depicts antibody 806 binding to A431 cells in the presence of increasing concentrations of AG1478 (0.5 μM and 5 μM).

FIGS. 14A-B illustrate (A) the nucleic acid sequence and (B) the amino acid translation thereof of the 806 VH gene (SEQ ID NO:1 and SEQ ID NO:2, respectively).

FIGS. 15A-B illustrate (A) the nucleic acid sequence and (B) the amino acid translation thereof of the 806 VL gene (SEQ ID NO:3 and SEQ ID NO:4, respectively).

FIG. 16 shows the VH sequence (SEQ ID NO:11) numbered according to Kabat, with the CDRs boxed. Key residues of the VH are 24, 37, 48, 67 and 78.

FIG. 17 shows the VL sequence (SEQ ID NO:12) numbered according to Kabat, with the CDRs boxed. Key residues of the VL are 36, 46, 57 and 71.

FIGS. 18A-D show the results of in vivo studies designed to determine the therapeutic effect of combination antibody therapy, particularly mAb806 and the 528 antibody. Mice received inoculations of U87MG.Δ2-7 (A and B), U87MG.DK (C), or A431 (D) cells.

FIGS. 19A-D show analysis of internalization by electron microscopy. U87MG.Δ2-7 cells were pre-incubated with MAb 806 or DH8.3 followed by gold conjugated anti-mouse IgG at 4° C., transferred to 37° C. and internalization examined at various time points by electron microscopy. (A) localization of the DH8.3 antibody to a coated pit (arrow) after 5 min; (B) internalization of MAb 806 by macropinocytosis (arrow) after 2 min; (C) localization of DH8.3 to lysosomes (arrow) after 20 min; (D) localization of MAb 806 to lysosomes (arrow) after 30 min. Original magnification for all images is ×30,000.

FIG. 20 shows autoradiography of a U87MG.Δ2-7 xenograft section collected 8 hr after injection of 125I-MAb 806.

FIGS. 21A-B show flow cytometric analysis of cell lines containing amplification of the EGFR gene. HN5 and MDA-468 cells were stained with an irrelevant IgG2b antibody (open histogram with dashed line), MAb 806 (black histogram) or 528 (open histogram with closed lines). The DH8.3 antibody was completely negative on both cell lines (data not shown).

FIG. 22 shows immunoprecipitation of EGFR from cell lines. The EGFR was immunoprecipitated from 35 S-labeled U87MG.Δ2-7 or A431 cells with MAb 806, sc-03 antibody or a IgG2b isotype control. Arrows at the side indicate the position of the de2-7 and wt EGFR. Identical banding patterns were obtained in 3 independent experiments.

FIG. 23 shows autoradiography of an A431 xenograft section collected 24 hr after injection of 125I-MAb 806, areas of localization to viable tissue are indicated (arrows).

FIGS. 24A-E illustrate: A and B, extended survival of nude mice bearing intracranial U87MG.ΔEGFR (A) and LN-Z308.ΔEGFR (B) xenografts with systemic mAb 806 treatment. U87MG.ΔGFR cells (1×105) or LN-Z308.ΔEGFR cells (5×105) were implanted into nude mice brains, and the animals were treated with either mAb 806, PBS, or isotype IgG from postimplantation days 0 through 14. C and D, growth inhibition of intracranial tumors by mAb 806 treatment. Nude mice (five per group), treated with either mAb 806 or the isotype IgG control, were euthanized on day 9 for U87MG.ΔEGFR(C) and on day 15 for LN-Z308.ΔEGFR (D), and their brains were harvested, fixed, and sectioned. Data were calculated by taking the tumor volume of control as 100%. Values are mean±SD. ***, P<0.001; control versus mAb 806. Arrowheads, tumor tissue. E, extended survival of nude mice bearing intracranial U87MG.ΔEGFR xenografts with intratumoral mAb 806 treatment. U87MG.ΔEGFR cells were implanted as described. Ten mg of mAb 806 or isotype IgG control in a volume of 5 μl were injected at the tumor-injection site every other day starting at day 1 for five times.

FIG. 25A-C mAb 806 extends survival of mice with U87.MG.wtEGFR brain tumors but not with U87MG.DK or U87MG brain tumors. U87MG (A), U87MG.DK (B), or U87MG.wtEGFR(C) cells (5×105) were implanted into nude mice brains, and the animals were treated with mAb 806 from postimplantation days 0 through 14 followed by observation after discontinuation of therapy.

FIGS. 26A-B illustrate: A, FACS analysis of mAb 806 reactivity with U87MG cell lines. U87MG, U87MGΔEGFR, U87MG.DK, and U87MG.wtEGFR cells were stained with anti-EGFR mAbs 528, EGFR.1, and anti-ΔEGFR antibody, mAb 806. Monoclonal EGFR.1 antibody recognized wtEGFR exclusively and monoclonal 528 antibody reacted with both wtEGFR and ΔEGFR. mAb 806 reacted intensively with U87MG.ΔEGFR and U87MG.DK and weakly with U87MG.wtEGFR. Bars on the abscissa, maximum staining of cells in the absence of primary antibody. Results were reproduced in three independent experiments. B, mAb 806 immunoprecipitation of EGFR forms. Mutant and wtEGFR were immunoisolated with anti-EGFR antibodies, 528, or EGFR.1, or anti-ΔEGFR antibody, mAb 806, from (Lane 1) U87MG, (Lane 2) U87Δ.EGFR, (Lane 3) U87MG.DK, and (Lane 4) U87MG.wtEGFR cells and were then detected by Western blotting with anti-pan EGFR antibody, C13.

FIGS. 27A-B illustrate systemic treatment with mAb 806 decreases the phosphorylation of ΔEGFR and Bcl-XL expression in U87MG.ΔEGFR brain tumors. U87MG.ΔEGFR tumors were resected at day 9 of mAb 806 treatment, immediately frozen in liquid nitrogen and stored at −80° C. before tumor lysate preparation. A, Western blot analysis of expression and the degree of autophosphorylation of ΔEGFR. Thirty μg of tumor lysates were subjected to SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with anti-phosphotyrosine mAb, then were stripped and re-probed with anti-EGFR antibody, C13. B, Western blotting of Bcl-XL by using the same tumor lysates as in A. Membranes were probed with anti-human Bcl-XL polyclonal antibody. Lanes 1 and 2, U87MG.ΔEGFR brain tumors treated with isotype control; Lanes 3 and 4, U87MG.ΔEGFR brain tumors treated with mAb 806.

FIG. 28 illustrates that mAb 806 treatment leads to a decrease in growth and vasculogenesis and to increases in apoptosis and accumulating macrophages in U87MG.ΔEGFR tumors. Tumor sections were stained for Ki-67. Cell proliferative index was assessed by the percentage of total cells that were Ki-67 positive from four randomly selected high-power fields (×400) in intracranial tumors from four mice of each group. Data are the mean±SE. Apoptotic cells were detected by TUNEL assay. Apoptotic index was assessed by the ratio of TUNEL-positive cells:total number of cells from four randomly selected high-power fields (×400) in intracranial tumors from four mice of each group. Data are the mean±SE. Tumor sections were immunostained with anti-CD31 antibody. MVAs were analyzed by computerized image analysis from four randomly selected fields (×200) from intracranial tumors from four mice of each group. Peritumoral infiltrates of macrophages in mAb 806-treated U87MG.ΔEGFR tumors. Tumor sections were stained with anti-F4/80 antibody.

FIGS. 29A-F show cytometric analysis of parental and transfected U87MG glioma cell lines. Cells were stained with either an irrelevant IgG2b antibody (open histograms) or the 528 antibody or mAb 806 (filled histograms) as indicated.

FIG. 30 show immunoprecipitation of EGFR from cell lines. The EGFR was immunoprecipitated from 35S-labeled U87MG.wtEGFR, U87MG.Δ2-7, and A431 cells with mAb806 (806), sc-03 antibody (c-term), or a IgG2b isotype control (con). Arrows, position of the de2-7 and wt EGFR.

FIG. 31 shows representative H&E-stained paraffin sections of U87MG.Δ2-7 and U87MG.wtEGFR xenografts. U87MG.Δ2-7 (collected 24 days after tumor inoculation) and U87MG.wtEGFR (collected 42 days after tumor inoculation) xenografts were excised from mice treated as described in FIGS. 10A-C above, and stained with H&E. Vehicle-treated U87MG.Δ2-7 (collected 18 days after tumor inoculation) and U87MG.wtEGFR (collected 37 days after tumor inoculation) xenografts showed very few areas of necrosis (left panel), whereas extensive necrosis (arrows) was observed in both U87MG.Δ2-7 and U87MG.wtEGFR xenografts treated with mAb 806 (right panel).

FIG. 32 shows immunohistochemical analysis of EGFR expression in frozen sections derived from U87MG, U87MG.Δ2-7, and U87MG.wtEGFR xenografts. Sections were collected at the time points described in FIG. 31 above. Xenograft sections were immunostained with the 528 antibody (left panel) and mAb 806 (right panel). No decreased immunoreactivity to either wt EGFR, amplified EGFR, or de2-7 EGFR was observed in xenografts treated with mAb 806. Consistent with the in vitro data, parental U87MG xenografts were positive for 528 antibody but were negative for mAb 806 staining.

FIGS. 33A-B show schematic representations of generated bicistronic expression constructs. Transcription of the chimeric antibody chains is initiated by Elongation Factor-1 promoter and terminated by a strong artificial termination sequence. IRES sequences were introduced between coding regions of light chain and NeoR and heavy chain and dhfr gene.

FIGS. 34A-B show biodistribution analysis of the ch806 radiolabeled with either A) 125I or B) 111In was performed in BALB/c nude mice bearing U87MG-de2-7 xenograft tumors. Mice were injected with 5 ug of radiolabeled antibody and in groups of 4 mice per time point, sacrificed at either 8, 28, 48 or 74 hours. Organs were collected, weighed and radioactivity measured in a gamma counter.

FIGS. 35A-B depict (A) the % ID gram tumor tissue and (B) the tumor to blood ratio. Indium-111 antibody shows approximately 30% ID/gram tissue and a tumor to blood ratio of 4.0.

FIG. 36 depicts the therapeutic efficacy of chimeric antibody ch806 in an established tumor model. 3×106 U87MG.Δ2-7 cells in 100 μl of PBS were inoculated s.c. into both flanks of 4-6 week old female nude mice. The mAb806 was included as a positive control. Treatment was started when tumors had reached a mean volume of 50 mm3 and consisted of 1 mg of ch806 or mAb806 given i.p. for a total of 5 injections on the days indicated. Data was expressed as mean tumor volume+/−S.E. for each treatment group.

FIGS. 37A-B show CDC Activity on Target A) U87MG.de2-7 and B) A431 cells for anti-EGFR chimeric IgG1 antibodies ch806 and control cG250. Mean (bars; ±SD) percent cytotoxicity of triplicate determinations are presented.

FIGS. 38A-B show ADCC on target A) U87MG.de2-7 and B) A431 cells at Effector:Target cell ratio of 50:1 mediated by ch806 and isotype control cG250 (0-10 ug/ml). Results are expressed as mean (bars; ±SD) percent cytotoxicity of triplicate determinations.

FIG. 39 shows ADCC mediated by 1 μg/ml parental mAb 806 and ch806 on target U87MG.de2-7 cells over a range of Effector:Target ratios. Mean (bars; ±SD) of triplicate determinations are presented.

FIGS. 40A-B illustrate that twenty-five hybridomas producing antibodies that bound ch806 but not huIgG were initially selected. Four of these anti-ch806 hybridomas with high affinity binding (clones 3E3, 5B8, 9D6 and 4D8) were subsequently pursued for clonal expansion from single cells by limiting dilution and designated Ludwig Institute for Cancer Research Melbourne Hybridoma (LMH)-11, -12, -13 and -14, respectively. In addition, two hybridomas that produced mAbs specific for huIgG were also cloned and characterized further: clones 2C10 (LMH-15) and 2B8 (LMH-16).

FIGS. 41A-C illustrate that after clonal expansion, the hybridoma culture supernatants were examined in triplicate by ELISA for the ability to neutralize ch806 or mAb 806 antigen binding activity with sEGFR621. Mean (±SD) results demonstrated the antagonist activity of anti-idiotype mAbs LMH-11, -12, -13 and -14 with the blocking in solution of both ch806 and murine mAb 806 binding to plates coated with sEGFR (LMH-14 not shown).

FIGS. 42A-C illustrate that microtitre plates were coated with 10 μg/ml purified A) LMH-11, B) LMH-12 and C) LMH-13. The three purified clones were compared for their ability to capture ch806 or mAb 806 in sera or 1% FCS/Media and then detect bound ch806 or mAb806. Isotype control antibodies hu3S193 and m3S193 in serum and 1% FCS/Media were included in addition to controls for secondary conjugate avidin-HRP and ABTS substrate. Results are presented as mean (±SD) of triplicate samples using biotinylated-LMH-12 (10 g/ml) for detection and indicate LMH-12 used for capture and detection had the highest sensitivity for ch806 in serum (3 ng/ml) with negligible background binding.

FIG. 43 illustrates validation of the optimal pharmacokinetic ELISA conditions using 1 μg/ml anti-idiotype LMH-12 and 1 μg/ml biotinylated LMH-12 for capture and detection, respectively. Three separate ELISAs were performed in quadruplicate to measure ch806 in donor serum (●) from three healthy donors or 1% BSA/media (▪) with isotype control hu3S193 in serum (σ) or 1% BSA/media (τ). Controls for secondary conjugate avidin-HRP (υ) and ABTS substrate (hexagon) alone were also included with each ELISA. Mean (±SD) results demonstrate highly reproducible binding curves for measuring ch806 (2 μg/ml-1.6 ng/ml) in sera with a 3 ng/ml limit of detection. (n=12; 1-100 ng/ml, Coefficient of Variation <25%; 100 ng/ml-5 μg/ml, Coefficient of Variation <15%). No background binding was evident with any of the three sera tested and negligible binding was observed with isotype control hu3S193.

FIG. 44 depicts an immunoblot of recombinant sEGFR expressed in CHO cells, blotted with mAb806. Recombinant sEGFR was treated with PNGaseF to remove N-linked glycosylation (deglycosylated), or untreated (untreated), the protein was run on SDS-PAGE, transferred to membrane and immunoblotted with mAb 806.

FIG. 45 depicts immunoprecipitation of EGFR from 35S-labelled cell lines (U87MG Δ2-7, U87MG-wtEGFR, and A431) with different antibodies (SC-03, 806 and 528 antibodies).

FIG. 46 depicts immunoprecipitation of EGFR from different cells (A431 and U87MGΔ2-7) at different time points (time 0 to 240 minutes) after pulse-labelling with 35S methionine/cysteine. Antibodies 528 and 806 are used for immunoprecipitation.

FIG. 47 depicts immunoprecipitation of EGFR from various cell lines (U87MGΔ2-7, U87MG-wtEGFR and A431) with various antibodies (SC-03, 806 and 528) in the absence of (−) and after Endo H digestion (+) to remove high mannose type carbohydrates.

FIG. 48 depicts cell surface iodination of the A431 and U87MGΔ2-7 cell lines followed by immunoprecipitation with the 806 antibody, and with or without Endo H digestion, confirming that the EGFR bound by mAb 806 on the cell surface of A431 cells is an EndoH sensitive form.

 DETAILED DESCRIPTION

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, “Molecular Cloning: A Laboratory Manual” (1989); “Current Protocols in Molecular Biology” Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A Laboratory Handbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocols in Immunology” Volumes I-III [Coligan, J. E., ed. (1994)]; “Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic Acid Hybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “Transcription And Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “Animal Cell Culture” [R.I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning” (1984).

Therefore, if appearing herein, the following terms shall have the definitions set out below.

A. Terminology

The term “specific binding member” describes a member of a pair of molecules which have binding specificity for one another. The members of a specific binding pair may be naturally derived or wholly or partially synthetically produced. One member of the pair of molecules has an area on its surface, or a cavity, which specifically binds to and is therefore complementary to a particular spatial and polar organisation of the other member of the pair of molecules. Thus the members of the pair have the property of binding specifically to each other. Examples of types of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. This application is concerned with antigen-antibody type reactions.

The term “aberrant expression” in its various grammatical forms may mean and include any heightened or altered expression or overexpression of a protein in a tissue, e.g. an increase in the amount of a protein, caused by any means including enhanced expression or translation, modulation of the promoter or a regulator of the protein, amplification of a gene for a protein, or enhanced half-life or stability, such that more of the protein exists or can be detected at any one time, in contrast to a non-overexpressed state. Aberrant expression includes and contemplates any scenario or alteration wherein the protein expression or post-translational modification machinery in a cell is taxed or otherwise disrupted due to enhanced expression or increased levels or amounts of a protein, including wherein an altered protein, as in mutated protein or variant due to sequence alteration, deletion or insertion, or altered folding is expressed.

It is important to appreciate that the term “aberrant expression” has been specifically chosen herein to encompass the state where abnormal (usually increased) quantities/levels of the protein are present, irrespective of the efficient cause of that abnormal quantity or level. Thus, abnormal quantities of protein may result from overexpression of the protein in the absence of gene amplification, which is the case e.g. in many cellular/tissue samples taken from the head and neck of subjects with cancer, while other samples exhibit abnormal protein levels attributable to gene amplification.

In this latter connection, certain of the work of the inventors that is presented herein to illustrate the invention includes the analysis of samples certain of which exhibit abnormal protein levels resulting from amplification of EFGR. This therefore accounts for the presentation herein of experimental findings where reference is made to amplification and for the use of the terms “amplification/amplified” and the like in describing abnormal levels of EFGR. However, it is the observation of abnormal quantities or levels of the protein that defines the environment or circumstance where clinical intervention as by resort to the binding members of the invention is contemplated, and for this reason, the present specification considers that the term “aberrant expression” more broadly captures the causal environment that yields the corresponding abnormality in EFGR levels.

Accordingly, while the terms “overexpression” and “amplification” in their various grammatical forms are understood to have distinct technical meanings, they are to be considered equivalent to each other, insofar as they represent the state where abnormal EFGR protein levels are present in the context of the present invention. Consequently, the term “aberrant expression” has been chosen as it is believed to subsume the terms “overexpression” and “amplification” within its scope for the purposes herein, so that all terms may be considered equivalent to each other as used herein.

The term “antibody” describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. CDR grafted antibodies are also contemplated by this term.

As antibodies can be modified in a number of ways, the term “antibody” should be construed as covering any specific binding member or substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023 and U.S. Pat. Nos. 4,816,397 and 4,816,567.

It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E. S. et al., Nature 341, 544-546 (1989)) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (viii) multivalent antibody fragments (scFv dimers, trimers and/or tetramers (Power and Hudson, J. Immunol. Methods 242: 193-204 9 (2000))(ix) bispecific single chain Fv dimers (PCT/US92/09965) and (x) “diabodies”, multivalent or multispecific fragments constructed by gene fusion (WO94/13804; P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, (1993)).

An “antibody combining site” is that structural portion of an antibody molecule comprised of light chain or heavy and light chain variable and hypervariable regions that specifically binds antigen.

The phrase “antibody molecule” in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab′, F(ab′)2 and F(v), which portions are preferred for use in the therapeutic methods described herein.

Antibodies may also be bispecific, wherein one binding domain of the antibody is a specific binding member of the invention, and the other binding domain has a different specificity, e.g. to recruit an effector function or the like. Bispecific antibodies of the present invention include wherein one binding domain of the antibody is a specific binding member of the present invention, including a fragment thereof, and the other binding domain is a distinct antibody or fragment thereof, including that of a distinct anti-EGFR antibody, for instance antibody 528 (U.S. Pat. No. 4,943,533), the chimeric and humanized 225 antibody (U.S. Pat. No. 4,943,533 and WO/9640210), an anti-de2-7 antibody such as DH8.3 (Hills, D. et al (1995) Int. J. Cancer 63(4):537-543), antibody L8A4 and Y10 (Reist, C J et al (1995) Cancer Res. 55(19):4375-4382; Foulon C F et al. (2000) Cancer Res. 60(16):4453-4460), ICR62 (Modjtahedi H et al (1993) Cell Biophys. January-June; 22(1-3):129-46; or the antibody of Wikstrand et al (Wikstrand C. et al (1995) Cancer Res. 55(14):3140-3148). The other binding domain may be an antibody that recognizes or targets a particular cell type, as in a neural or glial cell-specific antibody. In the bispecific antibodies of the present invention the one binding domain of the antibody of the invention may be combined with other binding domains or molecules which recognize particular cell receptors and/or modulate cells in a particular fashion, as for instance an immune modulator (e.g., interleukin(s)), a growth modulator or cytokine (e.g. tumor necrosis factor (TNF), and particularly, the TNF bispecific modality demonstrated in U.S. Ser. No. 60/355,838 filed Feb. 13, 2002 incorporated herein in its entirety) or a toxin (e.g., ricin) or anti-mitotic or apoptotic agent or factor.

Fab and F(ab′)2 portions of antibody molecules may be prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See for example, U.S. Pat. No. 4,342,566 to Theofilopolous et al. Fab′ antibody molecule portions are also well-known and are produced from F(ab′)2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide. An antibody containing intact antibody molecules is preferred herein.

The phrase “monoclonal antibody” in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may also contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.

The term “antigen binding domain” describes the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may bind to a particular part of the antigen only, which part is termed an epitope. An antigen binding domain may be provided by one or more antibody variable domains. Preferably, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

“Post-translational modification” may encompass any one of or combination of modification(s), including covalent modification, which a protein undergoes after translation is complete and after being released from the ribosome or on the nascent polypeptide cotranslationally. Post-translational modification includes but is not limited to phosphorylation, myristylation, ubiquitination, glycosylation, coenzyme attachment, methylation and acetylation. Post-translational modification can modulate or influence the activity of a protein, its intracellular or extracellular destination, its stability or half-life, and/or its recognition by ligands, receptors or other proteins Post-translational modification can occur in cell organelles, in the nucleus or cytoplasm or extracellularly.

The term “specific” may be used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s). The term is also applicable where e.g. an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the specific binding member carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.

The term “comprise” generally used in the sense of include, that is to say permitting the presence of one or more features or components.

The term “consisting essentially of” refers to a product, particularly a peptide sequence, of a defined number of residues which is not covalently attached to a larger product. In the case of the peptide of the invention referred to above, those of skill in the art will appreciate that minor modifications to the N- or C-terminal of the peptide may however be contemplated, such as the chemical modification of the terminal to add a protecting group or the like, e.g. the amidation of the C-terminus.

The term “isolated” refers to the state in which specific binding members of the invention, or nucleic acid encoding such binding members will be, in accordance with the present invention. Members and nucleic acid will be free or substantially free of material with which they are naturally associated such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practised in vitro or in vivo. Members and nucleic acid may be formulated with diluents or adjuvants and still for practical purposes be isolated—for example the members will normally be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy. Specific binding members may be glycosylated, either naturally or by systems of heterologous eukaryotic cells, or they may be (for example if produced by expression in a prokaryotic cell) unglycosylated.

Also, as used herein, the terms “glycosylation” and “glycosylated” includes and encompasses the post-translational modification of proteins, termed glycoproteins, by addition of oligosaccharides. Oligosaccharides are added at glycosylation sites in glycoproteins, particularly including N-linked oligosaccharides and O-linked oligosaccharides. N-linked oligosaccharides are added to an Asn residue, particularly wherein the Asn residue is in the sequence N—X—S/T, where X cannot be Pro or Asp, and are the most common ones found in glycoproteins. In the biosynthesis of N-linked glycoproteins, a high mannose type oligosaccharide (generally comprised of dolichol, N-Acetylglucosamine, mannose and glucose is first formed in the endoplasmic reticulum (ER). The high mannose type glycoproteins are then transported from the ER to the Golgi, where further processing and modification of the oligosaccharides occurs. O-linked oligosaccharides are added to the hydroxyl group of Ser or Thr residues. In O-linked oligosaccharides, N-Acetylglucosamine is first transferred to the Ser or Thr residue by N-Acetylglucosaminyltransferase in the ER. The protein then moves to the Golgi where further modification and chain elongation occurs. O-linked modifications can occur with the simple addition of the OGlcNAc monosaccharide alone at those Ser or Thr sites which can also under different conditions be phosphorylated rather than glycosylated.

As used herein, “pg” means picogram, “ng” means nanogram, “ug” or “μg” mean microgram, “mg” means milligram, “ul” or “μl” mean microliter, “ml” means milliliter, “l” means liter.

The terms “806 antibody”, “mAb806”, “ch806” and any variants not specifically listed, may be used herein interchangeably, and as used throughout the present application and claims refer to proteinaceous material including single or multiple proteins, and extends to those proteins having the amino acid sequence data described herein and presented in SEQ ID NO:2 and SEQ ID NO:4, and the chimeric antibody ch806 which is incorporated in and forms a part of SEQ ID NOS: 7 and 8, and the profile of activities set forth herein and in the Claims. Accordingly, proteins displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits. Also, the terms “806 antibody”, “mAb806” and “ch806” are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.

The amino acid residues described herein are preferred to be in the “L” isomeric form. However, residues in the “D” isomeric form can be substituted for any L-amino acid residue, as long as the desired fuctional property of immunoglobulin-binding is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969), abbreviations for amino acid residues are shown in the following Table of Correspondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyr tyrosine G Gly glycine F Phe phenylalanine M Met methionine A Ala alanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine V Val valine P Pro proline K Lys lysine H His histidine Q Gln glutamine E Glu glutamic acid W Trp tryptophan R Arg arginine D Asp aspartic acid N Asn asparagine C Cys cysteine

It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues. The above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.

A “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).

An “origin of replication” refers to those DNA sequences that participate in DNA synthesis.

A DNA “coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.

A “signal sequence” can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

The term “oligonucleotide,” as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.

The term “primer” as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

The primers herein are selected to be “substantially” complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5′ end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.

As used herein, the terms “restriction endonucleases” and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.

It should be appreciated that also within the scope of the present invention are DNA sequences encoding specific binding members (antibodies) of the invention which code for e.g. an antibody having the same amino acid sequence as SEQ ID NO:2 or SEQ ID NO:4, but which are degenerate to SEQ ID NO:2 or SEQ ID NO:4. By “degenerate to” is meant that a different three-letter codon is used to specify a particular amino acid. It is well known in the art that the following codons can be used interchangeably to code for each specific amino acid:

Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or L) UUA or UUG or CUU or CUC or CUA or CUG Isoleucine (Ile or I) AUU or AUC or AUA Methionine (Met or M) AUG Valine (Val or V) GUU or GUC of GUA or GUG Serine (Ser or S) UCU or UCC or UCA or UCG or AGU or AGC Proline (Pro or P) CCU or CCC or CCA or CCG Threonine (Thr or T) ACU or ACC or ACA or ACG Alanine (Ala or A) GCU or GCG or GCA or GCG Tyrosine (Tyr or Y) UAU or UAC Histidine (His or H) CAU or CAC Glutamine (Gln or Q) CAA or CAG Asparagine (Asn or N) AAU or AAC Lysine (Lys or K) AAA or AAG Aspartic Acid (Asp or D) GAU or GAC Glutamic Acid (Glu or E) GAA or GAG Cysteine (Cys or C) UGU or UGC Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG Glycine (Gly or G) GGU or GGC or GGA or GGG Tryptophan (Trp or W) UGG Termination codon UAA (ochre) or UAG (amber) or UGA (opal)

It should be understood that the codons specified above are for RNA sequences. The corresponding codons for DNA have a T substituted for U.

Mutations can be made in SEQ ID NO:2 or SEQ ID NO:4 such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible. A substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. A non-conservative change is more likely to alter the structure, activity or function of the resulting protein. The present invention should be considered to include sequences containing conservative changes which do not significantly alter the activity or binding characteristics of the resulting protein.

The following is one example of various groupings of amino acids:

Amino Acids with Nonpolar R Groups

Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine

Amino Acids with Uncharged Polar R Groups

Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine

Amino Acids with Charged Polar R Groups (Negatively Charged at pH 6.0)

Aspartic acid, Glutamic acid

Basic Amino Acids (Positively Charged at pH 6.0)

Lysine, Arginine, Histidine (at pH 6.0)

Another Grouping May be Those Amino Acids with Phenyl Groups:

Phenylalanine, Tryptophan, Tyrosine

Another Grouping May be According to Molecular Weight (i.e., Size of R Groups):

Glycine 75 Alanine 89 Serine 105 Proline 115 Valine 117 Threonine 119 Cysteine 121 Leucine 131 Isoleucine 131 Asparagine 132 Aspartic acid 133 Glutamine 146 Lysine 146 Glutamic acid 147 Methionine 149 Histidine (at pH 6.0) 155 Phenylalanine 165 Arginine 174 Tyrosine 181 Tryptophan 204

Particularly preferred substitutions are:

    • Lys for Arg and vice versa such that a positive charge may be maintained;
    • Glu for Asp and vice versa such that a negative charge may be maintained;
    • Ser for Thr such that a free —OH can be maintained; and
    • Gln for Asn such that a free NH2 can be maintained.

Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced a potential site for disulfide bridges with another Cys. A H is may be introduced as a particularly “catalytic” site (i.e., H is can act as an acid or base and is the most common amino acid in biochemical catalysis). Pro may be introduced because of its particularly planar structure, which induces β-turns in the protein's structure.

Two amino acid sequences are “substantially homologous” when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90 or 95%) are identical, or represent conservative substitutions.

A “heterologous” region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.

The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.

The phrase “therapeutically effective amount” is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, preferably by at least 50 percent, preferably by at least 70 percent, preferably by at least 80 percent, preferably by at least 90%, a clinically significant change in the growth or progression or mitotic activity of a target cellular mass, group of cancer cells or tumor, or other feature of pathology. For example, the degree of EGFR activation or activity or amount or number of EGFR positive cells, particularly of antibody or binding member reactive or positive cells may be reduced.

A DNA sequence is “operatively linked” to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence. The term “operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.

The term “standard hybridization conditions” refers to salt and temperature conditions substantially equivalent to 5×SSC and 65° C. for both hybridization and wash. However, one skilled in the art will appreciate that such “standard hybridization conditions” are dependent on particular conditions including the concentration of sodium and magnesium in the buffer, nucleotide sequence length and concentration, percent mismatch, percent formamide, and the like. Also important in the determination of “standard hybridization conditions” is whether the two sequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20° C. below the predicted or determined Tm with washes of higher stringency, if desired.

B. Detailed Disclosure

The present invention provides a novel specific binding member, particularly an antibody or fragment thereof, including immunogenic fragments, which recognizes an EGFR epitope which is found in tumorigenic, hyperproliferative or abnormal cells wherein the epitope is enhanced or evident upon aberrant post-translational modification and not detectable in normal or wild type cells. In a particular but non-limiting embodiment, the binding member, such as the antibody, recognizes an EGFR epitope which is enhanced or evident upon simple carbohydrate modification or early glycosylation and is reduced or not evident in the presence of complex carbohydrate modification or glycosylation. The specific binding member, such as the antibody or fragment thereof, does not bind to or recognize normal or wild type cells containing normal or wild type EGFR epitope in the absence of overexpression and in the presence of normal EGFR post-translational modification.

The invention relates to a specific binding member, particularly an antibody or a fragment thereof, which recognizes an EGFR epitope which is present in cells expressing amplified EGFR or expressing the de2-7 EGFR and not detectable in cells expressing normal or wild type EGFR, particularly in the presence of normal post-translational modification.

It is further noted and herein demonstrated that an additional non-limiting observation or characteristic of the antibodies of the present invention is their recognition of their epitope in the presence of high mannose groups, which is a characteristic of early glycosylation or simple carbohydrate modification. Thus, altered or aberrant glycosylation facilitates the presence and/or recognition of the antibody epitope or comprises a portion of the antibody epitope.

Glycosylation includes and encompasses the post-translational modification of proteins, termed glycoproteins, by addition of oligosaccharides. Oligosaccharides are added at glycosylation sites in glycoproteins, particularly including N-linked oligosaccharides and O-linked oligosaccharides. N-linked oligosaccharides are added to an Asn residue, particularly wherein the Asn residue is in the sequence N—X—S/T, where X cannot be Pro or Asp, and are the most common ones found in glycoproteins. In the biosynthesis of N-linked glycoproteins, a high mannose type oligosaccharide (generally comprised of dolichol, N-Acetylglucosamine, mannose and glucose is first formed in the endoplasmic reticulum (ER). The high mannose type glycoproteins are then transported from the ER to the Golgi, where further processing and modification of the oligosaccharides normally occurs. O-linked oligosaccharides are added to the hydroxyl group of Ser or Thr residues. In O-linked oligosaccharides, N-Acetylglucosamine is first transferred to the Ser or Thr residue by N-Acetylglucosaminyltransferase in the ER. The protein then moves to the Golgi where further modification and chain elongation occurs.

In a particular aspect of the invention and as stated above, the present inventors have discovered novel monoclonal antibodies, exemplified herein by the antibody designated mAb 806 and its chimeric ch806, which specifically recognize amplified wild type EGFR and the de2-7 EGFR, yet bind to an epitope distinct from the unique junctional peptide of the de2-7 EGFR mutation. The antibodies of the present invention specifically recognize overexpressed EGFR, including amplified EGFR and mutant EGFR (exemplified herein by the de2-7 mutation), particularly upon aberrant post-translational modification. Additionally, while mAb 806 does not recognize the normal, wild type EGFR expressed on the cell surface of glioma cells, it does bind to the extracellular domain of the EGFR immobilized on the surface of ELISA plates, indicating a conformational epitope with a polypeptide aspect. Importantly, mAb 806 did not bind significantly to normal tissues such as liver and skin, which express levels of endogenous wt EGFR that are higher than in most other normal tissues, but wherein EGFR is not overexpressed or amplified. Thus, mAb806 demonstrates novel and useful specificity, recognizing de2-7 EGFR and amplified EGFR, while not recognizing normal, wild type EGFR or the unique junctional peptide which is characteristic of de2-7 EGFR.

In a preferred aspect, the antibody is one which has the characteristics of the antibody which the inventors have identified and characterized, in particular recognizing amplified EGFR and de2-7EGFR. In a particularly preferred aspect the antibody is the mAb 806, or active fragments thereof. In a further preferred aspect the antibody of the present invention comprises the VH and VL amino acid sequences depicted in FIG. 14 (SEQ ID NO:2) and FIG. 15 (SEQ ID NO:4) respectively. The mature amino acid sequences (without the signal sequence) are depicted, respectively, in FIG. 16 with respect to the VH (SEQ ID NO:11) and in FIG. 17 with respect to the VL (SEQ ID NO:12).

Preferably the epitope of the specific binding member or antibody is located within the region comprising residues 273-501 of the mature normal or wild type EGFR sequence. Therefore, also provided are specific binding proteins, such as antibodies, which bind to the de2-7 EGFR at an epitope located within the region comprising residues 273-501 of the EGFR sequence. The epitope may be determined by any conventional epitope mapping techniques known to the person skilled in the art. Alternatively, the DNA sequence encoding residues 273-501 could be digested, and the resultant fragments expressed in a suitable host. Antibody binding could be determined as mentioned above.

In particular, the member will bind to an epitope comprising residues 273-501 of the mature normal or wild type EGFR. However other antibodies which show the same or a substantially similar pattern of reactivity also form an aspect of the invention. This may be determined by comparing such members with an antibody comprising the VH and VL domains shown in SEQ ID NO:2 and SEQ ID NO:4 respectively. The comparison will typically be made using a Western blot in which binding members are bound to duplicate blots prepared from a nuclear preparation of cells so that the pattern of binding can be directly compared.

In another aspect, the invention provides an antibody capable of competing with the 806 antibody, under conditions in which at least 10% of an antibody having the VH and VL sequences of the 806 antibody is blocked from binding to de2-7EGFR by competition with such an antibody in an ELISA assay. As set forth above, anti-idiotype antibodies are contemplated and are illustrated herein.

An isolated polypeptide consisting essentially of the epitope comprising residues 273-501 of the mature wild type EGFR (residues 6-234 of mature de2-7 EGFR) forms another aspect of the present invention. The peptide of the invention is particularly useful in diagnostic assays or kits and therapeutically or prophylactically, including as an anti-tumor or anti-cancer vaccine. Thus compositions of the peptide of the present invention include pharmaceutical composition and immunogenic compositions.

Diagnostic and Therapeutic Uses

The unique specificity of the specific binding members, particularly antibodies or fragments thereof, of the present invention, whereby the binding member(s) recognize an EGFR epitope which is found in tumorigenic, hyperproliferative or abnormal cells and not detectable in normal or wild type cells and wherein the epitope is enhanced or evident upon aberrant post-translational modification and wherein the member(s) bind to the de2-7 EGFR and amplified EGFR but not the wt EGFR, provides diagnostic and therapeutic uses to identify, characterize, target and treat, reduce or eliminate a number of tumorigenic cell types and tumor types, for example head and neck, breast, lung, bladder or prostate tumors and glioma, without the problems associated with normal tissue uptake that may be seen with previously known EGFR antibodies. Thus, cells overexpressing EGFR (e.g. by amplification or expression of a mutant or variant EGFR), particularly those demonstrating aberrant post-translational modification may be recognized, isolated, characterized, targeted and treated or eliminated utilizing the binding member(s), particularly antibody(ies) or fragments thereof of the present invention.

The antibodies of the present invention can thus specifically categorize the nature of EGFR tumors or tumorigenic cells, by staining or otherwise recognizing those tumors or cells wherein EGFR overexpression, particularly amplification and/or EGFR mutation, particularly de2-7EGFR, is present. Further, the antibodies of the present invention, as exemplified by mAb 806 and chimeric antibody ch806, demonstrate significant in vivo anti-tumor activity against tumors containing amplified EGFR and against de2-7 EGFR positive xenografts.

As outlined above, the inventors have found that the specific binding member of the invention recognises tumor-associated forms of the EGFR (de2-7 EGFR and amplified EGFR) but not the normal, wild-type receptor when expressed in normal cells. It is believed that antibody recognition is dependent upon an aberrant post-translational modification (e.g., a unique glycosylation, acetylation or phosphorylation variant) of the EGFR expressed in cells exhibiting overexpression of the EGFR gene.

As described below, mAb 806 and ch806 have been used in therapeutic studies. mAb 806 and ch806 are shown to inhibit growth of overexpressing (e.g. amplified) EGFR xenografts and human de2-7 EGFR expressing xenografts of human tumors and to induce significant necrosis within such tumors.

Moreover, the antibodies of the present invention inhibit the growth of intracranial tumors in a preventative model. This model involves injecting glioma cells expressing de2-7 EGFR into nude mice and then injecting the antibody intracranially either on the same day or within 1 to 3 days, optionally with repeated doses. The doses of antibody are suitably about 10 μg. Mice injected with antibody are compared to controls, and it has been found that survival of the treated mice is significantly increased.

Therefore, in a further aspect of the invention, there is provided a method of treatment of a tumor, a cancerous condition, a precancerous condition, and any condition related to or resulting from hyperproliferative cell growth comprising administration of a specific binding member of the invention.

Antibodies of the present invention are designed to be used in methods of diagnosis and treatment of tumors in human or animal subjects, particularly epithelial tumors. These tumors may be primary or secondary solid tumors of any type including, but not limited to, glioma, breast, lung, prostate, head or neck tumors.

Binding Member and Antibody Generation

The general methodology for making monoclonal antibodies by hybridomas is well known Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980); Hammerling et al., “Monoclonal Antibodies And T-cell Hybridomas” (1981); Kennett et al., “Monoclonal Antibodies” (1980); see also U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632; 4,493,890.

Panels of monoclonal antibodies produced against EFGR can be screened for various properties; i.e., isotype, epitope, affinity, etc. Of particular interest are monoclonal antibodies that mimic the activity of EFGR or its subunits. Such monoclonals can be readily identified in specific binding member activity assays. High affinity antibodies are also useful when immunoaffinity purification of native or recombinant specific binding member is possible.

Methods for producing polyclonal anti-EFGR antibodies are well-known in the art. See U.S. Pat. No. 4,493,795 to Nestor et al. A monoclonal antibody, typically containing Fab and/or F(ab′)2 portions of useful antibody molecules, can be prepared using the hybridoma technology described in Antibodies—A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, New York (1988), which is incorporated herein by reference. Briefly, to form the hybridoma from which the monoclonal antibody composition is produced, a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunized with an appropriate EGFR.

Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000. Fused hybrids are selected by their sensitivity to HAT. Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact with the present antibody or binding member and their ability to inhibit specified tumorigenic or hyperproliferative activity in target cells.

A monoclonal antibody useful in practicing the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity. The culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody-containing medium is then collected. The antibody molecules can then be further isolated by well-known techniques.

Media useful for the preparation of these compositions are both well-known in the art and commercially available and include synthetic culture media, inbred mice and the like. An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol. 8:396 (1959)) supplemented with 4.5 gm/l glucose, 20 mm glutamine, and 20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

Methods for producing monoclonal anti-EGFR antibodies are also well-known in the art. See Niman et al., Proc. Nall. Acad. Sci. USA, 80:4949-4953 (1983). Typically, the EGFR or a peptide analog is used either alone or conjugated to an immunogenic carrier, as the immunogen in the before described procedure for producing anti-EGFR monoclonal antibodies. The hybridomas are screened for the ability to produce an antibody that immunoreacts with the EGFR present in tumorigenic, abnormal or hyperproliferative cells. Other anti-EGFR antibodies include but are not limited to the HuMAX-EGFr antibody from Genmab/Medarex, the 108 antibody (ATCC HB9764) and U.S. Pat. No. 6,217,866, and antibody 14E1 from Schering A G (U.S. Pat. No. 5,942,602).

Recombinant Binding Members, Chimerics, Bispecifics and Fragments

In general, the CDR3 regions, comprising amino acid sequences substantially as set out as the CDR3 regions of SEQ ID NO:2 and SEQ ID NO:4 will be carried in a structure which allows for binding of the CDR3 regions to an tumor antigen. In the case of the CDR3 region of SEQ ID NO:4, this is preferably carried by the VL region of SEQ ID NO:4.

By “substantially as set out” it is meant that that CDR3 regions of the invention will be either identical or highly homologous to the specified regions of SEQ ID NO:2 and SEQ ID NO:4. By “highly homologous” it is contemplated that only a few substitutions, preferably from 1 to 8, preferably from 1 to 5, preferably from 1 to 4, or from 1 to 3 or 1 or 2 substitutions may be made in the CDRs.

The structure for carrying the CDR3s of the invention will generally be of an antibody heavy or light chain sequence or substantial portion thereof in which the CDR3 regions are located at locations corresponding to the CDR3 region of naturally occurring VH and VL antibody variable domains encoded by rearranged immunoglobulin genes. The structures and locations of immunoglobulin variable domains may be determined by reference to Kabat, E. A. et al, Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and updates thereof.

Preferably, the amino acid sequence substantially as set out as residues 93-102 of SEQ ID NO:2 is carried as the CDR3 in a human heavy chain variable domain or a substantial portion thereof, and the amino acid sequences substantially as set out as residues 24-34, 50-56 and 89-97 of SEQ ID NO:4 are carried as the CDRs 1-3 respectively in a human light chain variable domain or a substantial portion thereof.

The variable domains may be derived from any germline or rearranged human variable domain, or may be a synthetic variable domain based on consensus sequences of known human variable domains. The CDR3-derived sequences of the invention, as defined in the preceding paragraph, may be introduced into a repertoire of variable domains lacking CDR3 regions, using recombinant DNA technology.

For example, Marks et al (Bio/Technology, 1992, 10:779-783) describe methods of producing repertoires of antibody variable domains in which consensus primers directed at or adjacent to the 5′ end of the variable domain area are used in conjunction with consensus primers to the third framework region of human VH genes to provide a repertoire of VH variable domains lacking a CDR3. Marks et al further describe how this repertoire may be combined with a CDR3 of a particular antibody. Using analogous techniques, the CDR3-derived sequences of the present invention may be shuffled with repertoires of VH or VL domains lacking a CDR3, and the shuffled complete VH or VL domains combined with a cognate VL or VH domain to provide specific binding members of the invention. The repertoire may then be displayed in a suitable host system such as the phage display system of WO92/01047 so that suitable specific binding members may be selected. A repertoire may consist of from anything from 104 individual members upwards, for example from 106 to 108 or 1010 members.

Analogous shuffling or combinatorial techniques are also disclosed by Stemmer (Nature, 1994, 370:389-391), who describes the technique in relation to a β-lactamase gene but observes that the approach may be used for the generation of antibodies.

A further alternative is to generate novel VH or VL regions carrying the CDR3-derived sequences of the invention using random mutagenesis of, for example, the mAb806 VH or VL genes to generate mutations within the entire variable domain. Such a technique is described by Gram et al (1992, Proc. Natl. Acad. Sci., USA, 89:3576-3580), who used error-prone PCR.

Another method which may be used is to direct mutagenesis to CDR regions of VH or VL genes. Such techniques are disclosed by Barbas et al, (1994, Proc. Natl. Acad. Sci., USA, 91:3809-3813) and Schier et al (1996, J. Mol. Biol. 263:551-567).

All the above described techniques are known as such in the art and in themselves do not form part of the present invention. The skilled person will be able to use such techniques to provide specific binding members of the invention using routine methodology in the art.

A substantial portion of an immunoglobulin variable domain will comprise at least the three CDR regions, together with their intervening framework regions. Preferably, the portion will also include at least about 50% of either or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Additional residues at the N-terminal or C-terminal end of the substantial part of the variable domain may be those not normally associated with naturally occurring variable domain regions. For example, construction of specific binding members of the present invention made by recombinant DNA techniques may result in the introduction of N- or C-terminal residues encoded by linkers introduced to facilitate cloning or other manipulation steps. Other manipulation steps include the introduction of linkers to join variable domains of the invention to further protein sequences including immunoglobulin heavy chains, other variable domains (for example in the production of diabodies) or protein labels as discussed in more detail below.

Although in a preferred aspect of the invention specific binding members comprising a pair of binding domains based on sequences substantially set out in SEQ ID NO:2 and SEQ ID NO:4 are preferred, single binding domains based on either of these sequences form further aspects of the invention. In the case of the binding domains based on the sequence substantially set out in SEQ ID NO:2, such binding domains may be used as targeting agents for tumor antigens since it is known that immunoglobulin VH domains are capable of binding target antigens in a specific manner.

In the case of either of the single chain specific binding domains, these domains may be used to screen for complementary domains capable of forming a two-domain specific binding member which has in vivo properties as good as or equal to the mAb806 antibody disclosed herein.

This may be achieved by phage display screening methods using the so-called hierarchical dual combinatorial approach as disclosed in U.S. Pat. No. 5,969,108 in which an individual colony containing either an H or L chain clone is used to infect a complete library of clones encoding the other chain (L or H) and the resulting two-chain specific binding member is selected in accordance with phage display techniques such as those described in that reference. This technique is also disclosed in Marks et al, ibid.

Specific binding members of the present invention may further comprise antibody constant regions or parts thereof. For example, specific binding members based on SEQ ID NO:4 may be attached at their C-terminal end to antibody light chain constant domains including human Cκ or Cλ chains, preferably Cλ chains. Similarly, specific binding members based on SEQ ID NO:2 may be attached at their C-terminal end to all or part of an immunoglobulin heavy chain derived from any antibody isotype, e.g. IgG, IgA, IgE, IgD and IgM and any of the isotype sub-classes, particularly IgG1, IgG2b, and IgG4. IgG1 is preferred.

The advent of monoclonal antibody (mAb) technology 25 years ago has provide an enormous repertoire of useful research reagents and created the opportunity to use antibodies as approved pharmaceutical reagents in cancer therapy, autoimmune disorders, transplant rejection, antiviral prophylaxis and as anti-thrombotics (Glennie and Johnson 2000). The application of molecular engineering to convert murine mAbs into chimeric mAbs (mouse V-region, human C-region) and humanised reagents where only the mAb complementarity-determining regions (CDR) are of murine origin has been critical to the clinical success of mAb therapy. The engineered mAbs have markedly reduced or absent immunogenicity, increased serum half-life and the human Fc portion of the mAb increases the potential to recruit the immune effectors of complement and cytotoxic cells (Clark 2000). Investigations into the biodistribution, pharmacokinetics and any induction of an immune response to clinically administered mAbs requires the development of analyses to discriminate between the pharmaceutical and endogenous proteins.

The antibodies, or any fragments thereof, may also be conjugated or recombinantly fused to any cellular toxin, bacterial or other, e.g. pseudomonas exotoxin, ricin, or diphtheria toxin. The part of the toxin used can be the whole toxin, or any particular domain of the toxin. Such antibody-toxin molecules have successfully been used for targeting and therapy of different kinds of cancers, see e.g. Pastan, Biochim Biophys Acta. 1997 Oct. 24; 1333(2):C1-6; Kreitman et al., N Engl J. Med. 2001 Jul. 26; 345(4):241-7; Schnell et al., Leukemia. 2000 January; 14(1):129-35; Ghetie et al., Mol. Biotechnol. 2001 July; 18(3):251-68.

Bi- and tri-specific multimers can be formed by association of different scFv molecules and have been designed as cross-linking reagents for T-cell recruitment into tumors (immunotherapy), viral retargeting (gene therapy) and as red blood cell agglutination reagents (immunodiagnostics), see e.g. Todorovska et al., J Immunol Methods. 2001 Feb. 1; 248(1-2):47-66; Tomlinson et al., Methods Enzymol. 2000; 326:461-79; McCall et al., J. Immunol. 2001 May 15; 166(10):6112-7.

Fully human antibodies can be prepared by immunizing transgenic mice carrying large portions of the human immunoglobulin heavy and light chains. These mice, examples of such mice are the Xenomouse™ (Abgenix, Inc.) (U.S. Pat. Nos. 6,075,181 and 6,150,584), the HuMAb-Mouse™ (Medarex, Inc./GenPharm) (U.S. Pat. Nos. 5,545,806 and 5,569,825), the TransChromo Mouse™ (Kirin) and the KM Mouse™ (Medarex/Kirin), are well known within the art. Antibodies can then be prepared by, e.g. standard hybridoma technique or by phage display. These antibodies will then contain only fully human amino acid sequences.

Fully human antibodies can also be generated using phage display from human libraries. Phage display may be performed using methods well known to the skilled artisan, as in Hoogenboom et al and Marks et al (Hoogenboom H R and Winter G. (1992) J Mol. Biol. 227(2):381-8; Marks J D et al (1991) J Mol. Biol. 222(3):581-97; and also U.S. Pat. Nos. 5,885,793 and 5,969,108).

Therapeutic Antibodies and Uses

The in vivo properties, particularly with regard to tumor:blood ratio and rate of clearance, of specific binding members of the invention will be at least comparable to mAb806. Following administration to a human or animal subject such a specific binding member will show a peak tumor to blood ratio of >1:1. Preferably at such a ratio the specific binding member will also have a tumor to organ ratio of greater than 1:1, preferably greater than 2:1, more preferably greater than 5:1. Preferably at such a ratio the specific binding member will also have an organ to blood ratio of <1:1 in organs away from the site of the tumor. These ratios exclude organs of catabolism and secretion of the administered specific binding member. Thus in the case of scFvs and Fabs (as shown in the accompanying examples), the binding members are secreted via the kidneys and there is greater presence here than other organs. In the case of whole IgGs, clearance will be at least in part, via the liver. The peak localisation ratio of the intact antibody will normally be achieved between 10 and 200 hours following administration of the specific binding member. More particularly, the ratio may be measured in a tumor xenograft of about 0.2-1.0 g formed subcutaneously in one flank of an athymic nude mouse.

Antibodies of the invention may be labelled with a detectable or functional label. Detectable labels include, but are not limited to, radiolabels such as the isotopes 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 121I, 124I, 125I, 131I, 111In, 211At, 198Au, 67Cu, 225Ac, 213Bi, 99Tc and 186Re, which may be attached to antibodies of the invention using conventional chemistry known in the art of antibody imaging. Labels also include fluorescent labels and labels used conventionally in the art for MRI-CT imagine. They also include enzyme labels such as horseradish peroxidase. Labels further include chemical moieties such as biotin which may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin.

Functional labels include substances which are designed to be targeted to the site of a tumor to cause destruction of tumor tissue. Such functional labels include cytotoxic drugs such as 5-fluorouracil or ricin and enzymes such as bacterial carboxypeptidase or nitroreductase, which are capable of converting prodrugs into active drugs at the site of a tumor.

Also, antibodies including both polyclonal and monoclonal antibodies, and drugs that modulate the production or activity of the specific binding members, antibodies and/or their subunits may possess certain diagnostic applications and may for example, be utilized for the purpose of detecting and/or measuring conditions such as cancer, precancerous lesions, conditions related to or resulting from hyperproliferative cell growth or the like. For example, the specific binding members, antibodies or their subunits may be used to produce both polyclonal and monoclonal antibodies to themselves in a variety of cellular media, by known techniques such as the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells. Likewise, small molecules that mimic or antagonize the activity(ies) of the specific binding members of the invention may be discovered or synthesized, and may be used in diagnostic and/or therapeutic protocols.

The radiolabelled specific binding members, particularly antibodies and fragments thereof, are useful in in vitro diagnostics techniques and in in vivo radioimaging techniques and in radioimmunotherapy. In the instance of in vivo imaging, the specific binding members of the present invention may be conjugated to an imaging agent rather than a radioisotope(s), including but not limited to a magnetic resonance image enhancing agent, wherein for instance an antibody molecule is loaded with a large number of paramagnetic ions through chelating groups. Examples of chelating groups include EDTA, porphyrins, polyamines crown ethers and polyoximes. Examples of paramagnetic ions include gadolinium, iron, manganese, rhenium, europium, lanthanum, holmium and ferbium. In a further aspect of the invention, radiolabelled specific binding members, particularly antibodies and fragments thereof, particularly radioimmunoconjugates, are useful in radioimmunotherapy, particularly as radiolabelled antibodies for cancer therapy. In a still further aspect, the radiolabelled specific binding members, particularly antibodies and fragments thereof, are useful in radioimmuno-guided surgery techniques, wherein they can identify and indicate the presence and/or location of cancer cells, precancerous cells, tumor cells, and hyperproliferative cells, prior to, during or following surgery to remove such cells.

Immunoconjugates or antibody fusion proteins of the present invention, wherein the specific binding members, particularly antibodies and fragments thereof, of the present invention are conjugated or attached to other molecules or agents further include, but are not limited to binding members conjugated to a chemical ablation agent, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent or drug.

Radioimmunotherapy (RAIT) has entered the clinic and demonstrated efficacy using various antibody immunoconjugates. 131I labeled humanized anti-carcinoembryonic antigen (anti-CEA) antibody hMN-14 has been evaluated in colorectal cancer (Behr T M et al (2002) Cancer 94(4Suppl):1373-81) and the same antibody with 90Y label has been assessed in medullary thyroid carcinoma (Stein R et al (2002) Cancer 94(1):51-61). Radioimmunotherapy using monoclonal antibodies has also been assessed and reported for non-Hodgkin's lymphoma and pancreatic cancer (Goldenberg D M (2001) Crit Rev Oncol Hematol 39(1-2):195-201; Gold D V et al (2001) Crit Rev Oncol Hematol 39 (1-2) 147-54). Radioimmunotherapy methods with particular antibodies are also described in U.S. Pat. Nos. 6,306,393 and 6,331,175. Radioimmunoguided surgery (RIGS) has also entered the clinic and demonstrated efficacy and usefulness, including using anti-CEA antibodies and antibodies directed against tumor-associated antigens (Kim J C et al (2002) Int J Cancer 97(4):542-7; Schneebaum S et al (2001) World J Surg 25(12):1495-8; Avital S et al (2000) Cancer 89(8):1692-8; McIntosh D G et al (1997) Cancer Biother Radiopharm 12 (4):287-94).

Antibodies of the present invention may be administered to a patient in need of treatment via any suitable route, usually by injection into the bloodstream or CSF, or directly into the site of the tumor. The precise dose will depend upon a number of factors, including whether the antibody is for diagnosis or for treatment, the size and location of the tumor, the precise nature of the antibody (whether whole antibody, fragment, diabody, etc), and the nature of the detectable or functional label attached to the antibody. Where a radionuclide is used for therapy, a suitable maximum single dose is about 45 mCi/m2, to a maximum of about 250 mCi/m2. Preferable dosage is in the range of 15 to 40 mCi, with a further preferred dosage range of 20 to 30 mCi, or 10 to 30 mCi. Such therapy may require bone marrow or stem cell replacement. A typical antibody dose for either tumor imaging or tumor treatment will be in the range of from 0.5 to 40 mg, preferably from 1 to 4 mg of antibody in F(ab′)2 form. Naked antibodies are preferable administered in doses of 20 to 1000 mg protein per dose, or 20 to 500 mg protein per dose, or 20 to 100 mg protein per dose. This is a dose for a single treatment of an adult patient, which may be proportionally adjusted for children and infants, and also adjusted for other antibody formats in proportion to molecular weight. Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician.

These formulations may include a second binding protein, such as the EGFR binding proteins described supra. In an especially preferred form, this second binding protein is a monoclonal antibody such as 528 or 225, discussed infra.

Pharmaceutical and Therapeutic Compositions

Specific binding members of the present invention will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the specific binding member.

Thus pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. intravenous.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.

Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

A composition may be administered alone or in combination with other treatments, therapeutics or agents, either simultaneously or sequentially dependent upon the condition to be treated. In addition, the present invention contemplates and includes compositions comprising the binding member, particularly antibody or fragment thereof, herein described and other agents or therapeutics such as anti-cancer agents or therapeutics, hormones, anti-EGFR agents or antibodies, or immune modulators. More generally these anti-cancer agents may be tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post-translational modulators, cell growth or division inhibitors (e.g. anti-mitotics), or signal transduction inhibitors. Other treatments or therapeutics may include the administration of suitable doses of pain relief drugs such as non-steroidal anti-inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such as morphine, or anti-emetics. The composition can be administered in combination (either sequentially (i.e. before or after) or simultaneously) with tyrosine kinase inhibitors (including, but not limited to AG1478 and ZD1839, STI571, OSI-774, SU-6668), doxorubicin, temozolomide, cisplatin, carboplatin, nitrosoureas, procarbazine, vincristine, hydroxyurea, 5-fluoruracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, carmustine, lomustine, and/or other chemotherapeutic agents. Thus, these agents may be anti-EGFR specific agents, or tyrosine kinase inhibitors such as AG1478, ZD1839, STI571, OSI-774, or SU-6668 or may be more general anti-cancer and anti-neoplastic agents such as doxorubicin, cisplatin, temozolomide, nitrosoureas, procarbazine, vincristine, hydroxyurea, 5-fluoruracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, carmustine, or lomustine. In addition, the composition may be administered with hormones such as dexamethasone, immune modulators, such as interleukins, tumor necrosis factor (TNF) or other growth factors or cytokines which stimulate the immune response and reduction or elimination of cancer cells or tumors. An immune modulator such as TNF may be combined together with a member of the invention in the form of a bispecific antibody recognizing the 806 EGFR epitope as well as binding to TNF receptors. The composition may also be administered with, or may include combinations along with other anti-EGFR antibodies, including but not limited to the anti-EGFR antibodies 528, 225, SC-03, DH8.3, L8A4, Y10, ICR62 and ABX-EGF.

Previously the use of agents such as doxorubicin and cisplatin in conjunction with anti-EGFR antibodies have produced enhanced anti-tumor activity (Fan et al, 1993; Baselga et al, 1993). The combination of doxorubicin and mAb 528 resulted in total eradication of established A431 xenografts, whereas treatment with either agent alone caused only temporary in vivo growth inhibition (Baselga et al, 1993). Likewise, the combination of cisplatin and either mAb 528 or 225 also led to the eradication of well established A431 xenografts, which was not observed when treatment with either agent was used (Fan et al, 1993).

Conventional Radiotherapy

In addition, the present invention contemplates and includes therapeutic compositions for the use of the binding member in combination with conventional radiotherapy. It has been indicated that treatment with antibodies targeting EGF receptors can enhance the effects of conventional radiotherapy (Milas et al., Clin Cancer Res. 2000 February:6 (2):701 8, Huang et al., Clin Cancer Res. 2000 June: 6(6):2166 74).

As demonstrated herein, combinations of the binding member of the present invention, particularly an antibody or fragment thereof, preferably the mAb806, ch806 or a fragment thereof, and anti-cancer therapeutics, particularly anti-EGFR therapeutics, including other anti-EGFR antibodies, demonstrate effective therapy, and particularly synergy, against xenografted tumors. In the examples, it is demonstrated that the combination of AG1478 and mAb 806 results in significantly enhanced reduction of A431 xenograft tumor volume in comparison with treatment with either agent alone. AG1478 (4-(3-chloroanilino)-6,7-dimethoxyquinazoline) is a potent and selective inhibitor of the EGF receptor kinase and is particularly described in U.S. Pat. No. 5,457,105, incorporated by reference herein in its entirety (see also, Liu, W. et al (1999) J. Cell Sci. 112:2409; Eguchi, S. et al (1998) J. Biol. Chem. 273:8890; Levitsky, A. and Gazit, A. (1995) Science 267:1782). The specification examples further demonstrate therapeutic synergy of the 806 antibody with other anti-EGFR antibodies, particularly with the 528 anti-EGFR antibody.

The present invention further contemplates therapeutic compositions useful in practicing the therapeutic methods of this invention. A subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and one or more of a specific binding member, polypeptide analog thereof or fragment thereof, as described herein as an active ingredient. In a preferred embodiment, the composition comprises an antigen capable of modulating the specific binding of the present binding member/antibody with a target cell.

The preparation of therapeutic compositions which contain polypeptides, analogs or active fragments as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions. However, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.

A polypeptide, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The therapeutic polypeptide-, analog- or active fragment-containing compositions are conventionally administered intravenously, as by injection of a unit dose, for example. The term “unit dose” when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of EFGR binding capacity desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations of ten nanomolar to ten micromolar in the blood are contemplated.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Diagnostic Assays

The present invention also relates to a variety of diagnostic applications, including methods for detecting the presence of stimuli such as aberrantly expressed EGFR, by reference to their ability to be recognized by the present specific binding member. As mentioned earlier, the EGFR can be used to produce antibodies to itself by a variety of known techniques, and such antibodies could then be isolated and utilized as in tests for the presence of particular EGFR activity in suspect target cells.

Diagnostic applications of the specific binding members of the present invention, particularly antibodies and fragments thereof, include in vitro and in vivo applications well known and standard to the skilled artisan and based on the present description. Diagnostic assays and kits for in vitro assessment and evaluation of EGFR status, particularly with regard to aberrant expression of EGFR, may be utilized to diagnose, evaluate and monitor patient samples including those known to have or suspected of having cancer, a precancerous condition, a condition related to hyperproliferative cell growth or from a tumor sample. The assessment and evaluation of EGFR status is also useful in determining the suitability of a patient for a clinical trial of a drug or for the administration of a particular chemotherapeutic agent or specific binding member, particularly an antibody, of the present invention, including combinations thereof, versus a different agent or binding member. This type of diagnostic monitoring and assessment is already in practice utilizing antibodies against the HER2 protein in breast cancer (Hercep Test, Dako Corporation), where the assay is also used to evaluate patients for antibody therapy using Herceptin. In vivo applications include imaging of tumors or assessing cancer status of individuals, including radioimaging.

As suggested earlier, the diagnostic method of the present invention comprises examining a cellular sample or medium by means of an assay including an effective amount of an antagonist to an EFGR/protein, such as an anti-EFGR antibody, preferably an affinity-purified polyclonal antibody, and more preferably a mAb. In addition, it is preferable for the anti-EFGR antibody molecules used herein be in the form of Fab, Fab′, F(ab′)2 or F(v) portions or whole antibody molecules. As previously discussed, patients capable of benefiting from this method include those suffering from cancer, a pre-cancerous lesion, a viral infection, pathologies involving or resulting from hyperproliferative cell growth or other like pathological derangement. Methods for isolating EFGR and inducing anti-EFGR antibodies and for determining and optimizing the ability of anti-EFGR antibodies to assist in the examination of the target cells are all well-known in the art.

Preferably, the anti-EFGR antibody used in the diagnostic methods of this invention is an affinity purified polyclonal antibody. More preferably, the antibody is a monoclonal antibody (mAb). In addition, the anti-EFGR antibody molecules used herein can be in the form of Fab, Fab′, F(ab′)2 or F(v) portions of whole antibody molecules.

As described in detail above, antibody(ies) to the EGFR can be produced and isolated by standard methods including the well known hybridoma techniques. For convenience, the antibody(ies) to the EGFR will be referred to herein as Ab1 and antibody(ies) raised in another species as Ab2.

The presence of EGFR in cells can be ascertained by the usual in vitro or in vivo immunological procedures applicable to such determinations. A number of useful procedures are known. Three such procedures which are especially useful utilize either the EGFR labeled with a detectable label, antibody Ab1 labeled with a detectable label, or antibody Ab2 labeled with a detectable label. The procedures may be summarized by the following equations wherein the asterisk indicates that the particle is labeled, and “R” stands for the EGFR:
R*+Ab1=R*Ab1  A.
R+Ab*=RAb1*  B.
R+Ab1+Ab2*=RAb1Ab2*  C.

The procedures and their application are all familiar to those skilled in the art and accordingly may be utilized within the scope of the present invention. The “competitive” procedure, Procedure A, is described in U.S. Pat. Nos. 3,654,090 and 3,850,752. Procedure C, the “sandwich” procedure, is described in U.S. Pat. Nos. RE 31,006 and 4,016,043. Still other procedures are known such as the “double antibody,” or “DASP” procedure.

In each instance above, the EGFR forms complexes with one or more antibody(ies) or binding partners and one member of the complex is labeled with a detectable label.

The fact that a complex has formed and, if desired, the amount thereof, can be determined by known methods applicable to the detection of labels.

It will be seen from the above, that a characteristic property of Ab2 is that it will react with Ab1. This is because Ab1 raised in one mammalian species has been used in another species as an antigen to raise the antibody Ab2. For example, Ab2 may be raised in goats using rabbit antibodies as antigens. Ab2 therefore would be anti-rabbit antibody raised in goats. For purposes of this description and claims, Ab1 will be referred to as a primary or anti-EGFR antibody, and Ab2 will be referred to as a secondary or anti-Ab1 antibody.

The labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.

A number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red®, AMCA™ blue and Lucifer Yellow. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.

The EGFR or its binding partner(s) such as the present specific binding member, can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures. The preferred isotope may be selected from 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 121I, 124I, 125I, 131I, 111In, 211At, 198Au, 67Cu, 225Ac, 213Bi, 99Tc and 186Re.

Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques. The enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.

A particular assay system that may be advantageously utilized in accordance with the present invention, is known as a receptor assay. In a receptor assay, the material to be assayed such as the specific binding member, is appropriately labeled and then certain cellular test colonies are inoculated with a quantity of both the labeled and unlabeled material after which binding studies are conducted to determine the extent to which the labeled material binds to the cell receptors. In this way, differences in affinity between materials can be ascertained.

Accordingly, a purified quantity of the specific binding member may be radiolabeled and combined, for example, with antibodies or other inhibitors thereto, after which binding studies would be carried out. Solutions would then be prepared that contain various quantities of labeled and unlabeled uncombined specific binding member, and cell samples would then be inoculated and thereafter incubated. The resulting cell monolayers are then washed, solubilized and then counted in a gamma counter for a length of time sufficient to yield a standard error of <5%. These data are then subjected to Scatchard analysis after which observations and conclusions regarding material activity can be drawn. While the foregoing is exemplary, it illustrates the manner in which a receptor assay may be performed and utilized, in the instance where the cellular binding ability of the assayed material may serve as a distinguishing characteristic.

An assay useful and contemplated in accordance with the present invention is known as a “cis/trans” assay. Briefly, this assay employs two genetic constructs, one of which is typically a plasmid that continually expresses a particular receptor of interest when transfected into an appropriate cell line, and the second of which is a plasmid that expresses a reporter such as luciferase, under the control of a receptor/ligand complex. Thus, for example, if it is desired to evaluate a compound as a ligand for a particular receptor, one of the plasmids would be a construct that results in expression of the receptor in the chosen cell line, while the second plasmid would possess a promoter linked to the luciferase gene in which the response element to the particular receptor is inserted. If the compound under test is an agonist for the receptor, the ligand will complex with the receptor, and the resulting complex will bind the response element and initiate transcription of the luciferase gene. The resulting chemiluminescence is then measured photometrically, and dose response curves are obtained and compared to those of known ligands. The foregoing protocol is described in detail in U.S. Pat. No. 4,981,784 and PCT International Publication No. WO 88/03168, for which purpose the artisan is referred.

In a further embodiment of this invention, commercial test kits suitable for use by a medical specialist may be prepared to determine the presence or absence of aberrant expression of EGFR, including but not limited to amplified EGFR and/or an EGFR mutation, in suspected target cells. In accordance with the testing techniques discussed above, one class of such kits will contain at least the labeled EGFR or its binding partner, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g., “competitive,” “sandwich,” “DASP” and the like. The kits may also contain peripheral reagents such as buffers, stabilizers, etc.

Accordingly, a test kit may be prepared for the demonstration of the presence or capability of cells for aberrant expression or post-translational modification of EGFR, comprising:

    • (a) a predetermined amount of at least one labeled immunochemically reactive component obtained by the direct or indirect attachment of the present specific binding member or a specific binding partner thereto, to a detectable label;
    • (b) other reagents; and
    • (c) directions for use of said kit.

More specifically, the diagnostic test kit may comprise:

    • (a) a known amount of the specific binding member as described above (or a binding partner) generally bound to a solid phase to form an immunosorbent, or in the alternative, bound to a suitable tag, or plural such end products, etc. (or their binding partners) one of each;
    • (b) if necessary, other reagents; and
    • (c) directions for use of said test kit.

In a further variation, the test kit may be prepared and used for the purposes stated above, which operates according to a predetermined protocol (e.g. “competitive,” “sandwich,” “double antibody,” etc.), and comprises:

    • (a) a labeled component which has been obtained by coupling the specific binding member to a detectable label;
    • (b) one or more additional immunochemical reagents of which at least one reagent is a ligand or an immobilized ligand, which ligand is selected from the group consisting of:
      • (i) a ligand capable of binding with the labeled component (a);
      • (ii) a ligand capable of binding with a binding partner of the labeled component (a);
      • (iii) a ligand capable of binding with at least one of the component(s) to be determined; and
      • (iv) a ligand capable of binding with at least one of the binding partners of at least one of the component(s) to be determined; and
    • (c) directions for the performance of a protocol for the detection and/or determination of one or more components of an immunochemical reaction between the EFGR, the specific binding member, and a specific binding partner thereto.

In accordance with the above, an assay system for screening potential drugs effective to modulate the activity of the EFGR, the aberrant expression or post-translational modification of the EGFR, and/or the activity or binding of the specific binding member may be prepared. The receptor or the binding member may be introduced into a test system, and the prospective drug may also be introduced into the resulting cell culture, and the culture thereafter examined to observe any changes in the S-phase activity of the cells, due either to the addition of the prospective drug alone, or due to the effect of added quantities of the known agent(s).

Nucleic Acids

The present invention further provides an isolated nucleic acid encoding a specific binding member of the present invention. Nucleic acid includes DNA and RNA. In a preferred aspect, the present invention provides a nucleic acid which codes for a polypeptide of the invention as defined above, including a polypeptide as set out as residues 93-102 of SEQ ID NO:2 or 26-35A, 49-64 and 93-102 of SEQ ID NO:2, a polypeptide as set out in residues 24-34, 50-56 and 89-97 of SEQ ID NO:4, and the entire polypeptides of SEQ ID NO:2 and SEQ ID NO:4.

The present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one polynucleotide as above.

The present invention also provides a recombinant host cell which comprises one or more constructs as above. A nucleic acid encoding any specific binding member as provided itself forms an aspect of the present invention, as does a method of production of the specific binding member which method comprises expression from encoding nucleic acid therefor. Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression a specific binding member may be isolated and/or purified using any suitable technique, then used as appropriate.

Specific binding members and encoding nucleic acid molecules and vectors according to the present invention may be provided isolated and/or purified, e.g. from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes origin other than the sequence encoding a polypeptide with the required function. Nucleic acid according to the present invention may comprise DNA or RNA and may be wholly or partially synthetic.

Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others. A common, preferred bacterial host is E. coli.

The expression of antibodies and antibody fragments in prokaryotic cells such as E. coli is well established in the art. For a review, see for example Pliickthun, A. Bio/Technology 9: 545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of a specific binding member, see for recent reviews, for example Raff, M.E. (1993) Curr. Opinion Biotech. 4: 573-576; Trill J. J. et al. (1995) Curr. Opinion Biotech 6: 553-560.

Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. ‘phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference.

Thus, a further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein. A still further aspect provides a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.

The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene.

In one embodiment, the nucleic acid of the invention is integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.

The present invention also provides a method which comprises using a construct as stated above in an expression system in order to express a specific binding member or polypeptide as above.

As stated above, the present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes a specific binding member, particularly antibody or a fragment thereof, that possesses an amino acid sequence set forth in SEQ ID NO:2 and/or SEQ ID NO:4; preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the binding member or antibody has a nucleotide sequence or is complementary to a DNA sequence provided in SEQ ID NO:1 and/or SEQ ID NO:3.

Another feature of this invention is the expression of the DNA sequences disclosed herein. As is well known in the art, DNA sequences may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host.

Such operative linking of a DNA sequence of this invention to an expression control sequence, of course, includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence.

A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col E1, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e.g., the numerous derivatives of phage, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2 u plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

Any of a wide variety of expression control sequences—sequences that control the expression of a DNA sequence operatively linked to it—may be used in these vectors to express the DNA sequences of this invention. Such useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast-mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

A wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, YB/20, NSO, SP2/0, R1.1, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g., Sf9), and human cells and plant cells in tissue culture.

It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of this invention. For example, in selecting a vector, the host must be considered because the vector must function in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors will normally be considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the particular DNA sequence or gene to be expressed, particularly as regards potential secondary structures. Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products.

Considering these and other factors a person skilled in the art will be able to construct a variety of vector/expression control sequence/host combinations that will express the DNA sequences of this invention on fermentation or in large scale animal culture.

It is further intended that specific binding member analogs may be prepared from nucleotide sequences of the protein complex/subunit derived within the scope of the present invention. Analogs, such as fragments, may be produced, for example, by pepsin digestion of specific binding member material. Other analogs, such as muteins, can be produced by standard site-directed mutagenesis of specific binding member coding sequences. Analogs exhibiting “specific binding member activity” such as small molecules, whether functioning as promoters or inhibitors, may be identified by known in vivo and/or in vitro assays.

As mentioned above, a DNA sequence encoding a specific binding member can be prepared synthetically rather than cloned. The DNA sequence can be designed with the appropriate codons for the specific binding member amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al., Science, 223:1299 (1984); Jay et al., J. Biol. Chem., 259:6311 (1984).

Synthetic DNA sequences allow convenient construction of genes which will express specific binding member analogs or “muteins”. Alternatively, DNA encoding muteins can be made by site-directed mutagenesis of native specific binding member genes or cDNAs, and muteins can be made directly using conventional polypeptide synthesis.

A general method for site-specific incorporation of unnatural amino acids into proteins is described in Christopher J. Noren, Spencer J. Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, Science, 244:182-188 (April 1989). This method may be used to create analogs with unnatural amino acids.

The present invention extends to the preparation of antisense oligonucleotides and ribozymes that may be used to interfere with the expression of the EGFR at the translational level. This approach utilizes antisense nucleic acid and ribozymes to block translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule. (See Weintraub, 1990; Marcus-Sekura, 1988.) In the cell, they hybridize to that mRNA, forming a double stranded molecule. The cell does not translate an mRNA in this double-stranded form. Therefore, antisense nucleic acids interfere with the expression of mRNA into protein. Oligomers of about fifteen nucleotides and molecules that hybridize to the AUG initiation codon will be particularly efficient, since they are easy to synthesize and are likely to pose fewer problems than larger molecules when introducing them into—producing cells. Antisense methods have been used to inhibit the expression of many genes in vitro (Marcus-Sekura, 1988; Hambor et al., 1988).

Ribozymes are RNA molecules possessing the ability to specifically cleave other single stranded RNA molecules in a manner somewhat analogous to DNA restriction endonucleases. Ribozymes were discovered from the observation that certain mRNAs have the ability to excise their own introns. By modifying the nucleotide sequence of these RNAs, researchers have been able to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, 1988). Because they are sequence-specific, only mRNAs with particular sequences are inactivated.

Investigators have identified two types of ribozymes, Tetrahymena-type and “hammerhead”-type. (Hasselhoff and Gerlach, 1988) Tetrahymena-type ribozymes recognize four-base sequences, while “hammerhead”-type recognize eleven- to eighteen-base sequences. The longer the recognition sequence, the more likely it is to occur exclusively in the target mRNA species. Therefore, hammerhead-type ribozymes are preferable to Tetrahymena-type ribozymes for inactivating a specific mRNA species, and eighteen base recognition sequences are preferable to shorter recognition sequences.

The DNA sequences described herein may thus be used to prepare antisense molecules against, and ribozymes that cleave mRNAs for EFGRs and their ligands.

The invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention and should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLE 1 Isolation of Antibodies

Materials

Cell Lines

For immunization and specificity analyses, several cell lines, native or transfected with either the normal, wild type or “wtEGFR” gene or the ΔEGFR gene carrying the Δ2-7 deletion mutation were used: Murine fibroblast cell line NR6, NR6ΔEGFR (transfected with ΔEGFR) and NR6wtEGFR (transfected with wtEGFR), human glioblastoma cell line U87MG (expressing low levels of endogenous wtEGFR), U87MGwtEGFR (transfected with wtEGFR), U87MGΔEGFR (transfected with ΔEGFR), and human squamous cell carcinoma cell line A431 (expressing high levels of wtEGFR)[38]. Cell lines and transfections were described previously (Nishikawa R., et al. (1994) Proc. Natl. Acad. Sci. 91(16):7727-7731).

The U87MG astrocytoma cell line (20) (ATCC Accession No. HTB-14), which endogenously expresses low levels of the wt EGFR, was infected with a retrovirus containing the de2-7 EGFR to produce the U87MG.Δ2-7 cell line (10). The transfected cell line U87MG.wtEGFR was produced as described in Nagane et al 1996 (Cancer Res., 56: 5079-5086). Whereas U87MG cells express approximately 1×105 EGFR, U87MG.wtEGFR cells express approximately 1×106 EGFR, and thus mimic the situation seen with gene amplification.

Human squamous carcinoma A431 cells were obtained from ATCC (Rockville, Md.). All cell lines were cultured in DMEM/F-12 with GlutaMAX™ (Life Technologies, Melbourne, Australia) supplemented with 10% FCS(CSL, Melbourne, Australia).

Reagents

Biotinylated unique junctional peptides (Biotin-LEEKKGNYVVTDH (SEQ ID NO:5) and LEEKKGNYVVTDH-Biotin (SEQ ID NO:6)) from de2-7 EGFR were synthesized by standard Fmoc chemistry and purity (>96%) determined by reverse phase HPLC and mass spectral analysis (Auspep, Melbourne, Australia).

Antibodies Used in Studies

In order to compare our findings with other reagents, additional mAbs were included in our studies. These reagents were mAb 528 to the wtEGFR (Sato, J. D. et al. (1983) Mol. Biol. Med. 1(5):511-529) and DH8.3, which was generated against a synthetic peptide spanning the junctional sequence of the 42-7 EGFR deletion mutation. The DH8.3 antibody (IgG1) has been described previously (Hills et al, 1995, Int. J. Cancer 63(4); 537-543) and was obtained following immunization of mice with the unique junctional peptide found in de2-7 EGFR (16). The 528 antibody, which recognizes both de2-7 and wild type EGFR, has been described previously (21) and was produced in the Biological Production Facility (Ludwig Institute for Cancer Research, Melbourne) using a hybridoma obtained from ATCC HB-8509. SC-03 is an affinity purified rabbit polyclonal antibody raised against a carboxy terminal peptide of the EGFR (Santa Cruz Biotechnology Inc.).

Generation of Monoclonal Antibodies

The murine fibroblast line NR6ΔEGFR was used as immunogen. Mouse hybridomas were generated by immunizing BALB/c mice five times subcutaneously at 2- to 3-week intervals, with 5×105-2×106 cells in adjuvant. Complete Freund's adjuvant was used for the first injection. Thereafter, incomplete Freund's adjuvant (Difco) was used. Spleen cells from immunized mice were fused with mouse myeloma cell line SP2/0. Supernatants of newly generated clones were screened in hemadsorption assays for reactivity with cell line NR6, NR6wtEGFR, and NR6ΔEGFR and then analyzed by hemadsorption assays with human glioblastoma cell lines U87MG, U87MGwtEGFR, and U87MGΔEGFR. Selected hybridoma supernatants were subsequently tested by western blotting and further analyzed by immunohistochemistry. Newly generated mAbs showing the expected reactivity pattern were purified.

Five hybridomas were established and three, clones 124 (IgG2a), 806 (IgG2b) (hybridoma 806, which produced mAb806, was deposited with the American Type Culture Collection (1801 University Blvd., Manassas, Va. 20110) as ATCC Deposit Number PTA-3858 on Nov. 4, 2001 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms), and 1133 (IgG2a) were selected for further characterization based on high titer with NR6ΔEGFR and low background on NR6 and NR6wtEGFR cells in the hemagglutination assay. In a subsequent hemagglutination analysis, these antibodies showed no reactivity (undiluted supernatant <10%) with the native human glioblastoma cell line U87MG and U87MGwtEGFR, but were strongly reactive with U87MGΔEGFR; less reactivity was seen with A431. By contrast, in FACS analysis, 806 was unreactive with native U87MG and intensively stained U87MGΔEGFR and to a lesser degree U87MGwtEGFR indicating binding of 806 to both, ΔEGFR and wtEGFR (see below).

In Western blot assays, mAbs 124, 806 and 1133 were then analyzed for reactivity with wtEGFR and ΔEGFR. Detergent lysates were extracted from NR6ΔEGFR, U87MGΔEGFR as well as from A431. All three mAbs showed a similar reactivity pattern with cell lysates staining both the wtEGFR (170 kDa) and ΔEGFR protein (140 kDa). As a reference reagent, mAb R.I known to be reactive with the wtEGFR (Waterfield M. D. et al. (1982) J. Cell Biochem. 20(2):149-161) was used instead of mAb 528, which is known to be non-reactive in western blot analysis. Mab R.I showed reactivity with wt and ΔEGFR. All three newly generated clones showed reactivity with ΔEGFR and less intense with wtEGFR. DH8.3 was solely positive in the lysate of U87MGΔEGFR and NR6ΔEGFR.

The immunohistochemical analysis of clones 124, 806, and 1133 as well as mAb 528 and mAb DH8.3 on xenograft tumors U87MG, U87MGΔEGFR, and A431 are shown in Table 1. All mAbs showed strong staining of xenograft U87MGΔEGFR. Only mAb 528 showed weak reactivity in the native U87MG xenograft. In A431 xenografts, mAb 528 showed strong homogeneous reactivity. MAbs 124, 806, and 1133 revealed reactivity with mostly the basally located cells of the squamous cell carcinoma of A431 and did not react with the upper cell layers or the keratinizing component. DH8.3 was negative in A431 xenografts.

TABLE 1 Immunohistochemical Analysis of Antibodies 528, DH8.3, and 124, 806 and 1133 xenograft xenograft U87MG Mab ΔU87MGΔEGFR xenograft A431 (native) 528 pos. pos. pos. (focal staining) mAb-124 pos. pos. (predominantly basal − cells) mAb-806 pos. pos. (predominantly basal − cells) mAb-1133 pos. pos. (predominantly basal − cells) DH8.3 pos. − − minor stromal staining due to detection of endogenous mouse antibodies

EXAMPLE 2 Binding of Antibodies to Cell Lines by FACS

In order to determine the specificity of mAb 806, its binding to U87MG, U87MG.Δ2-7 and U87MG.wtEGFR cells was analyzed by flow activated cell sorting (FACS). Briefly, cells were labelled with the relevant antibody (10 μg/ml) followed by fluorescein-conjugated goat anti-mouse IgG (1:100 dilution; Calbiochem San Diego, USA). FACS data was obtained on a Coulter Epics Elite™ ESP by observing a minimum of 5,000 events and analysed using EXPO™ (version 2) for Windows. An irrelevant IgG2b was included as an isotype control for mAb 806 and the 528 antibody was included as it recognizes both the de2-7 and wt EGFR.

Only the 528 antibody was able to stain the parental U87MG cell line (FIG. 1) consistent with previous reports demonstrating that these cells express the wt EGFR (Nishikawa et al, 1994). MAb 806 and DH8.3 had binding levels similar to the control antibody, clearly demonstrating that they are unable to bind the wt receptor (FIG. 1). Binding of the isotype control antibody to U87MG.Δ2-7 and U87MG.wtEGFR cells was similar as that observed for the U87MG cells.

MAb 806 stained U87MG.Δ2-7 and U87MG.wtEGFR cells, indicating that mAb 806 specifically recognizes the de2-7 EGFR and amplified EGFR (FIG. 1). DH8.3 antibody stained U87MG.Δ2-7 cells, confirming that DH8.3 antibody specifically recognizes the de2-7 EGFR (FIG. 1). As expected, the 528 antibody stained both the U87MG.Δ2-7 and U87MG.wtEGFR cell lines (FIG. 1). As expected, the 528 antibody stained U87MG.Δ2-7 with a higher intensity than the parental cell as it binds both the de2-7 and wild type receptors that are co-expressed in these cells (FIG. 1). Similar results were obtained using a protein A mixed hemadsorption which detects surface bound IgG by appearance of Protein A coated with human red blood cells (group O) to target cells. Monoclonal antibody 806 was reactive with U87MG.Δ2-7 cells but showed no significant reactivity (undiluted supernatant less than 10%) with U87MG expressing wild type EGFR.

EXAMPLE 3 Binding of Antibodies in Assays

To further characterize the specificity of mAb 806 and the DH8.3 antibody, their binding was examined by ELISA. Two types of ELISA were used to determine the specificity of the antibodies. In the first assay, plates were coated with sEGFR (10 μg/ml in 0.1 M carbonate buffer pH 9.2) for 2 h and then blocked with 2% human serum albumin (HSA) in PBS. sEGFR is the recombinant extracellular domain (amino acids 1-621) of the wild type EGFR), and was produced as previously described (22). Antibodies were added to wells in triplicate at increasing concentration in 2% HSA in phosphate-buffered saline (PBS). Bound antibody was detected by horseradish peroxidase conjugated sheep anti-mouse IgG (Silenus, Melbourne, Australia) using ABTS (Sigma, Sydney, Australia) as a substrate and the absorbance measured at 405 nm.

Both mAb 806 and the 528 antibody displayed dose-dependent and saturating binding curves to immobilized wild type sEGFR (FIG. 2A). As the unique junctional peptide found in the de2-7 EGFR is not contained within the sEGFR, mAb 806 must be binding to an epitope located within the wild type EGFR sequence. The binding of the 528 antibody was lower than that observed for mAb 806. As expected the DH8.3 antibody did not bind the wild type sEGFR even at concentrations up to 10 μg/ml (FIG. 2A). While sEGFR in solution inhibited the binding of the 528 antibody to immobilized sEGFR in a dose-dependent fashion, it was unable to inhibit the binding of mAb 806 (FIG. 2B). This suggests that mAb 806 can only bind wild type EGFR once immobilized on ELISA plates, a process that may induce conformational changes. Similar results were observed using a BIAcore™ whereby mAb 806 bound immobilized sEGFR but immobilized mAb 806 was not able to bind sEGFR in solution (data not shown). The DH8.3 antibody exhibited dose-dependent and saturable binding to the unique de2-7 EGFR peptide (FIG. 2C).

In the second assay, the biotinylated de2-7 specific peptide (Biotin-LEEKKGNYVVTDH (SEQ ID NO:5)) was bound to ELISA plates precoated with streptavidin (Pierce, Rockford, Ill.). Antibodies were bound and detected as in the first assay. Neither mAb 806 nor the 528 antibody bound to the peptide, even at concentrations higher than those used to obtain saturation binding of DH8.3, further indicating that mAb 806 does not recognize an epitope determinant within this peptide.

To further demonstrate that mAb 806 recognizes an epitope distinct from the junction peptide, additional experiments were performed. C-terminal biotinylated de2-7 peptide (LEEKKGNYVVTDH-Biotin (SEQ ID NO:6)) was utilized in studies with mAb806 and mAb L8A4, generated against the de2-7 peptide (Reist, C J et al (1995) Cancer Res. 55(19):4375-4382; Foulon C F et al. (2000) Cancer Res. 60(16):4453-4460).

Reagents Used in Peptide Studies:

Junction Peptide: (SEQ ID NO: 13) LEEKKGNYVVTDH-OH (Biosource, Camarillo, CA). Peptide C: (SEQ ID NO: 14) LEEKKGNYVVTDH(K-Biot)-OH (Biosource, Camarillo, CA).
  • sEGFR: CHO-cell-derived recombinant soluble extracellular domain (aa 1-621) of the wild type EGFR (LICR Melbourne).
  • mAb 806: mouse monoclonal antibody, IgG2b (LICR NYB).
  • mAb L8A4: mouse monoclonal antibody, IgG1 (Duke University).
  • IgG1 isotype control mAb.
  • IgG2b isotype control mAb.

Peptide C was immobilized on a Streptavidin microsensor chip at a surface density of 350RU (+/−30RU). Serial dilutions of mAbs were tested for reactivity with the peptide. Blocking experiments using non-biotinylated peptide were performed to assess specificity.

MAb L8A4 showed strong reactivity with Peptide C even at low antibody concentrations (6.25 nM) (FIG. 2.D). mAb 806 did not show detectable specific reactivity with peptide C up to antibody concentrations of 100 nM (highest concentration tested) (FIGS. 2D AND 2E). It was expected that mAb L8A4 would react with Peptide C because the peptide was used as the immunogen in the generation of mAb L8A4. Addition of the Junction Peptide (non-biotinylated, 50 μg/ml) completely blocks the reactivity of mAb L8A4 with Peptide C, confirming the antibody's specificity for the junction peptide epitope.

In a second set of BIACORE™ experiments, sEGFR was immobilized on a CM microsensor chip at a surface density of ˜4000RU. Serial dilutions of mAbs were tested for reactivity with sEGFR.

MAb 806 was strongly reactive with denaturated sEGFR while mAb L8A4 did not react with denaturated sEGFR. Reactivity of mAb 806 with denaturated sEGFR decreases with decreasing antibody concentrations. It was expected that mAb L8A4 does not react with sEGFR because mAb L8A4 was generated using the junction peptide as the immunogen and sEGFR does not contain the junction peptide.

Dot-blot immune stain experiments were also performed. Serial dilutions of peptide were spotted in 0.5 μl onto a PVDF or nitrocellulose membranes. Membranes were blocked with 2% BSA in PBS, and then probed with 806, L8A4, DH8.3 and control antibodies. Antibodies L8A4 and DH8.3 bound to peptide on the membranes (data not shown). Mab806 did not bind peptide at concentrations where L8A4 clearly showed binding (data not shown). Control antibodies were also negative for peptide binding.

mAb 806 bound to the wtEGFR in cell lysates following immunoblotting (results not shown). This is different from the results obtained with DH8.3 antibody, which reacted with de2-7 EGFR but not wtEGFR. Thus, mAb 806 can recognize the wtEGFR following denaturation but not when the receptor is in its natural state on the cell surface.

EXAMPLE 4 Scatchard Analysis

A Scatchard analysis using U87MG.Δ2-7 cells was performed following correction for immunoreactivity in order to determine the relative affinity of each antibody. Antibodies were labelled with 125I (Amrad, Melbourne, Australia) by the chloramine T method and immunoreactivity determined by Lindmo assay (23). All binding assays were performed in 1% HSA/PBS on 1−2×106 live U87MG.Δ2-7 or A431 cells for 90 min at 4° C. with gentle rotation. A set concentration of 10 ng/ml 125I-labeled antibody was used in the presence of increasing concentrations of the appropriate unlabeled antibody. Non-specific binding was determined in the presence of 10,000-fold excess of unlabeled antibody. After the incubation was completed, cells were washed and counted for bound 125I-labeled antibody using a COBRA II gamma counter (Packard Instrument Company, Meriden, USA).

Both mAb 806 and the DH8.3 antibody retained high immunoreactivity when iodinated and was typically greater than 90% for mAb 806 and 45-50% for the DH8.3 antibody. mAb 806 had an affinity for the de2-7 EGFR receptor of 1.1×109 M−1 whereas the affinity of DH8.3 was some 10-fold lower at 1.0×108 M−1. Neither 125I-radiolabeled mAb 806 nor the 125I-radiolabeled DH8.3 antibody bound to parental U87MG cells. mAb 806 recognized an average of 2.4×105 binding sites per cell with the DH8.3 antibody binding an average of 5.2×105 sites. Thus, there was not only good agreement in receptor number between the antibodies, but also with a previous report showing 2.5×105 de2-7 receptors per cell as measured by a different de2-7 EGFR specific antibody on the same cell line (25).

EXAMPLE 5 Internalization of Antibodies by U87MG.Δ2-7 Cells

The rate of antibody internalization following binding to a target cell influences both its tumor targeting properties and therapeutic options. Consequently, the inventors examined the internalization of mAb 806 and the DH8.3 antibody following binding to U87MG.Δ2-7 cells by FACS. U87MG.Δ2-7 cells were incubated with either mAb 806 or the DH8.3 antibody (10 μg/ml) for 1 h in DMEM at 4° C. After washing, cells were transferred to DMEM pre-warmed to 37° C. and aliquots taken at various time points following incubation at 37° C. Internalization was stopped by immediately washing aliquots in ice-cold wash buffer (1% HSA/PBS). At the completion of the time course cells were stained by FACS as described above. Percentage internalization was calculated by comparing surface antibody staining at various time points to zero time using the formula: percent antibody internalized=(mean fluorescence at time×background fluorescence)/(mean fluorescence at time 0−background fluorescence)×100. This method was validated in one assay using an iodinated antibody (mAb 806) to measure internalization as previously described (24). Differences in internalization rate at different time points were compared using Student's t-test.

Both antibodies showed relatively rapid internalization reaching steady-state levels at 10 min for mAb 806 and 30 min for DH8.3 (FIG. 3). Internalization of DH8.3 was significantly higher both in terms of rate (80.5% of DH8.3 internalized at 10 min compared to 36.8% for mAb 806, p<0.01) and total amount internalized at 60 min (93.5% versus 30.4%, p<0.001). mAb 806 showed slightly lower levels of internalization at 30 and 60 min compared to 20 min in all 4 assays performed (FIG. 3). This result was also confirmed using an internalization assay based on iodinated mAb 806.

EXAMPLE 6 Electron Microscopy Analysis of Antibody Internalization

Given the above noted difference in internalization rates between the antibodies, a detailed analysis of antibody intracellular trafficking was performed using electron microscopy.

U87MG.Δ2-7 cells were grown on gelatin coated chamber slides (Nunc, Naperville, Ill.) to 80% confluence and then washed with ice cold DMEM. Cells were then incubated with mAb 806 or the DH8.3 antibody in DMEM for 45 min at 4° C. After washing, cells were incubated for a further 30 min with gold-conjugated (20 nm particles) anti-mouse IgG (BBInternational, Cardiff, UK) at 4° C. Following a further wash, pre-warmed DMEM/10% FCS was added to the cells, which were incubated at 37° C. for various times from 1-60 min. Internalization of the antibody was stopped by ice-cold media and cells fixed with 2.5% glutaraldehyde in PBS/0.1% HSA and then post-fixed in 2.5% osmium tetroxide. After dehydration through a graded series of acetone, samples were embedded in Epon/ARALDITE® resin, cut as ultra-thin sections with a Reichert Ultracut-S microtome (Leica) and collected on nickel grids. The sections were stained with uranyl acetate and lead citrate before being viewed on a Philips CM12 transmission electron microscope at 80 kV. Statistical analysis of gold grains contained within coated pits was performed using a Chi-square test.

While the DH8.3 antibody was internalized predominantly via coated-pits, mAb 806 appeared to be internalized by macropinocytosis (FIG. 19). In fact, a detailed analysis of 32 coated pits formed in cells incubated with mAb 806 revealed that none of them contained antibody. In contrast, around 20% of all coated-pits from cells incubated with DH8.3 were positive for antibody, with a number containing multiple gold grains. A statistical analysis of the total number of gold grains contained within coated-pits found that the difference was highly significant (p<0.01). After 20-30 min both antibodies could be seen in structures that morphologically resemble lysosomes. The presence of cellular debris within these structures was also consistent with their lysosome nature.

EXAMPLE 7 Biodistribution of Antibodies in Tumor Bearing Nude Mice

The biodistribution of mAb 806 and the DH8.3 antibody was compared in nude mice containing U87MG xenografts on one side and U87MG.Δ2-7 xenografts on the other. A relatively short time period was chosen for this study as a previous report demonstrated that the DH8.3 antibody shows peak levels of tumor targeting between 4-24 h (16).

Tumor xenografts were established in nude BALB/c mice by s.c. injection of 3×106 U87MG, U87MG.Δ2-7 or A431 cells. de2-7 EGFR expression in U87MG.Δ2-7 xenografts remained stable throughout the period of biodistribution. Also, A431 cells retained their mAb 806 reactivity when grown as tumor xenografts as determined by immunohistochemistry (data not shown). U87MG or A431 cells were injected on one side 7-10 days before U87MG.Δ2-7 cells were injected on the other side because of the faster growth rate observed for de2-7 EGFR expressing xenografts. Antibodies were radiolabeled and assessed for immunoreactivity as described above and were injected into mice by the retro-orbital route when tumors were 100-200 mg in weight. Each mouse received two different antibodies (2 μg per antibody): 2 μCi of 125I-labeled mAb 806 and 2 μCi of 131I-labelled DH8.3 or 528. Unless indicated, groups of 5 mice were sacrificed at various time points post-injection and blood obtained by cardiac puncture. The tumors, liver, spleen, kidneys and lungs were obtained by dissection. All tissues were weighed and assayed for 125I and 131I activity using a dual-channel counting Window. Data was expressed for each antibody as % ID/g tumor determined by comparison to injected dose standards or converted into tumor to blood/liver ratios (i.e. % ID/g tumor divided by % ID/g blood or liver). Differences between groups were analysed by Student's t-test.

In terms of % ID/g tumor, mAb 806 reached its peak level in U87MG.Δ2-7 xenografts of 18.6% m/g tumor at 8 h (FIG. 4A), considerably higher than any other tissue except blood. While DH8.3 also showed peak tumor levels at 8 h, the level was a statistically (p<0.001) lower 8.8% m/g tumor compared to mAb 806FIG. 4B). Levels of both antibodies slowly declined at 24 and 48 h. Neither antibody showed specific targeting of U87MG parental xenografts (FIGS. 4A-B). With regards to tumor to blood/liver ratios, mAb 806 showed the highest ratio at 24 h for both blood (ratio of 1.3) and liver (ratio of 6.1) (FIGS. 5 A-B). The DH8.3 antibody had its highest ratio in blood at 8 h (ratio of 0.38) and at 24 h in liver (ratio of 1.5) (FIGS. 5A-B), both of which are considerably lower than the values obtained for mAb 806.

As described above, levels of mAb 806 in the tumor peaked at 8 hours. While this peak is relatively early compared to many tumor-targeting antibodies, it is completely consistent with other studies using de2-7 EGFR specific antibodies which all show peaks at 4-24 hours post-injection when using a similar dose of antibody (16, 25, 33). Indeed, unlike the earlier reports, the 8 h time point was included on the assumption that antibody targeting would peak rapidly. The % ID/g tumor seen with mAb 806 was similar to that reported for other de2-7 EGFR specific antibodies when using standard iodination techniques (16, 24, 32). The reason for the early peak is probably two-fold. Firstly, tumors expressing the de2-7 EGFR, including the transfected U87MG cells, grow extremely rapidly as tumor xenografts. Thus, even during the relatively short period of time used in these biodistribution studies, the tumor size increases to such an extent (5-10 fold increase in mass over 4 days) that the % ID/g tumor is reduced compared with slow growing tumors. Secondly, while internalization of mAb 806 was relatively slow compared to DH8.3, it is still rapid with respect to many other tumor antibody/antigen systems. Internalized antibodies undergo rapid proteolysis with the degradation products being excreted from the cell (34). This process of internalization, degradation and excretion reduces the amount of iodinated antibody retained within the cell. Consequently, internalizing antibodies display lower levels of targeting than their non-internalizing counterparts. The electron microscopy data reported herein demonstrates that internalized mAb 806 is rapidly transported to lysosomes where rapid degradation presumably occurs. This observation is consistent with the swift expulsion of iodine from the cell.

The previously described L8A4 monoclonal antibody directed to the unique junctional peptide found in the de2-7 EGFR, behaves in a similar fashion to mAb 806 (35). Using U87MG cells transfected with the de2-7 EGFR, this antibody had a similar internalization rate (35% at 1 hour compared to 30% at 1 hour for mAb 806) and displayed comparable in vivo targeting when using 3T3 fibroblasts transfected with de2-7 EGFR (peak of 24% ID/g tumor at 24 hours compared to 18% ID/g tumor at 8 hours for mAb 806) (25). Interestingly, in vivo retention of this antibody in tumor xenografts was enhanced when labeled with N-succinimidyl 5-iodo-3-pyridine carboxylate (25). This labeled prosthetic group is positively charged at lysosomal pH and thus has enhanced cellular retention (33). Enhanced retention is potentially useful when considering an antibody for radioimmunotherapy and this method could be used to improve retention of iodinated mAb 806 or its fragments.

EXAMPLE 8 Binding of mAb 806 to Cells Containing Amplified EGFR

To examine if mAb 806 could recognize the EGFR expressed in cells containing an amplified receptor gene, its binding to A431 cells was analysed. As described previously, A431 cells are human squamous carcinoma cells and express high levels of wtEGFR. Low, but highly reproducible, binding of mAb 806 to A431 cells was observed by FACS analysis (FIG. 6). The DH8.3 antibody did not bind A431 cells, indicating that the binding of mAb 806 was not the result of low level de2-7 EGFR expression (FIG. 6). As expected, the anti-EGFR 528 antibody showed strong staining of A431 cells (FIG. 6). Given this result, binding of mAb 806 to A431 was characterized by Scatchard analysis. While the binding of iodinated mAb 806 was comparatively low, it was possible to get consistent data for Scatchard. The average of such experiments gave a value for affinity of 9.5×107 M−1 with 2.4×105 receptors per cell. Thus the affinity for this receptor was some 10-fold lower than the affinity for the de2-7 EGFR. Furthermore, mAb 806 appears to only recognize a small portion of EGFR found on the surface of A431 cells. Using the 528 antibody, the inventors measured approximately 2×106 receptors per cell which is in agreement with numerous other studies(26).

Recognition of the wild type sEGFR by mAb 806 clearly requires some denaturation of the receptor in order to expose the epitope. The extent of denaturation required is only slight as even absorption of the wild type sEGFR on to a plastic surface induced robust binding of mAb 806 in ELISA assays. As mAb 806 only bound approximately 10% of the EGFR on the surface of A431 cells, it is tempting to speculate that this subset of receptors may have an altered conformation similar to that induced by the de2-7 EGFR truncation. Indeed, the extremely high expression of the EGFR mediated by gene amplification in A431 cells, may cause some receptors to be incorrectly processed leading to altered conformation. Interestingly, semi-quantitative immunoblotting of A431 cell lysates with mAb 806 showed that it could recognize most of the A431 EGF receptors following SDS-PAGE and western transfer. This result further supports the argument that mAb 806 is binding to a subset of receptors on the surface of A431 cells that have an altered conformation. These observations in A431 cells are consistent with the immunohistochemistry data demonstrating that mAb 806 binds gliomas containing amplification of the EGFR gene. As mAb 806 binding was completely negative on parental U87MG cells it would appear this phenomenon may be restricted to cells containing amplified EGFR although the level of “denatured” receptor on the surface of U87MG cells may be below the level of detection. However, this would seem unlikely as iodinated mAb 806 did not bind to U87MG cell pellets containing up to 1×107 cells.

EXAMPLE 9 In Vivo Targeting of A431 Cells by MAb 806

A second biodistribution study was performed with mAb 806 to determine if it could target A431 tumor xenografts. The study was conducted over a longer time course in order obtain more information regarding the targeting of U87MG.Δ.2-7 xenografts by mAb 806, which were included in all mice as a positive control. In addition, the anti-EGFR 528 antibody was included as a positive control for the A431 xenografts, since a previous study demonstrated low but significant targeting of this antibody to A431 cells grown in nude mice (21).

During the first 48 h, mAb 806 displayed almost identical targeting properties as those observed in the initial experiments (FIG. 7A compared with FIG. 4A). In terms of % ID/g tumor, levels of mAb 806 in U87MG.Δ2-7 xenografts slowly declined after 24 h but always remained higher than levels detected in normal tissue. Uptake in the A431 xenografts was comparatively low, however there was a small increase in % ID/g tumor during the first 24 h not observed in normal tissues such as liver, spleen, kidney and lung (FIG. 7A). Uptake of the 528 antibody was very low in both xenografts when expressed as % ID/g tumor (FIG. 7B) partially due to the faster clearance of this antibody from the blood. In terms of tumor to blood ratio mAb 806 peaked at 72 h for U87MG.Δ2-7 xenografts and 100 h for A431 xenografts (FIGS. 8A and 8B). While the tumor to blood ratio for mAb 806 never surpassed 1.0 with respect to the A431 tumor, it did increase throughout the entire time course (FIG. 8) and was higher than all other tissues examined (data not shown) indicating low levels of targeting.

The tumor to blood ratio for the 528 antibody showed a similar profile to mAb 806 although higher levels were noted in the A431 xenografts (FIGS. 8A and 8B). mAb 806 had a peak tumor to liver ratio in U87MG.Δ2-7 xenografts of 7.6, at 72 h, clearly demonstrating preferential uptake in these tumors compared to normal tissue (FIG. 8C). Other tumor to organ ratios for mAb 806 were similar to those observed in the liver (data not shown). The peak tumor to liver ratio for mAb 806 in A431 xenografts was 2.0 at 100 h, again indicating a slight preferential uptake in tumor compared with normal tissue (FIG. 8D).

EXAMPLE 10 Therapy Studies

The effects of mAb806 were assessed in two xenograft models of disease—a preventative model and an established tumor model.

Xenograft Models

Consistent with previous reports (Nishikawa et al, Proc. Natl. Acad. Sci. USA, 91(16); 7727-7731) U87MG cells transfected with de2-7 EGFR grew more rapidly than parental cells and U87MG cells transfected with the wt EGFR. Therefore, it was not possible to grow both cell types in the same mice.

3×106 tumor cells in 100 ml of PBS were inoculated subcutaneously. into both flanks of 4-6 week old female nude mice (Animal Research Centre, Western Australia, Australia). Therapeutic efficacy of mAb 806 was investigated in both preventative and established tumor models. In the preventative model, 5 mice with 2 xenografts each were treated intraperitoneally. with either 1 or 0.1 mg of mAb 806 or vehicle (PBS) starting the day before tumor cell inoculation. Treatment was continued for a total of 6 doses, 3 times per week for 2 weeks. In the established model, treatment was started when tumors had reached a mean volume of 65±6.42 mm3 (U87MG.A.2-7), 84±9.07 mm3 (U87MG), 73±7.5 mm3 (U87MG.wtEGFR) or 201±19.09 mm3 (A431 tumors). Tumor volume in mm3 was determined using the formula (length×width2)/2, where length was the longest axis and width the measurement at right angles to the length (Scott et al, 2000). Data was expressed as mean tumor volume±S.E. for each treatment group. Statistical analysis was performed at given time points using Student's t test. Animals were euthanased when the xenografts reached an approximate volume of 1.5 cm3 and the tumors excised for histological examination. This research project was approved by the Animal Ethics Committee of the Austin and Repatriation Medical Centre.

Histological Examination of Tumor Xenografts

Xenografts were excised and bisected. One half was fixed in 10% formalin/PBS before being embedded in paraffin. Four micron sections were then cut and stained with haematoxylin and eosin (H&E) for routine histological examination. The other half was embedded in Tissue® O.C.T.™ compound (Sakura Finetek, Torrance, Calif.), frozen in liquid nitrogen and stored at −80° C. Thin (5 micron) cryostat sections were cut and fixed in ice-cold acetone for 10 min followed by air drying for a further 10 min. Sections were blocked in protein blocking reagent (Lipshaw Immunon, Pittsburgh U.S.A.) for 10 min and then incubated with biotinylated primary antibody (1 mg/ml), for 30 min at room temperature (RT). All antibodies were biotinylated using the ECL™ protein biotinylation module (Amersham, Baulkham Hills, Australia), as per the manufactures instructions. After rinsing with PBS, sections were incubated with a streptavidin horseradish peroxidase complex for a further 30 min (Silenus, Melbourne, Australia). Following a final PBS wash the sections were exposed to 3-amino-9-ethylcarbozole (AEC) substrate (0.1 M acetic acid, 0.1 M sodium acetate, 0.02 M AEC (Sigma Chemical Co., St Louis, Mo.)) in the presence of hydrogen peroxide for 30 min. Sections were rinsed with water and counterstained with hematoxylin for 5 min and mounted.

Efficacy of mAb 806 in Preventative Model

MAb 806 was examined for efficacy against U87MG and U87MG.Δ2-7 tumors in a preventative xenograft model. Antibody or vehicle were administered i.p. the day before tumor inoculation and was given 3 times per week for 2 weeks. MAb 806 had no effect on the growth of parental U87MG xenografts, which express the wt EGFR, at a dose of 1 mg per injection (FIG. 9A). In contrast, mAb 806 significantly inhibited the growth of U87MG.Δ2-7 xenografts in a dose dependent manner (FIG. 9B). At day 20, when control animals were sacrificed, the mean tumor volume was 1637±178.98 mm3 for the control group, a statistically smaller 526±94.74 mm3 for the 0.1 mg per injection group (p<0.0001) and 197±42.06 mm3 for the 1 mg injection group (p<0.0001). Treatment groups were sacrificed at day 24 at which time the mean tumor volumes was 1287±243.03 mm3 for the 0.1 mg treated group and 492±100.8 mm3 for the 1 mg group.

Efficacy of mAb 806 in Established Xenograft Model

Given the efficacy of mAb 806 in the preventative xenograft model, its ability to inhibit the growth of established tumor xenografts was then examined Antibody treatment was as described in the preventative model except that it commenced when tumors had reached a mean tumor volume of 65±6.42 mm3 for the U87MG.Δ2-7 xenografts and 84±9.07 mm3 for the parental U87MG xenografts. Once again, mAb 806 had no effect on the growth of parental U87MG xenografts at a dose of 1 mg per injection (FIG. 10A). In contrast, mAb 806 significantly inhibited the growth of U87MG.Δ2-7 xenografts in a dose dependent manner (FIG. 10B). At day 17, one day before control animals were sacrificed, the mean tumor volume was 935±215.04 mm3 for the control group, 386±57.51 mm3 for the 0.1 mg per injection group (p<0.01) and 217±58.17 mm3 for the 1 mg injection group (p<0.002).

To examine whether the growth inhibition observed with mAb 806 was restricted to cell expressing de2-7 EGFR, its efficacy against U87MG.wtEGFR tumor xenografts was examined in an established model. These cells serve as a model for tumors containing amplification of the EGFR gene without de2-7 EGFR expression. MAb 806 treatment commenced when tumors had reached a mean tumor volume of 73±7.5 mm3 MAb 806 significantly inhibited the growth of established U87MG.wtEGFR xenografts when compared to control tumors treated with vehicle (FIG. 10C). On the day control animals were sacrificed, the mean tumor volume was 960±268.9 mm3 for the control group and 468±78.38 mm3 for the group treated with 1 mg injections (p<0.04).

Histological and Immunohistochemical Analysis of Established Tumors

To evaluate potential histological differences between mAb 806-treated and control U87MG.Δ2-7 and U87MG.wtEGFR xenografts (collected at days 24 and 42 respectively), formalin-fixed, paraffin embedded sections were stained with H&E.

Areas of necrosis were seen in sections from both U87MG.Δ2-7 (collected 3 days after treatment finished), and U87MG.wtEGFR xenografts (collected 9 days after treatment finished) treated with mAb 806. This result was consistently observed in a number of tumor xenografts (n=4). However, analysis of sections from xenografts treated with control did not display the same areas of necrosis seen with mAb 806 treatment. Sections from mAb 806 or control treated U87MG xenografts were also stained with H&E and revealed no differences in cell viability between the two groups, further supporting the hypothesis that mAb 806 binding induces decreased cell viability/necrosis within tumor xenografts.

An immunohistochemical analysis of U87MG, U87MG.Δ2-7 and U87MG.wtEGFR xenograft sections was performed to determine the levels of de2-7 and wt EGFR expression following mAb806 treatment. Sections were collected at days 24 and 42 as above, and were immunostained with the 528 or 806 antibodies. As expected the 528 antibody stained all xenograft sections with no obvious decrease in intensity between treated and control tumors. Staining of U87MG sections was undetectable with the mAb 806, however positive staining of U87MG.Δ2-7 and U87MG.wtEGFR xenograft sections was observed. There was no difference in mAb 806 staining density between control and treated U87MG.Δ2-7 and U87MG.wtEGFR xenografts suggesting that antibody treatment does not down regulate de2-7 or wt EGFR expression.

Treatment of A431 Xenografts with mAb 806

To demonstrate that the anti-tumor effects of mAb 806 were not restricted to U87MG cells, the antibody was administered to mice with A431 xenografts. These cells contain an amplified EGFR gene and express approximately 2×106 receptors per cell. As described above, mAb 806 binds about 10% of these EGFR and targets A431 xenografts. MAb 806 significantly inhibited the growth of A431 xenografts when examined in the previously described preventative xenograft model (FIG. 11A). At day 13, when control animals were sacrificed, the mean tumor volume was 1385±147.54 mm3 in the control group and 260±60.33 mm3 for the 1 mg injection treatment group (p<0.0001).

In a separate experiment, a dose of 0.1 mg mAb also significantly inhibited the growth of A431 xenografts in a preventative model.

Given the efficacy of mAb 806 in the preventative A431 xenograft model, its ability to inhibit the growth of established tumor xenografts was examined Antibody treatment was as described in the preventative model except it was not started until tumors had reached a mean tumor volume of 201±19.09 mm3 MAb 806 significantly inhibited the growth of established tumor xenografts (FIG. 11B). At day 13, when control animals were sacrificed, the mean tumor volume was 1142±120.06 mm3 for the control group and 451±65.58 mm3 for the 1 mg injection group (p<0.0001).

In summary, the therapy studies with mAb 806 described here clearly demonstrated dose dependent inhibition of U87MG.Δ2-7 xenograft growth. In contrast, no inhibition of parental U87MG xenografts was observed despite the fact they continue to express the wt EGFR in vivo. MAb 806 not only significantly reduced xenograft volume, it also induced significant necrosis within the tumor. This is the first report showing the successful therapeutic use of such an antibody in vivo against a human de2-7 EGFR expressing glioma xenografts.

Gene amplification of the EGFR has been reported in a number of different tumors and is observed in approximately 50% of gliomas (Voldberg et al, 1997). It has been proposed that the subsequent EGFR over-expression mediated by receptor gene amplification may confer a growth advantage by increasing intracellular signalling and cell growth (Filmus et al, 1987). The U87MG cell line was transfected with the wt EGFR in order to produce a glioma cell that mimics the process of EGFR gene amplification. Treatment of established U87MG.wtEGFR xenografts with mAb 806 resulted in significant growth inhibition. Thus, mAb 806 also mediates in vivo anti-tumor activity against cells containing amplification of the EGFR gene. Interestingly, mAb 806 inhibition of U87MG.wtEGFR xenografts appears to be less effective than that observed with U87MG.Δ2-7 tumors. This probably reflects the fact that mAb 806 has a lower affinity for the amplified EGFR and only binds a small proportion of receptors expressed on the cell surface. However, it should be noted that despite the small effect on U87MG.wtEGFR xenograft volumes, mAb 806 treatment produced large areas of necrosis within these xenografts. To rule out the possibility that mAb 806 only mediates inhibition of the U87MG derived cell lines we tested its efficacy against A431 xenografts. This squamous cell carcinoma derived cell line contains significant EGFR gene amplification which is retained both in vitro and in vivo. Treatment of A431 xenografts with mAb 806 produced significant growth inhibition in both a preventative and established model, indicating the anti-tumor effects of mAb 806 are not restricted to transfected U87MG cell lines.

EXAMPLE 11 Combination Therapy Treatment of A431 Xenografts with MAb806 and AG1478

The anti-tumor effects of mAb 806 combined with AG1478 was tested in mice with A431 xenografts. AG1478 (4-(3-Chloroanilino)-6,7-dimethoxyquinazoline) is a potent and selective inhibitor of the EGFR kinase versus HER2-neu and platelet-derived growth factor receptor kinase (Calbiochem Cat. No. 658552). Three controls were included: treatment with vehicle only, vehicle+mAb806 only and vehicle+AG1478 only. The results are illustrated in FIG. 12. 0.1 mg mAb806 was administered at 1 day prior to xenograft and 1, 3, 6, 8 and 10 days post xenograft. 400 μg AG1478 was administered at 0, 2, 4, 7, 9, and 11 days post xenograft.

Both AG1478 and mAb806, when administered alone produced a significant reduction of tumor volume. However, in combination, the reduction of tumor volume was greatly enhanced.

In addition, the binding of mAb806 to EGFR of A431 cells was evaluated in the absence and presence of AG1478. Cells were placed in serum free media overnight, then treated with AG1478 for 10 min at 37° C., washed twice in PBS then lysed in 1% Triton and lysates prepared. The lysates were prepared as described in Example 20 herein. Lysate was then assessed for 806 reactivity by an ELISA is a modified version of an assay described by Schooler and Wiley, Analytical Biochemistry 277, 135-142 (2000). Plates were coated with 10 μg/ml of mAb 806 in PBS/EDTA overnight at room temperature and then washed twice. Plates were then blocked with 10% serum albumin/PBS for 2 hours at 37° C. and washed twice. A 1:20 cell lysate was added in 10% serum albumin/PBS for 1 hour at 37° C., then washed four times. Anti-EGFR(SC-03 (Santa Cruz Biotechnology Inc.)) in 10% serum albumin/PBS was reacted 90 min at room temperature, the plate washed four times, and anti-rabbit-HRP (1:2000 if from Silenus) in 10% serum albumin/PBS was added for 90 min at room temperature, washed four timed, and color developed using ABTS as a substrate. It was found that mAb806 binding is significantly increased in the presence of increasing amounts of AG1478 (FIG. 13).

EXAMPLE 12 Immunoreactivity in Human Glioblastomas Pre-Typed for EGFR Status

Given the high incidence of EGFR expression, amplification and mutation in glioblastomas, a detailed immunohistochemical study was performed in order to assess the specificity of 806 in tumors other than xenografts. A panel of 16 glioblastomas was analyzed by immunohistochemistry. This panel of 16 glioblastomas was pre-typed by RT-PCR for the presence of amplified wild-type EGFR and de2-7 EGFR expression. Six of these tumors expressed only the wt EGFR transcript, 10 had wtEGFR gene amplification with 5 of these showing wild-type EGFR transcripts only, and 5 both wild-type EGFR and de2-7 gene transcript. Immunohistochemical analysis was performed using 5 mm sections of fresh frozen tissue applied to histology slides and fixed for 10 minutes in cold acetone. Bound primary antibody was detected with biotinylated horse anti-mouse antibody followed by an avidin-biotin-complex reaction. Diaminobenzidine tetrahydrochloride (DAB) was used as chromogen. The extent of the immunohistochemical reactivity in tissues was estimated by light microscopy and graded according to the number of immunoreactive cells in 25% increments as follows:

    • Focal=less than 5%
    • +=5-25%
    • ++=25-50%
    • +++=50-75%
    • ++++=>75%

The 528 antibody showed intense reactivity in all tumors, while DH 8.3 immunostaining was restricted to those tumors expressing the de2-7 EGFR (Table 2). Consistent with the previous observations in FACS and rosetting assays, mAb 806 did not react with the glioblastomas expressing the wtEGFR transcript from nonamplified EGFR genes (Table 2). This pattern of reactivity for mAb 806 is similar to that observed in the xenograft studies and again suggests that this antibody recognizes the de2-7 and amplified EGFR but not the wtEGFR when expressed on the cell surface.

TABLE 2 Immunoreactivity of MAbs 528, DH8.3 and 806 on glioblastomas pretyped for the presence of wild type EGFR and mutated de2-7 EGFR and for their amplification status. de2-7 EGFR Amplification Expression 528 DH8.3 806 No ++++ − − No ++++ − −* No ++++ − − No ++ − − No +++ − − No ++++ − − Yes No ++++ − ++++ Yes No ++++ − + Yes No ++++ − +++ Yes No ++++ − ++++ Yes No ++++ − +−++++ Yes Yes ++++ ++++ ++++ Yes Yes ++++ ++++ ++++ Yes Yes ++++ ++++ ++++ Yes Yes ++++ ++++ ++++ Yes Yes ++++ ++ ++ *focal staining

EXAMPLE 13 EGFR Immunoreactivity in Normal Tissue

In order to determine if the de2-7 EGFR is expressed in normal tissue, an immunohistochemical study with mAb 806 and DH8.3 was conducted in a panel of 25 tissues. There was no strong immunoreactivity with either mAb 806 or DH8.3 in any tissue tested suggesting that the de2-7 EGFR is absent in normal tissues (Table 3). There was some variable staining present in tonsils with mAb 806 that was restricted to the basal cell layer of the epidermis and mucosal squamous cells of the epithelium. In placenta, occasional immunostaining of the trophoblast epithelium was observed. Interestingly, two tissues that express high endogenous levels of wtEGFR, the liver and skin, failed to show any significant mAb 806 reactivity. No reactivity was observed with the liver samples at all, and only weak and inconsistent focal reactivity was detected occasionally (in no more than 10% of all samples studied) in basal keratinocytes in skin samples and in the squamous epithelium of the tonsil mucosa, further demonstrating that this antibody does not bind the wtEGFR expressed on the surface of cells to any significant extent (Table 3). All tissues were positive for the wtEGFR as evidenced by the universal staining seen with the 528 antibody (Table 3).

TABLE 3 Reactivity of 582, DH8.3 and 806 on normal tissues. TISSUE 528 DH8.3 806 Esophagus pos − − Stomach pos − − Duodenum pos − − Small pos − − intestine/duodenum Colon pos − − Liver pos − − Salivary glands (parotid) pos − − Kidney pos − − Urinary bladder pos − − Prostate pos − − Testis pos − − Uterus (cx/endom) pos −* − Fallopian tube pos − − Ovary pos − − Breast pos −* − Placenta pos − − Peripheral nerve pos − − Skeletal muscle pos − − Thyroid gland pos − − Lymph node pos − − Spleen pos − − Tonsil pos − − occ. weak reactivity of basal layer of squamous epithelium Heart pos − − Lung pos − − Skin pos − − occ. weak reactivity of basal layer of squamous epithelium *some stromal staining in various tissue

EXAMPLE 14 EGFR Immunoreactivity in Various Tumors

The extent of de2-7 EGFR in other tumor types was examined using a panel of 12 different malignancies. The 528 antibody showed often homogeneous staining in many tumors analysed except melanoma and seminoma. When present, DH8.3 immunoreactivity was restricted to the occasional focal tumor cell indicating there is little if any de2-7 EGFR expression in tumors outside the brain using this detection system (Table 4). There was also focal staining of blood vessels and a varying diffuse staining of connective tissue with the DH8.3 antibody in some tumors (Table 4). This staining was strongly dependent on antibody concentration used and was considered nonspecific background reactivity. The mAb 806 showed positive staining in 64% of head and neck tumors and 50% of lung carcinomas (Table 4). There was little mAb 806 reactivity elsewhere except in urinary tumors that were positive in 30% of cases. Since the head and neck and lung cancers were negative for the DH8.3 antibody the reactivity seen with the mAb in these tumors maybe associated with EGFR gene amplification.

TABLE 4 Monoclonal antibodies 528, DH8.3 and 806 on tumor panel. Tumor 528 DH8.3 806 Malignant melanoma 0/10 0/10 0/10 metastases Urinary bladder (tcc, sqcc, 10/10 0/10* 3/10* adeno) (7x++++, 2x+++, 1x+) (2x++++, 1x++) Mammary gland 6/10 1/10 1/10 (3x++++, 3x++) (1x+) (foc) Head + neck cancer (sqcc) 11/11 0/11* 7/11 1x+++-10x++++) (3x++++, 3x+++, 1x+) Lung (sqcc, adeno, neuroend) 12/12 0/12* 6/12 10x++++-1x+++) (3x++++ 3x+++) Leiomyosarcoma 5/5 0/5 0/5 (4x++++, 1x+) Liposarcoma 5/5 0/5 0/5* (2x + 3x +++) Synovial sarcoma 4/5* 0/5 0/5* (4x ++++) Mfh Malignant fibrous histiocytoma 4/5* 0/5* 0/5* Colonic carcinoma 10/10 0/10* 0/10 (9x++++, 1x+) Seminoma 1/10* 1/10* 0/10 Ovary (serous-papillary) 4/5 0/5* 0/5 (3x++++, 1x+) *focal staining

EXAMPLE 15 Immunoreactivity in Human Glioblastomas Unselected for EGFR Status

In order to confirm the unique specificity and to evaluate the reactivity of mAb 806, it was compared to the 528 and DH8.3 antibodies in a panel of 46 glioblastomas not preselected for their EGFR status. The 528 antibody was strongly and homogeneously positive in all samples except two (No. 27 and 29) (44/46, 95.7%). These two cases were also negative for mAb806 and mAb DH8.3. The mAb 806 was positive in 27/46 (58.7%) cases, 22 of which displayed homogeneous immunoreactivity in more than 50% of the tumor. The DH8.3 antibody was positive in 15/46 (32.6%) glioblastomas, 9 of which showed homogeneous immunoreactivity. The immunochemical staining of these unselected tumors is tabulated in Table 5.

There was concordance between mAb 806 and DH8.3 in every case except one (No. 35).

A molecular analysis for the presence of EGFR amplification was done in 44 cases (Table 5). Of these, 30 cases co-typed with the previously established mAb 806 immunoreactivity pattern: e.g. 16 mAb 806-negative cases revealed no EGFR amplification and 14 EGFR-amplified cases were also mAb 806 immunopositive. However, 13 cases, which showed 806 immunoreactivity, were negative for EGFR amplification while 1 EGFR-amplified case was mAb 806 negative. Further analysis of the mutation status of these amplification negative and 806 positive cases is described below and provides explanation for most of the 13 cases which were negative for EGFR amplification and were recognized by 806.

Subsequently, a molecular analysis of the deletion mutation by RT-PCR was performed on 41/46 cases (Table 5). Of these, 34 cases co-typed with DH8.3 specific for the deletion mutation: 12 cases were positive in both RT-PCR and immunohistochemistry and 22 cases were negative/negative. Three cases (#2, #34, #40) were DH8.3 positive/RT-PCR negative for the deletion mutation and three cases (#12, #18, #39) were DH8.3 negative/RT-PCR positive. As expected based on our previous specificity analysis, mAb 806 immunoreactivity was seen in all DH8.3-positive tissues except in one case (#35).

Case #3 also revealed a mutation (designated A2 in Table 5), which included the sequences of the de2-7 mutation but this did not appear to be the classical de2-7 deletion with loss of the 801 bases (data not shown). This case was negative for DH8.3 reactivity but showed reactivity with 806, indicating that 806 may recognize an additional and possibly unique EGFR mutation.

TABLE 5 Immunohistochemical analysis of 46 unselected glioblastomas with mAbs 528, 806, and DH8.3 EGFR # 528 806 DH8.3 Amp.* 5′ MUT 1 ++++ ++++ ++ A 5′ MUT 2 ++++ ++++ ++++ N WT 3 ++++ ++++ neg. N A2 (det.) 4 ++++ ++++ neg. N WT 5 ++++ ++++ ++++ N 5′ MUT 6 ++++ ++++ neg. A WT 7 ++++ ++++ ++++ N 5′ MUT 8 ++++ ++++ ++++ A 5′ MUT 9 ++++ ++++ neg. A WT 10 ++++ neg. neg. N WT 11 ++ ++ ++ A 5′ MUT 12 ++++ ++ neg. A 5′ MUT 13 ++++ ++++ neg N WT 14 ++ neg. neg. Nd nd 15 ++ ++ neg N WT 16 + neg. neg. N nd 17 ++++ neg. neg. N WT 18 ++++ ++++ neg. A 5′ MUT 19 ++++ ++++ neg. N WT 20 ++++ neg. neg N WT 21 ++++ ++++ neg. N WT 22 +++ neg. neg. N WT 23 ++++ ++++ ++ N 5′ MUT 24 ++++ ++++ neg. A WT 25 ++++ neg. neg. N WT 26 ++++ ++++ +++ A 5′ MUT 27 neg. neg. neg. N WT 28 +++ neg. neg. N WT 29 neg. neg. neg. N WT 30 ++++ ++++ neg. N WT 31 ++++ neg. neg. N nd par det 32 ++ +++ ++ N 5′ MUT 33 +++ ++++ ++++ A 5′ MUT 34 ++++ +++ ++++ N WT 35 ++++ neg. ++++ A 5′ MUT 36 +++ ++ +++ A 5′ MUT 37 ++++ + + A 5′ MUT 38 ++++ neg. neg. N WT 39 ++ neg. neg. N 5′ MUT 40 ++++ ++++ + A WT 41 ++ neg. neg. N WT 42 ++++ ++++ neg. A WT 43 ++++ neg. neg. nd nd 44 ++++ neg. neg. N WT 45 ++++ neg. neg. N WT 46 ++++ neg. neg. N nd *N = not amplified, A—amplified, + WT = wildtype, 5′-mut nd = not done

The 806 antibody reactivity co-typed with amplified or de2-7 mutant EGFR in 19/27 or over 70% of the cases. It is notable that 2 of these 8 cases were also DH8.3 reactive.

EXAMPLE 16 Systemic Treatment and Analysis of Intracranial Glioma Tumors

To test the efficacy of the anti-ΔEGFR monoclonal antibody, mAb 806, we treated nude mice bearing intracranial ΔEGFR-overexpressing glioma xenografts with intraperitoneal injections of mAb806, the isotype control IgG or PBS.

The human glioblastoma cell lines U87MG, LN-Z308 and A1207 (gift from Dr. S. Aaronson, Mount Sinai Medical Center, New York, N.Y.) were infected with ΔEGFR, kinase-deficient ΔEGFR (DK), or wild-type EGFR (wtEGFR) viruses. Populations expressing similar high levels of EGFRs were selected by fluorescence-activated cell sorting and designated as U87MG.ΔEGFR, U87MG.DK, U87MG.wtEGFR, LN-Z308.ΔEGFR, LN-Z308.DK, LN-Z308. wtEGFR, A1207.ΔEGFR, A1207.DK and A1207.wtEGFR, respectively. Each was maintained in medium containing G418 (U87MG cell lines, 400 μg/ml; LN-Z308 and A1207 cell lines, 800 μg/ml).

U87MG.ΔEGFR cells were implanted intracranially into nude mice and the treatments began on the same day. 105 cells in 5 μl PBS were implanted into the right corpus striatum of nude mice brains. Systemic therapy with mAb 806, or the IgG2b isotype control, was accomplished by i.p. injection of 1 mg of mAbs in a volume of 100 μl every other day from post-implantation day 0 through 14. For direct therapy of intracerebral U87MG.ΔEGFR tumors, 10 μg of mAb 806, or the IgG2b isotype control, in a volume of 5 μl were injected at the tumor-injection site every other day starting at day 1 for 5 days.

Animals treated with PBS or isotype control IgG had a median survival of 13 days, whereas mice treated with mAb 806 had a 61.5% increase in median survival up to 21 days (P<0.001).

Treatment of mice 3 days post-implantation, following tumor establishment, also extended the median survival of the mAb 806 treated animals by 46.1% (from 13 days to 19 days; P<0.01) compared to that of the control groups.

To determine whether these antitumor effects of mAb 806 extended beyond U87MG.ΔEGFR xenografts, similar treatments were administered to animals bearing other glioma cell xenografts of LN-Z308.ΔEGFR and A 1207.ΔEGFR. The median survival of mAb 806 treated mice bearing LN-Z308.ΔEGFR xenografts was extended from 19 days for controls to 58 days (P<0.001). Remarkably, four of eight mAb 806 treated animals survived beyond 60 days. The median survival of animals bearing A1207.ΔEGFR xenografts was also extended from 24 days for controls to 29 days (P<0.01).

MAb 806 Treatment Inhibits ΔEGFR-Overexpressing Brain Tumor Growth.

Mice bearing U87MG.ΔEGFR and LN-Z308.ΔEGFR xenografts were euthanized at day 9 and day 15, respectively. Tumor sections were histopathologically analyzed and tumor volumes were determined Consistent with the results observed for animal survival, mAb 806 treatment significantly reduced the volumes by about 90% of U87MG.ΔEGFR. (P<0.001) and LN-Z308.ΔEGFR by more than 95% (P<0.001) xenografts in comparison to that of the control groups. Similar results were obtained for animals bearing A1207.ΔEGFR tumors (65% volume reduction, P<0.01).

Intratumoral Treatment with mAb 806 Extends Survival of Mice Bearing U87MG.ΔEGFR Brain Tumors.

The efficacy of direct intratumoral injection of mAb 806 for the treatment of U87MG.ΔEGFR xenografts was also determined Animals were given intratumoral injections of mAb 806 or isotype control IgG one day post-implantation. Control animals survived for 15 days, whereas mAb 806 treated mice remained alive for 18 days (P<0.01). While the intratumoral treatment with mAb 806 was somewhat effective, it entailed the difficulties of multiple intracranial injections and increased risk of infection. We therefore focused on systemic treatments for further studies.

MAb 806 Treatment Slightly Extends Survival of Mice Bearing U87MG.wtEGFR but not U87MG or U87MG.DK Intracranial Xenografts.

To determine whether the growth inhibition by mAb 806 was selective for tumors expressing ΔEGFR, we treated animals bearing U87MG, U87MG.DK (kinase-deficient ΔEGFR) and U87MG.wtEGFR brain xenografts. MAb 806 treatment did not extend survival of mice implanted with U87MG tumors which expressed a low level of endogenous wild-type EGFR (wtEGFR), or animals bearing U87MG.DK xenografts which overexpressed a kinase-deficient ΔEGFR in addition to a low level of endogenous wtEGFR. The mAb 806 treatment slightly extended the survival of mice bearing U87MG.wtEGFR tumors (P<0.05,median survival 23 days versus 26 days for the control groups) which overexpressed wtEGFR

MAb 806 Reactivity Correlates with In Vivo Anti-Tumor Efficacy.

To understand the differential effect of mAb 806 on tumors expressing various levels or different types of EGFR, we determined mAb 806 reactivity with various tumor cells by FACS analysis. Consistent with previous reports, the anti-EGFR monoclonal antibody 528 recognized both ΔEGFR and wtEGFR, and demonstrated stronger staining for U87MG.ΔEGFR cells compared to U87MG cells. In contrast, antibody EGFR.1 reacted with wtEGFR but not ΔEGFR, as U87MG.ΔEGFR cells were as weakly reactive as U87MG cells. This EGFR.1 antibody reacted with U87MG.wtEGFR more intensively than U87MG cells, as U87MG.wtEGFR cells overexpressed wtEGFR. While mAb 806 reacted intensely with U87MG.ΔEGFR and U87MG.DK cells and not with U87MG cells, it reacted weakly with U87MG.wtEGFR, indicating that mAb 806 is selective for ΔEGFR with a weak cross-activity to overexpressed wtEGFR. This level of reactivity with U87MG.wtEGFR was quantitatively and qualitatively similar to the extension of survival mediated by the antibody treatment.

We further determined mAb 806 specificity by immunoprecipitation. EGFRs in various cell lines were immunoprecipitated with antibody 528, EGFR.1 and mAb 806. Blots of electrophoretically-separated proteins were then probed with the anti-EGFR antibody, C13, which recognizes wtEGFR as well as ΔEGFR and DK. Consistent with the FACS analysis antibody 528 recognized wtEGFR and mutant receptors, while antibody EGFR.1 reacted with wtEGFR but not the mutant species. Moreover, the levels of mutant receptors in U87MG.ΔEGFR and U87MG.DK cells are comparable to those of wtEGFR in the U87MG.wtEGFR cells. However, antibody mAb 806 was able to precipitate only a small amount of the wtEGFR from the U87MG.wtEGFR cell lysates as compared with the larger amount of mutant receptor precipitated from U87MG.ΔEGFR and U87MG.DK cells, and an undetectable amount from the U87MG cells. Collectively, these data suggest that mAb 806 recognizes an epitope in ΔEGFR which also exists in a small fraction of wtEGFR only when it is overexpressed on cell surface.

MAb 806 Treatment Reduces ΔEGFR Autophosphorylation and Down-Regulates Bcl.XL Expression in U87MG.ΔEGFR Brain Tumors.

We next investigated the mechanisms underlying the growth inhibition by mAb 806. Since the constitutively active kinase activity and autophosphorylation of the carboxyl terminus of ΔEGFR are essential for its biological functions we determined ΔEGFR phosphorylation status in tumors from treated and control animals. It was found that mAb 806 treatment dramatically reduced ΔEGFR autophosphorylation, even though receptor levels were only slightly decreased in the mAb 806 treated xenografts. We have previously shown that receptor autophosphorylation causes up-regulation of the antiapoptotic gene, Bcl-XL, which plays a key role in reducing apoptosis of ΔEGFR overexpressing tumors. Therefore, we next determined the effect of mAb 806 treatment on Bcl-XL, expression. ΔEGFR tumors from mAb 806 treated animals did indeed show reduced levels of Bcl-XL.

MAb 806 Treatment Decreases Growth and Angiogenesis, and Increases Apoptosis in U87MG.ΔEGFR Tumors.

In light of the in vivo suppression caused by mAb 806 treatment and its biochemical effects on receptor signaling, we determined the proliferation rate of tumors from control or treated mice. The proliferative index, measured by Ki-67 staining of the mAb 806-treated tumors, was significantly lower than that of the control tumors (P<0.001). In addition, analysis of the apoptotic index through TUNEL staining demonstrated a significant increase in the number of apoptotic cells in mAb 806 treated tumors as compared with the control tumors (P<0.001). The extent of tumor vascularization was also analyzed by immunostaining of tumors from treated and control specimens for CD31. To quantify tumor vascularization, microvascular areas (MVA) were measured using computerized image analysis. MAb 806 treated tumors showed 30% less MVA than control tumors (P<0.001). To understand whether interaction between receptor and antibody may elicit an inflammatory response, we stained tumor sections for the macrophage marker, F4/80, and the NK cell marker, asialo GM1. Macrophages were identified throughout the tumor matrix and especially accumulated around the mAb 806 treated-U87MG.ΔEGFR tumor periphery. We observed a few NK cells infiltrated in and around the tumors and no significant difference between mAb 806 treated and isotype-control tumors.

EXAMPLE 17 Combination Immunotherapy with mAb806 and mAb528

The experiments set forth herein describe in vivo work designed to determine the efficacy of antibodies in accordance with this invention.

Female nude mice, 4-6 weeks old, were used as the experimental animals. Mice received subcutaneous inoculations of 3×106 tumor cells in each of their flanks. The animals received either U87MG.Δ2-7, U87MG.DK, or A431 cells, all of which are described, supra. Therapy began when tumors had grown to a sufficient size.

Mice then received injections of one of (i) phosphate buffered saline, (ii) mAb 806 (0.5 mg/injection), (iii) mAb 528 (0.5 mg/injection), or (iv) a combination of both mAbs. With respect to “(iv),” different groups of mice received either 0.5 mg/injection of each mAb, or 0.25 mg/injection of each mAb.

The first group of mice examined were those which had received U87MG.Δ2-7 injections. The treatment protocol began 9 days after inoculation, and continued, 3 times per week for 2 weeks (i.e., the animals were inoculated 9, 11, 13, 16, 18 and 20 days after they were injected with the cells). At the start of the treatment protocol, the average tumor diameter was 115 mm3. Each group contained 50 mice, each with two tumors.

Within the group of mice which received the combination of antibodies (0.5 mg/injection of each), there were three complete regressions. There were no regressions in any of the other groups. FIG. 18A shows the results graphically.

In a second group of mice, the injected materials were the same, except the combination therapy contained 0.25 mg of each antibody per injection. The injections were given 10, 12, 14, 17, 19 and 21 days after inoculation with the cells. At the start of the therapy the average tumor size was 114 mm3 Results are shown in FIG. 18B.

The third group of mice received inoculations of U87MG.DK. Therapeutic injections started 18 days after inoculation with the cells, and continued on days 20, 22, 25, 27 and 29. The average tumor size at the start of the treatment was 107 mm3 FIG. 18C summarizes the results. The therapeutic injections were the same as in the first group.

Finally, the fourth group of mice, which had been inoculated with A431 cells, received injections as in groups I and III, at 8, 10, 12 and 14 days after inoculation. At the start, the average tumor size was 71 mm3. Results are shown in FIG. 18D.

The results indicated that the combination antibody therapy showed a synergistic effect in reducing tumors. See FIG. 18A. A similar effect was seen at a lower dose, as per FIG. 18B, indicating that the effect is not simply due to dosing levels.

The combination therapy did not inhibit the growth of U87MG.DK (FIG. 18C), indicating that antibody immune function was not the cause for the decrease seen in FIGS. 18A and 18B.

It is noted that, as shown in FIG. 18D, the combination therapy also exhibited synergistic efficacy on A431 tumors, with 4 doses leading to a 60% complete response rate. These data suggest that the EGFR molecule recognized by mAb806 is functionally different from that inhibited by 528.

References

  • 1. Wikstrand, C. J., McLendon, R. E., Friedman, A. H., and Bigner, D. D. Cell surface localization and density of the tumor-associated variant of the epidermal growth factor receptor, EGFRvIII, Cancer Res. 57: 4130-40, 1997.
  • 2. Olapade-Olaopa, E. O., Moscatello, D. K., MacKay, E. H., Horsburgh, T., Sandhu, D. P., Terry, T. R., Wong, A. J., and Habib, F. K. Evidence for the differential expression of a variant EGF receptor protein in human prostate cancer, Br J. Cancer. 82: 186-94, 2000.
  • 3. Wikstrand, C. J., Hale, L. P., Batra, S. K., Hill, M. L., Humphrey, P. A., Kurpad, S, N., McLendon, R. E., Moscatello, D., Pegram, C. N., Reist, C. J., and et al. Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas, Cancer Res. 55: 3140-8, 1995.
  • 4. Garcia de Palazzo, I. E., Adams, G. P., Sundareshan, P., Wong, A. J., Testa, J. R., Bigner, D. D., and Weiner, L. M. Expression of mutated epidermal growth factor receptor by non-small cell lung carcinomas, Cancer Res. 53: 3217-20, 1993.
  • 5. Ekstrand, A. J., Sugawa, N., James, C. D., and Collins, V. P. Amplified and rearranged epidermal growth factor receptor genes in human glioblastomas reveal deletions of sequences encoding portions of the N- and/or C-terminal tails, Proc Natl Acad Sci USA. 89: 4309-13, 1992.
  • 6. Wong, A. J., Ruppert, J. M., Bigner, S. H., Grzeschik, C. H., Humphrey, P. A., Bigner, D. S., and Vogelstein, B. Structural alterations of the epidermal growth factor receptor gene in human gliomas, Proc Natl Acad Sci USA. 89: 2965-9, 1992.
  • 7. Yamazaki, H., Ohba, Y., Tamaoki, N., and Shibuya, M. A deletion mutation within the ligand binding domain is responsible for activation of epidermal growth factor receptor gene in human brain tumors, Jpn J Cancer Res. 81: 773-9, 1990.
  • 8. Yamazaki, H., Fukui, Y., Ueyama, Y., Tamaoki, N., Kawamoto, T., Taniguchi, S., and Shibuya, M. Amplification of the structurally and functionally altered epidermal growth factor receptor gene (c-erbB) in human brain tumors, Mol Cell Biol. 8: 1816-20, 1988.
  • 9. Sugawa, N., Ekstrand, A. J., James, C. D., and Collins, V. P. Identical splicing of aberrant epidermal growth factor receptor transcripts from amplified rearranged genes in human glioblastomas, Proc Natl Acad Sci USA. 87: 8602-6, 1990.
  • 10. Nishikawa, R., Ji, X. D., Harmon, R. C., Lazar, C. S., Gill, G. N., Cavenee, W. K., and Huang, H. J. A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity, Proc Natl Acad Sci USA. 91: 7727-31, 1994.
  • 11. Batra, S. K., Castelino-Prabhu, S., Wikstrand, C. J., Zhu, X., Humphrey, P. A., Friedman, H. S., and Bigner, D. D. Epidermal growth factor ligand-independent, unregulated, cell-transforming potential of a naturally occurring human mutant EGFRvIII gene, Cell Growth Differ. 6: 1251-9, 1995.
  • 12. Nagane, M., Coufal, F., Lin, H., Bogler, O., Cavenee, W. K., and Huang, H. J. A common mutant epidermal growth factor receptor confers enhanced tumorigenicity on human glioblastoma cells by increasing proliferation and reducing apoptosis, Cancer Res. 56: 5079-86, 1996.
  • 13. Wikstrand, C. J., Reist, C. J., Archer, G. E., Zalutsky, M. R., and Bigner, D. D. The class III variant of the epidermal growth factor receptor (EGFRvIII): characterization and utilization as an immunotherapeutic target, J. Neurovirol. 4: 148-58, 1998.
  • 14. Humphrey, P. A., Wong, A. J., Vogelstein, B., Zalutsky, M. R., Fuller, G. N., Archer, G. E., Friedman, H. S., Kwatra, M. M., Bigner, S. H., and Bigner, D. D. Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma, Proc Natl Acad Sci USA. 87: 4207-11, 1990.
  • 15. Okamoto, S., Yoshikawa, K., Obata, Y., Shibuya, M., Aoki, S., Yoshida, J., and Takahashi, T. Monoclonal antibody against the fusion junction of a deletion-mutant epidermal growth factor receptor, Br J. Cancer. 73: 1366-72, 1996.
  • 16. Hills, D., Rowlinson-Busza, G., and Gullick, W. J. Specific targeting of a mutant, activated EGF receptor found in glioblastoma using a monoclonal antibody, Int J. Cancer. 63: 537-43, 1995.
  • 17. Moscatello, D. K., Holgado-Madruga, M., Godwin, A. K., Ramirez, G., Gunn, G., Zoltick, P. W., Biegel, J. A., Hayes, R. L., and Wong, A. J. Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors, Cancer Res. 55: 5536-9, 1995.
  • 18. Baselga, J., Pfister, D., Cooper, M. R., Cohen, R., Burtness, B., Bos, M., D'Andrea, G., Seidman, A., Norton, L., Gunnett, K., Falcey, J., Anderson, V., Waksal, H., and Mendelsohn, J. Phase I Studies of Anti-Epidermal Growth Factor Receptor Chimeric Antibody C225 Alone and in Combination With Cisplatin, J Clin Oncol. 18: 904, 2000.
  • 19. Faillot, T., Magdelenat, H., Mady, E., Stasiecki, P., Fohanno, D., Gropp, P., Poisson, M., and Delattre, J. Y. A phase I study of an anti-epidermal growth factor receptor monoclonal antibody for the treatment of malignant gliomas, Neurosurgery. 39: 478-83, 1996.
  • 20. Ponten, J. and Macintyre, E. H. Long term culture of normal and neoplastic human glia, Acta Pathol Microbiol Scand. 74: 465-86, 1968.
  • 21. Masui, H., Kawamoto, T., Sato, J. D., Wolf, B., Sato, G., and Mendelsohn, J. Growth inhibition of human tumor cells in athymic mice by anti-epidermal growth factor receptor monoclonal antibodies, Cancer Res. 44: 1002-7, 1984.
  • 22. Domagala, T., Konstantopoulos, N., Smyth, F., Jorissen, R. N., Fabri, L., Geleick, D., Lax, I., Schlessinger, J., Sawyer, W., Howlett, G. J., Burgess, A. W., and Nice, E. C. Stoichiometry, kinetic and binding analysis of the interaction between Epidermal Growth Factor (EGF) and the Extracellular Domain of the EGF receptor., Growth Factors. 18: 11-29, 2000.
  • 23. Lindmo, T., Boven, E., Cuttitta, F., Fedorko, J., and Bunn, P. A., Jr. Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess, J Immunol Methods. 72: 77-89, 1984.
  • 24. Huang, H. S., Nagane, M., Klingbeil, C. K., Lin, H., Nishikawa, R., Ji, X. D., Huang, C. M., Gill, G. N., Wiley, H. S., and Cavenee, W. K. The enhanced tumorigenic activity of a mutant epidermal growth factor receptor common in human cancers is mediated by threshold levels of constitutive tyrosine phosphorylation and unattenuated signaling, J Biol. Chem. 272: 2927-35, 1997.
  • 25. Reist, C. J., Archer, G. E., Wikstrand, C. J., Bigner, D. D., and Zalutsky, M. R. Improved targeting of an anti-epidermal growth factor receptor variant III monoclonal antibody in tumor xenografts after labeling using N-succinimidyl 5-iodo-3-pyridinecarboxylate, Cancer Res. 57: 1510-5, 1997.
  • 26. Santon, J. B., Cronin, M. T., MacLeod, C. L., Mendelsohn, J., Masui, H., and Gill, G. N. Effects of epidermal growth factor receptor concentration on tumorigenicity of A431 cells in nude mice, Cancer Res. 46: 4701-5, 1986.
  • 27. Voldborg, B. R., Damstrup, L., Spang-Thomsen, M., and Poulsen, H. S. Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials, Ann Oncol. 8: 1197-206, 1997.
  • 28. den Eynde, B. and Scott, A. M. Tumor Antigens. In: P. J. Delves and I. M. Roitt (eds.), Encyclopedia of Immunology, Second Edition, pp. 2424-31. London: Academic Press, 1998.
  • 29. Seymour, L. Novel anti-cancer agents in development: exciting prospects and new challenges, Cancer Treat Rev. 25: 301-12, 1999.
  • 30. Sturgis, E. M., Sacks, P. G., Masui, H., Mendelsohn, J., and Schantz, S. P. Effects of antiepidermal growth factor receptor antibody 528 on the proliferation and differentiation of head and neck cancer, Otolaryngol Head Neck Surg. 111: 633-43, 1994.
  • 31. Goldstein, N. I., Prewett, M., Zuklys, K., Rockwell, P., and Mendelsohn, J. Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in a human tumor xenograft model, Clin Cancer Res. 1: 1311-8, 1995.
  • 32. Reist, C. J., Archer, G. E., Kurpad, S, N., Wikstrand, C. J., Vaidyanathan, G., Willingham, M. C., Moscatello, D. K., Wong, A. J., Bigner, D. D., and Zalutsky, M. R. Tumor-specific anti-epidermal growth factor receptor variant III monoclonal antibodies: use of the tyramine-cellobiose radioiodination method enhances cellular retention and uptake in tumor xenografts, Cancer Res. 55: 4375-82, 1995.
  • 33. Reist, C. J., Garg, P. K., Alston, K. L., Bigner, D. D., and Zalutsky, M. R. Radioiodination of internalizing monoclonal antibodies using N-succinimidyl 5-iodo-3-pyridinecarboxylate, Cancer Res. 56: 4970-7, 1996.
  • 34. Press, O. W., DeSantes, K., Anderson, S. K., and Geissler, F. Inhibition of catabolism of radiolabeled antibodies by tumor cells using lysosomotropic amines and carboxylic ionophores, Cancer Res. 50: 1243-50, 1990.
  • 35. Reist, C. J., Batra, S. K., Pegram, C. N., Bigner, D. D., and Zalutsky, M. R. In vitro and in vivo behavior of radiolabeled chimeric anti-EGFRvIII monoclonal antibody: comparison with its murine parent, Nucl Med. Biol. 24: 639-47, 1997.
  • 36. Mineo, C., Gill, G. N., and Anderson, R. G. Regulated migration of epidermal growth factor receptor from caveolae, J Biol. Chem. 274: 30636-43, 1999.
  • 37. Gunther, N., Betzel, C., and Weber, W. The secreted form of the epidermal growth factor receptor. Characterization and crystallization of the receptor-ligand complex, J Biol. Chem. 265: 22082-5, 1990.

EXAMPLE 18 Novel Monoclonal Antibody Specific for the De2-7 Epidermal Growth Factor Receptor (EGFR) that Also Recognizes the EGFR Expressed in Cells Containing Amplification of the EGFR Gene

The following experiments were presented in Johns et al, (2002) Int. J. Cancer, 98, and in co-pending application Ser. No. 60/342,258 filed Dec. 21, 2001, the entire disclosure of both of which are incorporated herein by reference with cross referencing to the Figures herein where appropriate. The monoclonal antibody mAb 806 was studied and additional data respecting its binding characteristics as to the EGF receptor were developed, which is in addition to and corroborative of the data presented earlier herein. Accordingly, the following represents a review and presentation of the material set forth in the patent application and corresponding publication.

Monoclonal antibody (MAb 806) potentially overcomes the difficulties associated with targeting the EGFR expressed on the surface of tumor cells. MAb 806 bound to de2-7 EGFR transfected U87MG glioma cells (U87MG.Δ2-7) with high affinity (˜1×109 M−1), but did not bind parental cells that express the wild type EGFR. Consistent with this observation, MAb 806 was unable to bind a soluble version of the wild type EGFR containing the extracellular domain. In contrast, immobilization of this extracellular domain to ELISA plates induced saturating and dose response binding of MAb 806, suggesting that MAb 806 can bind the wild type EGFR under certain conditions. MAb 806 also bound to the surface of A431 cells, which due to an amplification of the EGFR gene express large amounts of the EGFR. Interestingly, MAb 806 only recognized 10% of the total EGFR molecules expressed by A431 cells and the binding affinity was lower than that determined for the de2-7 EGFR. MAb 806 specifically targeted U87MG.Δ2-7 and A431 xenografts grown in nude mice with peak levels in U87MG.Δ2-7 xenografts detected 8 h after injection. No specific targeting of parental U87MG xenografts was observed. Following binding to U87MG.Δ2-7 cells, MAb 806 was rapidly internalized by macropinocytosis and subsequently transported to lysosomes, a process that probably contributes to the early targeting peak observed in the xenografts. Thus, MAb 806 can be used to target tumor cells containing amplification of the EGFR gene or de2-7 EGFR but does not bind to the wild type EGFR when expressed on the cell surface.

As discussed above, MAb 806 is specific for the de2-7 EGFR yet binds to an epitope distinct from the unique junctional peptide. Interestingly, while MAb 806 did not recognize the wild type EGFR expressed on the cell surface of glioma cells, it did bind to the extracellular domain of the wild type EGFR immobilized on the surface of ELISA plates. Furthermore, MAb 806 bound to the surface of A431 cells, which have an amplification of the EGFR gene but do not express the de2-7 EGFR. Therefore, it is possible that MAb 806 could be used to specifically target tumors with amplified EGFR regardless of their de2-7 EGFR status, although our results suggest tumors coexpressing the mutated receptor would still show preferential targeting. As MAb 806 does not bind wild type receptor in the absence of gene amplification, there would be no uptake in normal tissue, a potential problem associated with EGFR antibodies currently being developed.18,19

Material and Methods

MAbs and Cell Lines

The U87MG astrocytoma cell line has been described in detail previously.20 This cell line was infected with a retrovirus containing the de2-7 EGFR to produce the U87MG.Δ2-7 cell line. 10 Human squamous carcinoma A431 cells were obtained from ATCC (Rockville, Md.). These cell lines were cultured in DMEM/F-12 with GlutaMAX™ (Life Technologies, Melbourne, Australia) supplemented with 10% FCS(CSL, Melbourne, Australia). The murine pro-B cell line BaF/3, which does not express any known EGFR related molecules, was transfected with de2-7 EGFR as described above. The DH8.3 antibody (IgG1) has been described previously and was obtained following immunization of mice with the unique junctional peptide found in de2-7 EGFR.16 MAb 806 (IgG2b) was produced following immunization of mice with NR6 mouse fibroblasts transfected with the de2-7 EGFR. It was selected for further characterization as hemagglutination assays showed a high titer against NR6.ΔEGFR cells but low backgrounds on NR6.wtEGFR cells. The 528 antibody, which recognizes both de2-7 and wild type EGFR, has been described previously21 and was produced in the Biological Production Facility (Ludwig Institute for Cancer Research, Melbourne) using a hybridoma obtained from ATCC. The polyclonal antibody sc-03 directed to the COOH-terminal domain of the EGFR was purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).

Other Reagents

The recombinant extracellular domain (amino acids 1-621) of the wild type EGFR (sEGFR) was produced as previously described.22 The biotinylated unique junctional peptide (Biotin-LEEKKGNYVVTDH) (SEQ ID NO:5) from de2-7 EGFR was synthesized by standard Fmoc chemistry and purity (>96%) determined by reverse phase HPLC and mass spectral analysis (Auspep, Melbourne, Australia).

FACS Analysis

Cells were labeled with the relevant antibody (10 μg/ml) followed by fluorescein-conjugated goat anti-mouse IgG (1:100 dilution; Calbiochem, San Diego, Calif.). FACS data was obtained on a Coulter Epics ELITE™ ESP by observing a minimum of 5,000 events and analyzed using EXPO™ (version 2) for Windows.

ELISA Assays

Two types of ELISA were used to determine the specificity of the antibodies. In the first assay, plates were coated with Segfr (10 μg/ml in 0.1 M carbonate buffer pH 9.2) for 2 hr and then blocked with 2% human serum albumin (HSA) in PBS. Antibodies were added to wells in triplicate at increasing concentration in 2% HSA in phosphate-buffered saline (PBS). Bound antibody was detected by horseradish peroxidase conjugated sheep anti-mouse IgG (Silenus, Melbourne, Australia) using ABTS (Sigma, Sydney, Australia) as a substrate and the absorbance measured at 405 nm. In the second assay, the biotinylated de2-7 specific peptide was bound to ELISA plates precoated with streptavidin (Pierce, Rock-ford, Ill.). Antibodies were bound and detected as in the first assay.

Scatchard Analysis

Antibodies were labeled with 125I (Amrad, Melbourne, Australia) by the chloramine T method and immunoreactivity determined by Lindmo assay. 23 All binding assays were performed in 1% HSA/PBS on 1−2×106 live U87MG.Δ2-7 or A431 cells for 90 min at 4° C. with gentle rotation. A set concentration of 10 ng/ml 125I-labeled antibody was used in the presence of increasing concentrations of the appropriate unlabeled antibody. Non-specific binding was determined in the presence of 10,000-fold excess of unlabeled antibody. Neither 125I-radiolabeled MAb 806 or the DH8.3 antibody bound to parental U87MG cells. After the incubation was completed, cells were washed and counted for bound 125I-labeled antibody using a COBRA II™ gamma counter (Packard Instrument Company, Meriden, Conn.). Scatchard analysis was done following correction for immunoreactivity.

Internalization Assay

U87MG.Δ2-7 cells were incubated with either MAb 806 or the DH8.3 antibody (10 μg/ml) for 1 hr in DMEM at 4° C. After washing, cells were transferred to DMEM pre-warmed to 37° C. and aliquots taken at various time points following incubation at 37° C. Internalization was stopped by immediately washing aliquots in ice-cold wash buffer (1% HSA/PBS). At the completion of the time course cells were stained by FACS as described above. Percentage internalization was calculated by comparing surface antibody staining at various time points to zero time using the formula: percent antibody internalized=(mean fluorescence at timex−background fluorescence)/(mean fluorescence at time0_background fluorescence)×100. This method was validated in 1 assay using an iodinated antibody (MAb 806) to measure internalization as previously described.24 Differences in internalization rate at different time points were compared using Student's t-test.

Electron Microscopy of U87MG.Δ2-7 Cells

U87MG.Δ2-7 cells were grown on gelatin coated chamber slides (Nunc, Naperville, Ill.) to 80% confluence and then washed with ice cold DMEM. Cells were then incubated with MAb 806 or the DH8.3 antibody in DMEM for 45 min at 4° C. After washing, cells were incubated for a further 30 min with gold-conjugated (20 nm particles) anti-mouse IgG (BBInternational, Cardiff, UK) at 4° C. Following a further wash, pre-warmed DMEM/10% FCS was added to the cells, which were incubated at 37° C. for various times from 1-60 min. Internalization of the antibody was stopped by ice-cold media and cells fixed with 2.5% glutaraldehyde in PBS/0.1% HSA and then postfixed in 2.5% osmium tetroxide. After dehydration through a graded series of acetone, samples were embedded in Epon/Araldite resin, cut as ultrathin sections with a Reichert Ultracut-S microtome (Leica) and collected on nickel grids. The sections were stained with uranyl acetate and lead citrate before being viewed on a Philips CM12 transmission electron microscope at 80 kV. Statistical analysis of gold grains contained within coated pits was performed using a χ2 test.

Immunoprecipitation Studies

Cells were labeled for 16 hr with 100 μCi/ml of Tran35 S-Label (ICN Biomedicals, CA) in DMEM without methionine/cysteine supplemented with 5% dialyzed FCS. After washing with PBS, cells were placed in lysis buffer (1% Triton X-100, 30 mM HEPES, 150 mM NaCl, 500 μM AEBSF, 150 nM aprotinin, 1 μM E-64 protease inhibitor, 0.5 mM EDTA and 1 μM leupeptin, pH 7.4) for 1 hr at 4° C. Lysates were clarified by centrifugation for 10 min at 12,000 g, then incubated with 5 μg of appropriate antibody for 30 min at 4° C. before the addition of Protein A-Sepharose™ Immunoprecipitates were washed 3 times with lysis buffer, mixed with SDS sample buffer, separated by gel electrophoresis using a 4-20% Tris/glycine gel that was then dried and exposed to X-ray film.

Biodistribution in Tumor Bearing Nude Mice

Tumor xenografts were established in nude BALB/c mice by s.c. injection of 3×106 U87MG, U87MG.Δ2-7 or A431 cells. de2-7 EGFR expression in U87MG.Δ2-7 xenografts remains stable throughout the period of biodistribution as measured by immunohistochemistry at various time points (data not shown). A431 cells also retained their MAb 806 reactivity when grown as tumor xenografts as determined by immunohistochemistry. U87MG or A431 cells were injected on 1 side 7-10 days before U87MG.Δ2-7 cells were injected on the other side because of the faster growth rate observed for de2-7 EGFR expressing xenografts. Antibodies were radiolabeled and assessed for immunoreactivity as described above and were injected into mice by the retro-orbital route when tumors were 100-200 mg in weight. Each mouse received 2 different antibodies (2 μg per antibody): 2 μCi of 125I-labeled MAb 806 and 2 μCi of 131I-labeled DH8.3 or 528. Unless indicated, groups of 5 mice were sacrificed at various time points post-injection and blood obtained by cardiac puncture. The tumors, liver, spleen, kidneys and lungs were obtained by dissection. All tissues were weighed and assayed for 125I and 131I activity using a dual-channel counting window. Data was expressed for each antibody as percentage injected dose per gram tumor (% ID/g tumor) determined by comparison to injected dose standards or converted into tumor to blood/liver ratios (i.e., % ID/g tumor÷% ID/g blood or liver). Differences between groups were analyzed by Student's t-test. After injection of radiolabeled MAb 806, some tumors were fixed in formalin, embedded in paraffin, cut into 5 μm sections and then exposed to X-ray film (AGFA, Mortsel, Belgium) to determine antibody localization by autoradiography.

Results

Binding of Antibodies to Cell Lines

In order to confirm the specificity of MAb 806 and the DH8.3 antibody, binding to U87MG and U87MG.Δ2-7 cells was analyzed by FACS. An irrelevant murine IgG2b was included as an isotype control for MAb 806 and the 528 antibody was included as it recognizes both the de2-7 and wild type EGFR. Only the 528 antibody was able to stain the U87MG cell line (FIG. 1) consistent with previous reports demonstrating that these cells express the wild type EGFR.10 Both MAb 806 and the DH8.3 antibody had binding levels similar to the irrelevant antibody, clearly demonstrating they are unable to bind the wild type receptor (FIG. 1). Binding of the isotype control antibody to U87MG.Δ2-7 cells was similar as that observed for the U87MG cells. MAb 806 and the DH8.3 antibody immunostained U87MG.Δ2-7 cells, indicating that these antibodies specifically recognize the de2-7 EGFR (FIG. 1). The 528 antibody stained U87MG.Δ2-7 with a higher intensity than the parental cell as it binds both the de2-7 and wild type receptors that are co-expressed in these cells (FIG. 1). Importantly, MAb 806 also bound the BaF/3.Δ2-7 cell line, demonstrating that the co-expression of wild type EGFR is not a requirement for MAb 806 reactivity (FIG. 1 but data not shown herein).

Binding of Antibodies in ELISA Assays

To further characterize the specificity of MAb 806 and the DH8.3 antibody, their binding was examined by ELISA. Both MAb 806 and the 528 antibody displayed dose-dependent and saturating binding curves to immobilized wild type sEGFR (FIG. 2A). As the unique junctional peptide found in the de2-7 EGFR is not contained within the sEGFR, MAb 806 must be binding to an epitope located within the wild type EGFR sequence. The binding of the 528 antibody was probably lower than that observed for MAb 806 as it recognizes a conformational determinant. As expected the DH8.3 antibody did not bind the wild type sEGFR even at concentrations up to 10 μg/ml (FIG. 2A). Although sEGFR in solution inhibited the binding of the 528 antibody to immobilized sEGFR in a dose-dependent fashion, it was unable to inhibit the binding of MAb 806 (FIG. 2B). This suggests that MAb 806 can only bind wild type EGFR once immobilized on ELISA plates, a process that may induce conformational changes. Similar results were observed using a BIAcore™ whereby MAb 806 bound immobilized sEGFR but immobilized MAb 806 was not able to bind sEGFR in solution (data not shown). Following denaturation by heating for 10 min at 95° C., sEGFR in solution was able to inhibit the binding of MAb 806 to immobilized sEGFR (FIG. 2C but data not shown herein), confirming that MAb 806 can bind the wild type EGFR under certain conditions. Interestingly, the denatured sEGFR was unable to inhibit the binding of the 528 antibody (FIG. 2C but data not shown herein, demonstrating that this antibody recognizes a conformational epitope. The DH8.3 antibody exhibited dose-dependent and saturable binding to the unique de2-7 EGFR peptide (FIG. 2D). Neither MAb 806 or the 528 antibody bound to the peptide, even at concentrations higher than those used to obtain saturation binding of DH8.3, further indicating MAb 806 does not recognize an epitope determinant within this peptide.

Scatchard Analysis of Antibodies

A Scatchard analysis was performed using U87MG.Δ2-7 cells in order to determine the relative affinity of each antibody. Both MAb 806 and the DH8.3 antibody retained high immunoreactivity when iodinated and was typically greater than 90% for MAb 806 and 45-50% for the DH8.3 antibody. MAb 806 had an affinity for the de2-7 EGFR receptor of 1.1×109 M−1 whereas the affinity of DH8.3 was some 10-fold lower at 1.0×108M−1. Neither iodinated antibody bound to U87MG parental cells. MAb 806 recognized an average of 2.4×105 binding sites per cell with the DH8.3 antibody binding an average of 5.2×105 sites. Thus, there was not only good agreement in receptor number between the antibodies, but also with a previous report showing 2.5×105 de2-7 receptors per cell as measured by a different de2-7 EGFR specific antibody on the same cell line.25

Internalization of Antibodies by U87MG.Δ2-7 Cells

The rate of antibody internalization following binding to a target cell influences both its tumor targeting properties and therapeutic options. Consequently, we examined the internalization of MAb 806 and the DH8.3 antibody following binding to U87MG.Δ2-7 cells by FACS. Both antibodies showed relatively rapid internalization reaching steady-state levels at 10 min for MAb 806 and 30 min for DH8.3 (FIG. 3).

Internalization of DH8.3 was significantly higher both in terms of rate (80.5% of DH8.3 internalized at 10 min compared to 36.8% for MAb 806,p<0.01) and total amount internalized at 60 min (93.5% vs. 30.4%, p<0.001). MAb 806 showed slightly lower levels of internalization at 30 and 60 min compared to 20 min in all 4 assays performed (FIG. 3). This result was also confirmed using an internalization assay based on iodinated MAb 806 (data not shown).

Electron Microscopy Analysis of Antibody Internalization

Given this difference in internalization rates between the antibodies, a detailed analysis of antibody intracellular trafficking was performed using electron microscopy. Although the DH8.3 anti-body was internalized predominantly via coated-pits (FIG. 19A), MAb 806 appeared to be internalized by macropinocytosis (FIG. 19B). In fact, a detailed analysis of 32 coated pits formed in cells incubated with MAb 806 revealed that none of them contained antibody. In contrast, around 20% of all coated-pits from cells incubated with DH8.3 were positive for antibody, with a number containing multiple gold grains. A statistical analysis of the total number of gold grains contained within coated-pits found that the difference was highly significant (p<0.01). After 20-30 min both antibodies could be seen in structures that morphologically resemble lysosomes (FIG. 19C). The presence of cellular debris within these structures is also consistent with their lysosome nature.

Biodistribution of Antibodies in Tumor Bearing Nude Mice

The biodistribution of MAb 806 and the DH8.3 antibody was compared in nude mice containing U87MG xenografts on 1 side and U87MG.Δ2-7 xenografts on the other. A relatively short time period was chosen for this study as a previous report demonstrated that the DH8.3 antibody shows peak levels of tumor targeting between 4-24 hr.16 In terms of % ID/g tumor, MAb 806 reached its peak level in U87MG.Δ2-7 xenografts of 18.6% ID/g tumor at 8 hr (FIG. 4A), considerably higher than any other tissue except blood. Although DH 8.3 also showed peak tumor levels at 8 hr, the level was a statistically (p<0.001) lower 8.8% ID/g tumor compared to MAb 806 (FIG. 4B). Levels of both antibodies slowly declined at 24 and 48 hr. Autoradiography of U87MG.Δ2-7 xenograft tissue sections collected 8 hr after injection with 125I-labeled MAb 806 alone, clearly illustrates localization of antibody to viable tumor (FIG. 20). Neither antibody showed specific targeting of U87MG parental xenografts (FIGS. 4A and 4B). With regards to tumor to blood/liver ratios, MAb 806 showed the highest ratio at 24 hr for both blood (ratio of 1.3) and liver (ratio of 6.1) (FIGS. 5A and 5B). The DH8.3 antibody had its highest ratio in blood at 8 hr (ratio of 0.38) and at 24 hr in liver (ratio of 1.5) (FIGS. 5A and 5B), both of which are considerably lower than the values obtained for MAb 806.

Binding of MAb 806 to Cells Containing Amplified EGFR

To examine if MAb 806 could recognize the EGFR expressed in cells containing an amplified receptor gene, its binding to A431 cells was analyzed. Low, but highly reproducible, binding of MAb 806 to A431 cells was observed by FACS analysis (FIG. 6). The DH8.3 antibody did not bind A431 cells, indicating that the binding of MAb 806 was not the result of low level de2-7 EGFR expression (FIG. 6). As expected, the anti-EGFR 528 antibody showed strong staining of A431 cells (FIG. 6). The average of 3 such experiments gave a value for affinity of 9.5×107 M−1 with 2.4×105 receptors per cell. Thus the affinity for this receptor was some 10-fold lower than the affinity for the de2-7 EGFR. Furthermore, MAb 806 appears to only recognize a small portion of EGFR found on the surface of A431 cells. Using the 528 antibody approximately 2×106 receptors per cell were measured, which is in agreement with numerous other studies.26 To ensure that these results were not simply restricted to the A431 cell line, MAb 806 reactivity was examined in 2 other cells lines exhibiting amplification of the EGFR gene. Both the HN5 head and neck cell line27 and the MDA-468 breast cancer cell line 28 have been reported to contain multiple copies of the EGFR gene. Consistent with these reports, the 528 antibody displayed intense staining of both cell lines (FIG. 21). As with the A431 cell line, the MAb 806 clearly stained both cell lines but at a lower level than that observed with the 528 antibody (FIG. 21). Thus, MAb 806 binding is not simply restricted to A431 cells but appears to be a general observation for cells containing amplification of the EGFR gene.

Immunoprecipitations

MAb 806 reactivity was further characterized by immunoprecipitation using 35S-labeled cells. The sc-03 antibody (a commercial polyclonal antibody specific for the c-terminal domain of the EGFR) immunoprecipitated 3 bands from U87MG.Δ2-7 cells; a doublet corresponding to the 2 de2-7 EGFR bands observed in these cells and a higher molecular weight band corresponding to the wt EGFR (FIG. 22). In contrast, while MAb 806 immunoprecipitated the 2 de2-7 EGFR bands, the wt EGFR was completely absent. The sc-03 antibody immunoprecipitated a single band corresponding to the wt EGFR from A431 cells (FIG. 22). The MAb 806 also immunoprecipitated a single band corresponding to the wt EGFR from A431 cells (FIG. 22) but consistent with the FACS and Scatchard data, the amount of EGFR immunoprecipitated by MAb 806 was substantially less than the total EGFR present on the cell surface. Given that MAb 806 and the sc-03 immunoprecipitated similar amounts of the de2-7 EGFR, this result supports the notion that the MAb 806 antibody only recognizes a portion of the EGFR in cells overexpressing the receptor. An irrelevant IgG2b (an isotype control for MAb 806) did not immunoprecipitate EGFR from either cell line (FIG. 22). Using identical conditions, MAb 806 did not immunoprecipitate the EGFR from the parental U87MG cells (data not shown).

In Vivo Targeting of A431 Cells by MAb 806

A second biodistribution study was performed with MAb 806 to determine if it could target A431 tumor xenografts. The study was conducted over a longer time course in order to obtain more information regarding the targeting of U87MG.Δ2-7 xenografts by MAb 806, which were included in all mice as a positive control. In addition, the anti-EGFR 528 antibody was included as a positive control for the A431 xenografts, since a previous study demonstrated low but significant targeting of this antibody to A431 cells grown in nude mice.21 During the first 48 hr, MAb 806 displayed almost identical targeting properties as those observed in the initial experiments (FIG. 7A compared with FIG. 4A). In terms of % ID/g tumor, levels of MAb 806 in U87MG.Δ2-7 xenografts slowly declined after 24 hr but always remained higher than levels detected in normal tissue. Uptake in the A431 xenografts was comparatively low, however, there was a small increase in % ID/g tumor during the first 24 hr not observed in normal tissues such as liver, spleen, kidney and lung (FIG. 7A). Uptake of the 528 antibody was low in both xenografts when expressed as % ID/g tumor (FIG. 7B). Autoradiography of A431 xenograft tissue sections collected 24 hr after injection with 125I-labeled MAb 806 alone, clearly illustrates localization of antibody to viable tumor around the periphery of the tumor and not central areas of necrosis (FIG. 23). In terms of tumor to blood ratio MAb 806 peaked at 72 hr for U87MG.42-7 xenografts and 100 hr for A431 xenografts (FIGS. 8A AND 8B). Although the tumor:blood ratio for MAb 806 never surpassed 1.0 with respect to the A431 tumor, it did increase through-out the entire time course (FIG. 8B) and was higher than all other tissues examined (data not shown) indicating low levels of targeting. The tumor to blood ratio for the 528 antibody showed a similar profile to MAb 806 although higher levels were noted in the A431 xenografts (FIGS. 8A AND 8B). MAb 806 had a peak tumor to liver ratio in U87MG.Δ2-7 xenografts of 7.6 at 72 hr, clearly demonstrating preferential uptake in these tumors compared to normal tissue (FIG. 8C). Other tumor to organ ratios for MAb 806 were similar to those observed in the liver (data not shown). The peak tumor to liver ratio for MAb 806 in A431 xenografts was 2.0 at 1 od hr, again indicating a slight preferentially uptake in tumor compared with normal tissue (FIG. 8D).

Discussion

The previously described L8A4 monoclonal antibody directed to the unique junctional peptide found in the de2-7 EGFR, behaves in a similar fashion to MAb 806.38 Using U87MG cells transfected with the de2-7 EGFR, this antibody had a similar internalization rate (35% at 1 hr compared to 30% at 1 hr for MAb 806) and displayed comparable in vivo targeting when using 3T3 fibroblasts transfected with de2-7 EGFR (peak of 24% ID/g tumor at 24 hr compared to 18% ID/g tumor at 8 hr for MAb 806).25.

Perhaps the most important advantage of MAb 806 compared to current EGFR antibodies, is that MAb 806 can be directly conjugated to cytotoxic agents. This approach is not feasible with current EGFR specific antibodies as they target the liver and cytotoxic conjugation would almost certainly induce severe toxicity. Conjugation of cytotoxic agents such as drugs41 or radioisotopes42 to antibodies has the potential to improve efficacy and reduce the systemic toxicity of these agents. The ability of a conjugated antibody to mediate tumor kill is dependent upon its potential to be internalized. Thus, the rapid internalization observed with MAb 806 in U87MG.Δ2-7 cells, suggests MAb 806 is an ideal candidate for this type of approach.

MAb 806 is novel in that it is the first de2-7 EGFR specific antibody directed to an epitope not associated with the unique junctional peptide. It has superior affinity and better tumor targeting properties than DH8.3, a previously described de2-7 EGFR antibody. An important property, however, is its ability to recognize a subset of EGFR molecules expressed on the surface of tumor cells exhibiting amplification of the EGFR gene. This suggests that MAb 806 may possess a unique clinical property; the ability to target both de2-7 and amplified EGFR but not wild type receptors. If proven correct, this antibody would not target organs such as liver and therefore would be more versatile than current antibodies directed to the EGFR,18,19 which cannot be used for the coupling of cytotoxic agents. Finally, MAb 806 may be a useful reagent for analyzing the conformational changes induced by the truncation found in de2-7 EGFR.

References

  • 1. Wikstrand C J, McLendon R E, Friedman A H, et al. Cell surface localization and density of the tumor-associated variant of the epidermal growth factor receptor, EGFRvIII. Cancer Res 1997; 57:4130-40.
  • 2. Olapade-Olaopa E O, Moscatello D K, MacKay E H, et al. Evidence for the differential expression of a variant EGF receptor protein in human prostate cancer. Br J Cancer 2000; 82:186-94.
  • 3. Wikstrand C J, Hale L P, Batra S K, et al. Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas. Cancer Res 1995; 55:3140-8.
  • 4. Garcia de Palazzo I E, Adams G P, Sundareshan P, et al. Expression of mutated epidermal growth factor receptor by non-small cell lung carcinomas. Cancer Res 1993; 53:3217-20.
  • 5. Ekstrand A J, Sugawa N, James C D, et al. Amplified and rearranged epidermal growth factor receptor genes in human glioblastomas reveal deletions of sequences encoding portions of the N- or C-terminal tails. Proc Natl Acad Sci USA 1992; 89:4309-13.
  • 6. Wong A J, Ruppert J M, Bigner S H, et al. Structural alterations of the epidermal growth factor receptor gene in human gliomas. Proc Natl Acad Sci USA 1992; 89:2965-9.
  • 7. Yamazaki H, Ohba Y, Tamaoki N, et al. A deletion mutation within the ligand binding domain is responsible for activation of epidermal growth factor receptor gene in human brain tumors. Jpn J Cancer Res 1990; 81:773-9.
  • 8. Yamazaki H, Fukui Y, Ueyama Y, et al. Amplification of the structurally and functionally altered epidermal growth factor receptor gene (c-erbB) in human brain tumors. Mol Cell Biol 1988; 8:1816-20.
  • 9. Sugawa N, Ekstrand A J, James C D, et al. Identical splicing of aberrant epidermal growth factor receptor transcripts from amplified re-arranged genes in human glioblastomas. Proc Natl Acad Sci USA 1990; 87:8602-6.
  • 10. Nishikawa R, Ji X D, Harmon R C, et al. A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc Natl Acad Sci USA 1994; 91:7727-31.
  • 11. Batra S K, Castelino-Prabhu S, Wikstrand C J, et al. Epidermal growth factor ligand-independent, unregulated, cell-transforming potential of a naturally occurring human mutant EGFRvIII gene. Cell Growth Differ 1995; 6:1251-9.
  • 12. Nagane M, Coufal F, Lin H, et al. A common mutant epidermal growth factor receptor confers enhanced tumorigenicity on human glioblastoma cells by increasing proliferation and reducing apoptosis. Cancer Res 1996; 56:5079-86.
  • 13. Wikstrand C J, Reist C J, Archer G E, et al. The class III variant of the epidermal growth factor receptor (EGFRvIII): characterization and utilization as an immunotherapeutic target. J Neurovirol 1998; 4:148-58.
  • 14. Humphrey P A, Wong A J, Vogelstein B, et al. Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma. Proc Natl Acad Sci USA 1990; 87:4207-11.
  • 15. Okamoto S, Yoshikawa K, Obata Y, et al. Monoclonal antibody against the fusion junction of a deletion-mutant epidermal growth factor receptor. Br J Cancer 1996; 73:1366-72.
  • 16. Hills D, Rowlinson-Busza G, Gullick W J. Specific targeting of a mutant, activated EGF receptor found in glioblastoma using a monoclonal antibody. Int J Cancer 1995; 63:537-43.
  • 17. Moscatello D K, Holgado-Madruga M, Godwin A K, et al. Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors. Cancer Res 1995; 55:5536-9.
  • 18. Baselga J, Pfister D, Cooper M R, et al. Phase I studies of anti-epidermal growth factor receptor chimeric antibody C225 alone and in combination with cisplatin. J Clin Oncol 2000; 18:904-14.
  • 19. Faillot T, Magdelenat H, Mady E, et al. A phase I study of an anti-epidermal growth factor receptor monoclonal antibody for the treatment of malignant gliomas. Neurosurgery 1996; 39:478-83.
  • 20. Ponten J, Macintyre E H. Long term culture of normal and neoplastic human glia. Acta Pathol Microbiol Scand 1968; 74:465-86.
  • 21. Masui H, Kawamoto T, Sato J D, et al. Growth inhibition of human tumor cells in athymic mice by anti-epidermal growth factor receptor monoclonal antibodies. Cancer Res 1984; 44:1002-7.
  • 22. Domagala T, Konstantopoulos N, Smyth F, et al. Stoichiometry, kinetic and binding analysis of the interaction between epidermal growth factor (EGF) and the extracellular domain of the EGF recep-tor. Growth Factors 2000; 18:11-29.
  • 23. Lindmo T, Boven E, Cuttitta F, et al. Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. J Immunol Methods 1984; 72:77-89.
  • 24. Huang H S, Nagane M, Klingbeil C K, et al. The enhanced tumorigenic activity of a mutant epidermal growth factor receptor common in human cancers is mediated by threshold levels of constitutive tyrosine phosphorylation and unattenuated signaling. J Biol Chem 1997; 272:2927-35.
  • 25. Reist C J, Archer G E, Wikstrand C J, et al. Improved targeting of an anti-epidermal growth factor receptor variant III monoclonal antibody in tumor xenografts after labeling using N-succinimidyl 5-iodo-3-pyridinecarboxylate. Cancer Res 1997; 57:1510-5.
  • 26. Santon J B, Cronin M T, MacLeod C L, et al. Effects of epidermal growth factor receptor concentration on tumorigenicity of A431 cells in nude mice. Cancer Res 1986; 46:4701-5.
  • 27. Kwok T T, Sutherland R M. Differences in EGF related radiosensitisation of human squamous carcinoma cells with high and low num-bers of EGF receptors. Br J Cancer 1991; 64:251-4.
  • 28. Filmus J, Pollak M N, Cailleau R, et al. MDA-468, a human breast cancer cell line with a high number of epidermal growth factor (EGF) receptors, has an amplified EGF receptor gene and is growth inhibited by EGF. Biochem Biophys Res Commun 1985; 128:898-905.
  • 29. Voldborg B R, Damstrup L, Spang-Thomsen M, et al. Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials. Ann Oncol 1997; 8:1197-206.
  • 30. den Eynde B, Scott A M. Tumor antigens. In: Delves P J, Roitt I M, eds. Encyclopedia of immunology. London: Academic Press, 1998.2424-31.
  • 31. Seymour L. Novel anti-cancer agents in development: exciting prospects and new challenges. Cancer Treat Rev 1999; 25:301-12.
  • 32. Sturgis E M, Sacks P G, Masui H, et al. Effects of antiepidermal growth factor receptor antibody 528 on the proliferation and differentiation of head and neck cancer. Otolaryngol Head Neck Surg 1994; 111:633-43.
  • 33. Goldstein N I, Prewett M, Zuklys K, et al. Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in a human tumor xenograft model. Clin Cancer Res 1995; 1:1311-8.
  • 34. Fernandes H, Cohen S, Bishayee S. Glycosylation-induced conformational modification positively regulates receptor-receptor association: a study with an aberrant epidermal growth factor receptor (EGFRvIII/DEGFR) expressed in cancer cells. J Biol Chem 2001; 276:5375-83.
  • 35. Reist C J, Garg P K, Alston K L, et al. Radioiodination of internalizing monoclonal antibodies using N-succinimidyl 5-iodo-3-pyridinecar-boxylate. Cancer Res 1996; 56:4970-7.
  • 36. Reist C J, Archer G E, Kurpad S N, et al. Tumor-specific anti-epidermal growth factor receptor variant III monoclonal antibodies: use of the tyramine-cellobiose radio iodination method enhances cellular retention and uptake in tumor xenografts. Cancer Res 1995; 55:4375-82.
  • 37. Press O W, DeSantes K, Anderson S K, et al. Inhibition of catabolism of radiolabeled antibodies by tumor cells using lysosomotropic amines and carboxylic ionophores. Cancer Res 1990; 50:1243-50.
  • 38. Reist C J, Batra S K, Pegram C N, et al. In vitro and in vivo behavior of radiolabeled chimeric anti-EGFRvIII monoclonal antibody: comparison with its murine parent. Nucl Med Biol 1997; 24:639-47.
  • 39. Mineo C, Gill G N, Anderson R G. Regulated migration of epidermal growth factor receptor from caveolae. J Biol Chem 1999; 274:30636-43.
  • 40. Luwor R B, Johns T G, Murone C, et al. Monoclonal antibody 806 inhibits the growth of tumor xenografts expressing either the de2-7 or amplified epidermal growth factor receptor (EGFR) but not wild-type EGFR. Cancer Res 2001; 61:5355-61.
  • 41. Trail P A, Bianchi A B. Monoclonal antibody drug conjugates in the treatment of cancer. Curr Opin Immunol 1999; 11:584-8.
  • 42. DeNardo S J, Kroger L A, DeNardo G L. A new era for radiolabeled antibodies in cancer? Curr Opin Immunol 1999; 11:563-9.

EXAMPLE 19 Growth Suppression of Intracranial Xenografted Glioblastomas Overexpressing Mutant Epidermal Growth Factor Receptors by Systemic Administration of Monoclonal Antibody (mAb) 806, a Novel Monoclonal Antibody Directed to the Receptor

This example presents the evaluation of mAb 806 on the growth of intracranial xenografted gliomas in nude mice. The following corresponds to and was presented in Mishima et al, (2001) Cancer Research, 61:5349-5354, and the entire publication is incorporated herein by reference with cross referencing to the Figures herein where appropriate. and made a part hereof. The data and findings of Mishima et al. are set forth below.

Systemic treatment with mAb 806 significantly reduced the volume of tumors and increased the survival of mice bearing xenografts of U87MG.ΔEGFR, LN-Z308.ΔEGFR, or A1207 gliomas, each of which expresses high levels of ΔEGFR. In contrast, mAb 806 treatment was ineffective with mice bearing the parental U87MG tumors, which expressed low levels of endogenous wild-type EGFR, or U87MG.DK tumors, which expressed high levels of kinase-deficient ΔEGFR. A slight increase of survival of mice xenografted with a wild-type EGFR over-expressing U87MG glioma (U87MG.wtEGFR) was effected by mAb 806 concordant with its weak cross-reactivity with such cells. Treatment of U87MG.ΔEGFR tumors in mice with mAb 806 caused decreases in both tumor growth and angiogenesis, as well as increased apoptosis. Mechanistically, in vivo mAb 806 treatment resulted in reduced phosphorylation of the constitutively active ΔEGFR and caused down-regulated expression of the apoptotic protector, BclXL. These data provide preclinical evidence that mAb 806 treatment may be a useful biotherapeutic agent for those aggressive gliomas that express ΔEGFR.

The present example demonstrates that systemic treatment with the novel ΔEGFR-specific mAb, mAb 806, causes reduced phosphorylation of the constitutively active ΔEGFR and thereby suppresses growth of intracranially implanted gliomas overexpressing this mutant receptor in nude mice and extends their survival. The inhibition of tumor growth was mediated by a decrease in proliferation and angiogenesis and increased apoptosis of the tumor cells. This suppression affected active signaling by ΔEGFR because intracranial xenografts that were derived from cells overexpressing kinase-deficient ΔEGFR (DK), which are recognized equally well by mAb 806, were not significantly suppressed after the same therapy.

Materials and Methods

Cell Lines.

Because primary explants of human glioblastomas rapidly lose expression of amplified, rearranged receptors in culture, no existing glioblastoma cell lines exhibit such expression. To force maintenance of expression levels comparable with those seen in human tumors, U87MG, LN-Z308, and A1207 (gift from Dr. S. Aaronson, Mount Sinai Medical Center, New York, N.Y.) cells were infected with ΔEGFR, kinase-deficient ΔEGFR (DK), or wtEGFR viruses which also conferred resistance to G418 as described previously (21). Populations expressing similar levels of the various EGFR alleles (these expression levels correspond approximately to an amplification level of 25 gene copies; human glioblastomas typically have amplification levels from 10 to 50 gene copies of the truncated receptor) were selected by FACS as described previously (21) and designated asU87MG.ΔEGFR, U87MG.DK, U87MG.wtEGFR, LN-Z308.ΔEGFR, LN-Z308.DK, LN-Z308.wtEGFR, A1207.ΔEGFR, A1207.DK, and A1207.wtEGFR, respectively. Each was maintained in medium containing G418 (U87MG cell lines, 400 mg/ml; LN-Z308 and A1207 cell lines, 800 mg/ml). mAbs. mAb 806 (IgG2b, k), a ΔEGFR specific mAb, was produced after immunization of mice with NR6 mouse fibroblasts expressing the ΔEGFR. It was selected from several clones because hemagglutination assays showed that it had a high reactivity against NR6.ΔEGFR cells, low reactivity for NR6.wtEGFR cells, and none for NR6 cells.

Immunoprecipitation and Western Blot Analysis.

Cells were lysed with lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate, 10 mM sodium PPi, 1 mM phenylmethylsulfonyl fluoride, 2 mM Na3 VO4, 5 μg/m1leupeptin, and 5 mg/ml aprotinin. Antibodies were incubated with cell lysates at 4° C. for 1 h before the addition of protein-A and -G Sepharose™Immuno-precipitates were washed twice with lysis buffer and once with HNTG buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol], electrophoresed, and transferred to nitrocellulose membranes. Blots were probed with the anti-EGFR antibody, C13, and proteins were visualized using the ECL™ chemiluminescent detection system (Amersham Pharmacia Biotech.). The mAbs used for precipitation were mAb 806, anti-EGFR mAb clone 528 (Oncogene Research Products, Boston, Mass.), or clone EGFR.1 (Oncogene Research Products). A mAb, C13, used for detection of both wild-type and ΔEGFR on immunoblots was provided by Dr. G. N. Gill (University of California, San Diego, Calif.). Antibodies to Bcl-XL (rabbit poly-clonal antibody; Transduction Laboratories, Lexington, Ky.) and phosphotyrosine (4G10, Upstate Biotechnology, Lake Placid, N.Y.) were used for Western blot analysis as described previously (26).

Flow Cytometry Analysis.

Cells were labeled with the relevant antibody followed by fluorescein-conjugated goat anti-mouse IgG (1:100 dilution; Becton-Dickinson PharMingen, San Diego, Calif.) as described previously (21). Stained cells were analyzed with a FACSCalibur™ using Cell Quest™ software (Becton-Dickinson PharMingen). For the first antibody, the following mAbs were used: mAb 806, anti-EGFR mAb clone 528, and clone EGFR.1. Mouse IgG2a or IgG2b was used as an isotype control.

Tumor Therapy.

U87MG.ΔEGFR cells 1×105) or 5×105 LN-Z308.ΔEGFR, A1207.ΔEGFR, U87MG, U87MG.DK, and U87MG.wtEGFR cells in 5 μl of PBS were implanted into the right corpus striatum of nude mice brains as described previously (27). Systemic therapy with mAb 806, or the IgG2b isotype control, was accomplished by i.p. injection of 1 μg of mAbs in a volume of 100 μl every other day from postimplantation day 0 through 14. For direct therapy of intracerebral U87MG.ΔEGFR tumors, 10 μg of mAb 806, or the IgG2b isotype control, in a volume of 5 μl were injected at the tumor-injection site every other day starting at day 1 for 5 days.

Immunohistochemistry.

To assess angiogenesis in tumors, they were fixed in a solution containing zinc chloride, paraffin embedded, sectioned, and immunostained using a monoclonal rat anti-mouse CD31 antibody (Becton-Dickinson PharMingen; 1:200). Assessment of tumor cell proliferation was performed by Ki-67 immunohistochemistry on formalin-fixed paraffin-embedded tumor tissues. After deparaffinization and rehydration, the tissue sections were incubated with 3% hydrogen peroxide in methanol to quench endogenous peroxidase. The sections were blocked for 30 min with goat serum and incubated overnight with the primary antibody at 4° C. The sections were then washed with PBS and incubated with a biotinylated secondary antibody for 30 min. After several washes with PBS, products were visualized using streptavidin horseradish peroxidase with diaminobenzidine as chromogen and hematoxylin as the counterstain. As a measure of proliferation, the Ki-67 labeling index was determined as the ratio of labeled:total nuclei in high-power (3400) fields. Approximately 2000 nuclei were counted in each case by systematic random sampling. For macrophage and NK cell staining, frozen sections, fixed with buffered 4% paraformaldehyde solution, were immunostained using biotinylated mAb F4/80 (Serotec, Raleigh, N.C.) and polyclonal rabbit anti-asialo GM1 antibody (Dako Chemicals, Richmond, Va.), respectively. Angiogenesis was quantitated as vessel area using computerized analysis. For this purpose, sections were immunostained using anti-CD31 and were analyzed using a computerized image analysis system without counterstain. MVAs were determined by capturing digital images of the sections at 3200 magnification using a CCD color camera as described previously (27). Images were then analyzed using Image Pro® Plus version 4.0 software (Media Cybernetics, Silver Spring, Md.) and MVA was determined by measuring the total amount of staining in each section. Four fields were evaluated for each slide. This value was represented as a percentage of the total area in each field. Results were confirmed in each experiment by at least two observers (K. M., H-J. S. H.).

TUNEL Assay.

Apoptotic cells in tumor tissue were detected by using the TUNEL method as described previously (27). TUNEL-positive cells were counted at ×400. The apoptotic index was calculated as a ratio of apoptotic cell number:total cell number in each field.

Statistical Analysis.

The data were analyzed for significance by Student's t test, except for the in vivo survival assays, which were analyzed by Wilcoxon analysis.

Results

Systemic Treatment of mAb 806 Extends the Survival of Mice Bearing ΔEGFR-Overexpressing Intracranial Glioma Tumors.

To test the efficacy of the anti-ΔEGFR mAb, mAb 806, we treated nude mice bearing intracranial ΔEGFR-overexpressing glioma xenografts with i.p. injections of mAb 806, the isotype control IgG, or PBS. U87MG.ΔEGFR cells were implanted intracranially into nude mice, and the treatments began on the same day as described in “Materials and Methods.”

Animals treated with PBS or isotype control IgG had a median survival of 13 days, whereas mice treated with mAb 806 had a 61.5% increase in median survival up to 21 days (P<0.001; FIG. 24A). Treatment of mice 3 days postimplantation, after tumor establishment, also extended the median survival of the mAb 806-treated animals by 46.1% (from 13 days to 19 days; P<0.01) compared with that of the control groups (data not shown). To determine whether these antitumor effects of mAb 806 extended beyond U87MG.ΔEGFR xenografts, we also did similar treatments of animals bearing other glioma cell xenografts of LN-Z308.ΔEGFR and A1207.ΔEGFR. The median survival of mAb 806-treated mice bearing LN-Z308.ΔEGFR xenografts was extended from 19 days for controls to 58 days (P<0.001; FIG. 24B). Remarkably, four of eight mAb 806-treated animals survived beyond 60 days (FIG. 24B). The median survival of animals bearing A1207.ΔEGFR xenografts was also extended from 24 days for controls to 29 days (P<0.01; data not shown).

mAb 806 Treatment Inhibits ΔEGFR-Overexpressing Brain Tumor Growth.

Mice bearing U87MG.ΔEGFR and LN-Z308.ΔEGFR xenografts were killed at day 9 and day 15, respectively. Tumor sections were histopathologically analyzed, and tumor volumes were determined as described in “Materials and Methods.” Consistent with the results observed for animal survival, mAb 806 treatment significantly reduced the volumes of U87MG.ΔEGFR by 90% (P<0.001; FIG. 24C), and of LN-Z308.ΔEGFR by 0.95% (P<0.001; FIG. 24D), of xenografts in comparison with those of the control groups. Similar results were obtained for animals bearing A1207.ΔEGFR tumors (65% volume reduction; P<0.01; data not shown).

Intratumoral Treatment with mAb 806 Extends Survival of Mice Bearing U87 MG.ΔEGFR Brain Tumors.

We also determined the efficacy of direct intratumoral injection of mAb 806 for the treatment of U87MG.ΔEGFR xenografts. Animals were given intratumoral injections of mAb 806 or isotype control IgG at 1 day post implantation, as described in “Materials and Methods.” Control animals survived for 15 days, whereas mAb 806 treated mice remained alive for 18 days (P<0.01; FIG. 24E). Although the intratumoral treatment with mAb 806 was somewhat effective, it entailed the difficulties of multiple intracranial injections and of increased risk of infection. We, therefore, focused on systemic treatments for additional studies.

mAb 806 Treatment Slightly Extends Survival of Mice Bearing U87 MG.wtEGFR but not of Mice Bearing U87 MG or U87 MG.DK Intracranial Xenografts.

To determine whether the growth inhibition by mAb 806 was selective for tumors expressing ΔEGFR, we treated animals bearing U87MG, U87MG.DK (kinase-deficient ΔEGFR) or U87MG.wtEGFR brain xenografts. mAb 806 treatment did not extend the survival of mice implanted with U87 mg tumors (FIG. 25A), which expressed a low level of endogenous wtEGFR (22), or of animals bearing U87MG.DK xenografts, which overexpressed a kinase-deficient ΔEGFR in addition to a low level of endogenous wtEGFR (FIG. 25B). The mAb 806 treatment slightly extended the survival of mice bearing U87MG.wtEGFR tumors (P<0.05; median survival, 23 days versus 26 days for the control groups), which overexpressed wtEGFR (FIG. 25C).

mAb 806 Reactivity Correlates with in Vivo Antitumor Efficacy.

To understand the differential effect of mAb 806 on tumors expressing various levels or different types of EGFR, we determined mAb 806 reactivity with various tumor cells by FACS analysis. Consistent with previous reports (21), the anti-EGFR mAb 528 recognized both ΔEGFR and wtEGFR and demonstrated stronger staining for U87MG.ΔEGFR cells compared with U87MG cells (FIG. 26A, 528). In contrast, antibody EGFR.1 reacted with wtEGFR but not with ΔEGFR (21), because U87MG.ΔEGFR cells were as weakly reactive as U87MG cells (FIG. 26A, panel EGFR.1). This EGFR.1 antibody reacted with U87MG.wt.EGFR more intensively than with U87MG cells, because U87MG.wt.EGFR cells overexpressed wtEGFR (FIG. 26A, panel EGFR.1). Although mAb 806 reacted intensely with U87MG.ΔEGFR and U87MG.DK cells and not with U87MG cells, it reacted weakly U87MG.wtEGFR, which indicated that mAb 806 is selective for ΔEGFR with a weak cross-activity to overexpressed wtEGFR (FIG. 26A, panel mAb 806). This level of reactivity with U87MG.wtEGFR was quantitatively and qualitatively similar to the extension of survival mediated by the antibody treatment (FIG. 25C).

We further determined mAb 806 specificity by immunoprecipitation. EGFRs in various cell lines were immunoprecipitated with antibody 528, EGFR.1, and mAb 806. Blots of electrophoretically separated proteins were then probed with the anti-EGFR antibody, C13, which recognizes wtEGFR as well as ΔEGFR and DK (22). Consistent with the FACS analysis, antibody 528 recognized wtEGFR and mutant receptors (FIG. 26B-panel IP: 528), whereas antibody EGFR.1 reacted with wtEGFR but not with the mutant species (FIG. 26B, panel IP: EGFR.1). Moreover, the levels of mutant receptors in U87MG.ΔEGFR and U87MG.DK cells are comparable with those of wtEGFR in the U87MG.wtEGFR cells (FIG. 26B, panel IP: 528).

However, antibody mAb 806 was able to precipitate only a small amount of the wtEGFR from the U87MG.wtEGFR cell lysates as compared with the larger amount of mutant receptor precipitated from U87MG.ΔEGFR and U87MG.DK cells, and an undetectable amount from the U87MG cells (FIG. 26B, panel IP: mAb 806). Collectively, these data suggest that mAb 806 recognizes an epitope in ΔEGFR that also exists in a small fraction of wtEGFR only when it is overexpressed on the cell surface.

mAb 806 Treatment Reduces ΔEGFR Autophosphorylation and Down-Regulates Bcl-XL Expression in U87MG.ΔEGFR Brain Tumors.

The mechanisms underlying the growth inhibition by mAb 806 were next investigated. Because the constitutively active kinase activity and autophosphorylation of the COOH terminus of ΔEGFR are essential for its biological functions (21, 22, 28, 29), ΔEGFR phosphorylation status was determined in tumors from treated and control animals. As shown in FIG. 27A, mAb 806 treatment dramatically reduced ΔEGFR autophosphorylation, although receptor levels were only slightly decreased in the mAb 806-treated xenografts. We have previously shown that receptor autophosphorylation causes up-regulation of the antiapoptotic gene, Bcl-XL, which plays a key role in reducing apoptosis of ΔEGFR-overexpressing tumors (28, 29). Therefore, the effect of mAb 806 treatment on Bcl-XL expression was next determined ΔEGFR tumors from mAb 806-treated animals did indeed show reduced levels of Bcl-XL (FIG. 27A).

mAb 806 Treatment Decreases Growth and Angiogenesis and Increases Apoptosis in U87 MG.ΔEGFR Tumors.

In light of the in vivo suppression caused by mAb 806 treatment and its biochemical effects on receptor signaling, we determined the proliferation rate of tumors from control or treated mice. The proliferative index, measured by Ki-67 staining of the mAb 806-treated tumors, was significantly lower than that of the control tumors (P<0.001; FIG. 28). In addition, analysis of the apoptotic index through TUNEL staining demonstrated a significant increase in the number of apoptotic cells in mAb 806-treated tumors as compared with the control tumors (P<0.001; FIG. 28). The extent of tumor vascularization was also analyzed by immunostaining of tumors from treated and control specimens for CD31. To quantify tumor vascularization, MVAs were measured using computerized image analysis. mAb 806-treated tumors showed 30% less MVA than did control tumors (P<0.001; FIG. 28). To understand whether interaction between receptor and antibody may elicit an inflammatory response, we stained tumor sections for the macrophage marker, F4/80, and the NK cell marker, asialo GM1. Macrophages were identified throughout the tumor matrix and especially accumulated around the mAb 806-TREATED-U87MG.ΔEGFR-tumor periphery (FIG. 28). We observed few NK cells infiltrated in and around the tumors and no significant difference between mAb 806-treated and isotype-control tumors (data not shown).

Discussion

ΔEGFR appears to be an attractive potential therapeutic target for cancer treatment of gliomas. It is correlated with poor prognosis (25), whereas its genetic or pharmacological inhibition effectively suppresses growth of ΔEGFR-overexpressing cells both in vitro and in vivo (29, 30). Because this mutant EGFR is expressed on the cell surface, it represents a potential target for antibody-based therapy, and, here, we tested the efficacy of a novel anti-ΔEGFR mAb, mAb 806, on the treatment of intracranial xenografts of ΔEGFR-overexpressing gliomas of different cellular backgrounds in nude mice. The systemic administration of mAb 806 inhibited tumor growth and extended animal survival. The effect of mAb 806 was evident for each cell line and was independent of the p53 status of the tumors, because U87MG.ΔEGFR and A1207.ΔEGFR expressed wild-type p53, whereas LN-Z308.ΔEGFR was p53-null.

The enhanced tumorigenicity of ΔEGFR is mediated through its constitutively active kinase activity and tyrosine autophosphorylation at the COOH terminus (22, 28, 29). Phosphorylation of ΔEGFR in mAb 806-treated tumors was significantly decreased, proliferation was reduced, and apoptosis was elevated, which suggests that the antitumor effect of mAb 806 is, at least in part, attributable to the inhibition of the intrinsic function of the receptor. The ΔEGFR signaling caused up-regulation of the antiapoptotic gene, Bcl-XL (28), and treatment with mAb 806 resulted in down-regulation of Bcl-XL expression, which further suggests that the antitumor effect of mAb 806 is mediated through the inhibition of ΔEGFR signaling. The level of ΔEGFR in the mAb 806-treated tumors was also slightly reduced (FIG. 27A), but not to a degree that was consistent with the degree of dephosphorylation of the mutant receptor or sufficient to explain the magnitude of its biological effect. The antitumor effect of mAb 806 is likely to result, at least in part, from the inhibition of the intrinsic signaling function of ΔEGFR. This assertion is also supported by the lack of antitumor effects on DK tumors, which bind to the antibody but are kinase deficient.

Intratumoral injection of a different anti-ΔEGFR antibody, mAbY10, inhibited the growth of ΔEGFR-expressing B16 melanoma tumors in mouse brains through a Fc/Fc receptor-dependent mechanism (31). In conjunction with this, mAbY10 was shown to mediate antibody-dependent macrophage cytotoxicity in vitro with both murine and human effector cells (17), although it had little effect with macro-phage infiltration found in our mAb 806-treated tumors raises the question as to whether the antitumor effect of mAb 806 may be accomplished by macrophage-mediated cytotoxicity. We believe this to be unlikely, because macrophage infiltration also occurred on mAb 806 treatment of U87MG.DK (kinase-deficient ΔEGFR) tumors, in which it was ineffective in regulating tumor growth.

mAb 806 appears to be selective for ΔEGFR with a weak cross-reactivity with overexpressed wtEGFR. Consistent with the in vitro specificity, mAb 806 treatment was very effective in ΔEGFR-over-expressed tumors, whereas it showed a much less robust, but reproducible, growth inhibition for tumors overexpressing wtEGFR. However, the simple interaction between mAb 806 and its target molecules is insufficient to inhibit tumor growth because, although mAb 806 is capable of binding equally well to kinase-deficient ΔEGFR (DK) receptors and ΔEGFR, it is ineffective in affecting DK-expressing tumor growth. The inability of mAb 806 to interact with the low-level of wtEGFR normally present in cells suggests a large therapeutic window for ΔEGFR-overexpressed as well as, to a lesser extent, wtEGFR-overexpressed cancers when compared with normal tissues.

Although the mAb 806 treatment was effective for suppression of intracranial xenografts, it should be noted that the ΔEGFR-tumors eventually grew, and durable remissions were not achieved. This may have resulted from inefficient distribution of antibody in the tumor mass. mAbs in combination with other therapeutic modalities such as toxins, isotopes or drugs, for cancer treatments have been shown to be more effective than antibody alone in many cases (2, 3, 32-34). Chemotherapeutic drugs such as doxorubicin and cisplatin in conjunction with wtEGFR antibodies have also shown enhanced antitumor activity (35, 36). Combination treatments targeted at tumor growth as well as angiogenic development have more effectively inhibited glioblastoma growth than either treatment alone (27). This raises the possibility that mAb 806 in combination with chemotherapeutic drugs or compounds modulating angiogenesis may be even more effective than mAb 806 alone.

References

  • 1. Old, L. J. Immunotherapy for cancer. Sci. Am., 275: 102-109, 1996.
  • 2. Weiner, L. M. An overview of monoclonal antibody therapy of cancer. Semin. Oncol., 26 (Suppl. 12): 41-50, 1999.
  • 3. Green, M. C., Murray, J. L., and Hortobagyi, G. N. Monoclonal antibody therapy for solid tumors. Cancer Treat. Rev., 26: 269-286, 2000.
  • 4. Ashley, D. M., Batra, S. K., and Bigner, D. D. Monoclonal antibodies to growth factors and growth factor receptors: their diagnostic and therapeutic potential in brain tumors. J. Neurooncol., 35: 259-273, 1997.
  • 5. Fan, Z., and Mendelsohn, J. Therapeutic application of anti-growth factor receptor antibodies. Curr. Opin. Oncol., 10: 67-73, 1998.
  • 6. Slamon, D. J., Godolphin, W., Jones, L. A., Holt, J. A., Wong, S. G., Keith, D. E., Levin, W. J., Stuart, S. G., Udove, J., Ullrich, A., and Press, M. F. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science (Wash. D.C.), 244: 707-712, 1989.
  • 7. Harari, D., and Yarden, Y. Molecular mechanisms underlying ErbB2/HER2 action in breast cancer. Oncogene, 19: 6102-6114, 2000.
  • 8. Sliwkowski, M. X., Lofgren, J. A., Lewis, G. D., Hotaling, T. E., Fendly, B. M., and Fox, J. A. Nonclinical studies addressing the mechanism of action of trastuzumab (Herceptin). Semin. Oncol., 26 (Suppl. 12): 60-70, 1999.
  • 9. Baselga, J., Tripathy, D., Mendelsohn, J., Baughman, S., Benz, C. C, Dantis, L., Sklarin, N. T., Seidman, A. D., Hudis, C. A., Moore, J., Rosen, P. P., Twaddell, T., Henderson, I. C., and Norton, L. Phase II study of weekly intravenous recombinant humanized anti-p185HER2 monoclonal antibody in patients with HER2/neu-overexpressing metastatic breast cancer. J. Clin. Oncol., 14: 737-744, 1996.
  • 10. Baselga, J. Clinical trials of Herceptin® (trastuzumab). Eur. J. Cancer, 37: 18-24, 2001.
  • 11. Salomon, D. S., Brandt, R., Ciardiello, F., and Normanno, N. Epidermal growth factor-related peptides and their receptors in human malignancies. Crit. Rev. Oncol. Hematol., 19: 183-232, 1995.
  • 12. Mendelsohn, J. Epidermal growth factor receptor inhibition by a monoclonal antibody as anticancer therapy. Clin. Cancer Res., 3: 2703-2707, 1997.
  • 13. Waksal, H. W. Role of an anti-epidermal growth factor receptor in treating cancer. Cancer Metastasis Rev., 18: 427-436, 1999.
  • 14. Faillot, T., Magdelenat, H., Mady, E., Stasiecki, P., Fohanno, D., Gropp, P., Poisson, M., and Delattre, J. Y. A Phase I study of an anti-epidermal growth factor receptor monoclonal antibody for the treatment of malignant gliomas. Neurosurgery (Baltimore), 39: 478-483, 1996.
  • 15. Wikstrand, C. J., Reist, C. J., Archer, G. E., Zalutsky, M. R., and Bigner, D. D. The class III variant of the epidermal growth factor receptor (EGFRvIII): characterization and utilization as an immunotherapeutic target. J. Neurovirol., 4: 148-158, 1998.
  • 16. Wong, A. J., Ruppert, J. M., Bigner, S. H., Grzeschik, C. H., Humphrey, P. A., Bigner, D. S., and Vogelstein, B. Structural alterations of the epidermal growth factor receptor gene in human gliomas. Proc. Natl. Acad. Sci. USA, 89: 2965-2969, 1992.
  • 17. Wikstrand, C. J., Cokgor, I., Sampson, J. H., and Bigner, D. D. Monoclonal antibody therapy of human gliomas: current status and future approaches. Cancer Metastasis Rev., 18: 451-464, 1999.
  • 18. Garcia de Palazzo, I. E., Adams, G. P., Sundareshan, P., Wong, A. J, Testa, J. R., Bigner, D. D., and Weiner, L. M. Expression of mutated epidermal growth factor receptor by non-small cell lung carcinomas. Cancer Res., 53: 3217-3220, 1993.
  • 19. Moscatello, D. K., Holgado-Madruga, M., Godwin, A. K., Ramirez, G., Gunn, G., Zoltick, P. W., Biegel, J. A., Hayes, R. L., and Wong, A. J. Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors. Cancer Res., 55: 5536-5539, 1995.
  • 20. Olapade-Olaopa, E. O., Moscatello, D. K., MacKay, E. H., Horsburgh, T., Sandhu, D. P., Terry, T. R., Wong, A. J., and Habib, F. K. Evidence for the differential expression of a variant EGF receptor protein in human prostate cancer. Br. J. Cancer, 82: 186-194, 2000.
  • 21. Nishikawa, R., Ji, X. D., Harmon, R. C., Lazar, C. S, Gill, G. N., Cavenee, W. K., and Huang, H. J. A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc. Natl. Acad. Sci. USA, 91: 7727-7731, 1994.
  • 22. Huang, H-J. S., Nagane, M., Klingbeil, C. K., Lin, H., Nishikawa, R., Ji, X. D., Huang, C. M., Gill, G. N., Wiley, H. S., and Cavenee, W. K. The enhanced tumorigenic activity of a mutant epidermal growth factor receptor common in human cancers is mediated by threshold levels of constitutive tyrosine phosphorylation and unattenuated signaling. J. Biol. Chem., 272: 2927-2935, 1997.
  • 23. Mishima, K. Nagane, M., Lin, H., Cavenee, W. K., and Huang, H-J. S. Expression of a tumor-specific mutant epidermal growth factor receptor mediates glioma cell invasion in vivo. Proc. Am. Assoc. Cancer Res., 40: 519, 1999.
  • 24. Han, Y., Caday, C. G., Nanda, A., Cavenee, W. K., and Huang, H. J. Tyrphostin AG1478 preferentially inhibits human glioma cells expressing truncated rather than wild-type epidermal growth factor receptors. Cancer Res., 56: 3859-3861, 1996.
  • 25. Feldkamp, M. M., Lala, P., Lau, N., Roncari, L., and Guha, A. Expression of activated epidermal growth factor receptors, Ras-guanosine triphosphate, and mitogen-activated protein kinase in human glioblastoma multiforme specimens. Neurosurgery (Baltimore), 45: 1442-1453, 1999.
  • 26. Nagane, M., Levitzki, A., Gazit, A., Cavenee, W. K., and Huang, H-J. S. Drug resistance of human glioblastoma cells conferred by a tumor-specific mutant epidermal growth factor receptor through modulation of Bcl-XL and caspase-3-like proteases. Proc. Natl. Acad. Sci. USA, 95: 5724-5729, 1998.
  • 27. Mishima, K., Mazar, A. P., Gown, A., Skelly, M., Ji, X. D., Wang, X. D., Jones, T. R., Cavenee, W. K., and Huang, H-J. S. A peptide derived from the non-receptor-binding region of urokinase plasminogen activator inhibits glioblastoma growth and angiogenesis in vivo in combination with cisplatin. Proc. Natl. Acad. Sci. USA, 97: 8484-8489, 2000.
  • 28. Nagane, M., Coufal, F., Lin, H., Bogler, O., Cavenee, W. K., and Huang, H-J. S. A common mutant epidermal growth factor receptor confers enhanced tumorigenicity on human glioblastoma cells by increasing proliferation and reducing apoptosis. Cancer Res., 56: 5079-5086, 1996.
  • 29. Nagane, M., Lin, H., Cavenee, W. K., and Huang, H-J. S. Aberrant receptor signaling in human malignant gliomas: mechanisms and therapeutic implications. Cancer Lett., 162 (Suppl. 1): S17-S21, 2001.
  • 30. Halatsch, M. E., Schmidt, U., Botefur, I. C., Holland, J. F., and Ohnuma, T. Marked inhibition of glioblastoma target cell tumorigenicity in vitro by retrovirus-mediated transfer of a hairpin ribozyme against deletion-mutant epidermal growth factor receptor messenger RNA. J. Neurosurg., 92: 297-305, 2000.
  • 31. Sampson, J. H, Crotty, L. E., Lee, S., Archer, G. E., Ashley, D. M., Wikstrand, C. J., Hale, L. P., Small, C., Dranoff, G., Friedman, A. H., Friedman, H. S., and Bigner, D. D. Unarmed, tumor-specific monoclonal antibody effectively treats brain tumors. Proc. Natl. Acad. Sci. USA, 97: 7503-7508, 2000.
  • 32. Trail, P. A, and Bianchi, A. B. Monoclonal antibody drug conjugates in the treatment of cancer. Curr. Opin. Immunol., 11: 584-588, 1999.
  • 33. Pietras, R. J., Pegram, M. D., Finn, R. S., Maneval, D. A., and Slamon, D. J. Remission of human breast cancer xenografts on therapy with humanized monoclonal antibody to HER-2 receptor and DNA-reactive drugs. Oncogene, 17: 2235-2249, 1998.
  • 34. Baselga, J., Norton, L., Albanell, J., Kim, Y. M., and Mendelsohn, J. Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of pacli-taxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts. Cancer Res., 58: 2825-2831, 1998.
  • 35. Baselga, J., Norton, L., Masui, H., Pandiella, A., Coplan, K., Miller, W. H., and Mendelsohn, J. Antitumor effects of doxorubicin in combination with anti-epidermal growth factor receptor monoclonal antibodies. J. Natl. Cancer Inst. (Bethesda), 85: 1327-1333, 1993.
  • 36. Fan, Baselga, J., Masui, H., and Mendelsohn, J. Antitumor effect of anti-epidermal growth factor receptor monoclonal antibodies plus cis-diamminedichloroplatinum on well established A431 cell xenografts. Cancer Res., 53: 4637-4642, 1993.

EXAMPLE 20 Monoclonal Antibody 806 Inhibits the Growth of Tumor Xenografts Expressing Either the De2-7 or Amplified Epidermal Growth Factor Receptor (EGFR) but not Wild-Type EGFR

The following example presents findings by the present inventors that is also set forth in Luwor et al., (2001) Cancer Research, 61:5355-5361. The disclosure of this publication is incorporated herein in its entirety with cross referencing to the Figures herein where appropriate. and made a part hereof.

The monoclonal antibody (mAb) 806 was raised against the delta2-7 epidermal growth factor receptor (de2-7 EGFR or EGFRvIII), a truncated version of the EGFR commonly expressed in glioma. Unexpectedly, mAb 806 also bound the EGFR expressed by cells exhibiting amplification of the EGFR gene but not to cells or normal tissue expressing the wild-type receptor in the absence of gene amplification. The unique specificity of mAb 806 offers an advantage over current EGFR antibodies, which all display significant binding to the liver and skin in humans. Therefore, we examined the antitumor activity of mAb 806 against human tumor xenografts grown in nude mice. The growth of U87MG xenografts, a glioma cell line that endogenously expresses ˜105 EGFRs in the absence of gene amplification, was not inhibited by mAb 806. In contrast, mAb 806 significantly inhibited the growth of U87MG xenografts transfected with the de2-7 EGFR in a dose-dependent manner using both preventative and established tumor models. Significantly, U87MG cells transfected with the wild-type EGFR, which increased expression to ˜106 EGFRs/cell and mimics the situation of gene amplification, were also inhibited by mAb 806 when grown as xenografts in nude mice. Xenografts treated with mAb 806 all displayed large areas of necrosis that were absent in control tumors. This reduced xenograft viability was not mediated by receptor down-regulation or clonal selection because levels of antigen expression were similar in control and treated groups. The antitumor effect of mAb 806 was not restricted to U87MG cells because the antibody inhibited the growth of new and established A431 xenografts, a cell line expressing >106 EGFRs/cell. This study demonstrates that mAb 806 possesses significant antitumor activity.

The de2-7 EGFR specific mAb 806 was produced after immunization of mice with NR6 mouse fibroblasts expressing the truncated de2-7 EGFR. mAb 806 binds the U87MG glioma cell line transfected with the de2-7 EGFR but not the parental U87MG cell line, which expresses the wt EGFR without gene amplification.3 Similar results were observed in vivo with mAb 806 showing specific targeting of de2-7 EGFR expressing U87MG xenografts but not parental U87MG tumors.3 Interestingly, mAb 806 was capable of binding an EGFR subset (−10%) on the surface of the A431 cell line, which contains an amplified EGFR gene. Therefore, unlike all other de2-7 EGFR-specific antibodies, which recognize the unique peptide junction that is generated by the de2-7 EGFR truncation, mAb 806 binds to an epitope also found in overexpressed wt EGFR. However, it would appear that this epitope is preferentially exposed in the de2-7 EGFR and a small proportion of receptors expressed in cells containing wt EGFR gene amplification. Importantly, normal tissues that expresses high levels of endogenous wt EFGR, such as liver and skin, show no significant mAb 806 binding. On the basis of the unique property of the mAb 806 to bind both the de2-7 and amplified wt EGFR but not the native wt EGFR when expressed at normal levels, we decided to examine the efficacy of mAb 806 against several tumor cell lines grown as xenografts in nude mice.

Materials and Methods

Cell Lines and Monoclonal Antibodies.

The human glioblastoma cell line U87MG, which endogenously expresses the wt EGFR, and the transfected cell lines U87MG.Δ2-7 and U87MG.wtEGFR, which express the de2-7 EGFR and overexpress the wt EGFR, respectively, have been described previously (16, 23). The epidermoid carcinoma cell line A431 has been described previously (24).

All cell lines were maintained in DMEM (DMEM/F12; Life Technologies, Inc., Grand Island, N.Y.) containing 10% FCS (CSL, Melbourne, Victoria, Australia), 2 mM glutamine (Sigma Chemical Co., St. Louis, Mo.), and penicillin/streptomycin (Life Technologies, Inc., Grand Island, N.Y.). In addition, the U87MG.Δ2-7 and U87MG.wtEGFR cell lines were maintained in 400 mg/ml of GENETICIN™ (Life Technologies, Inc., Melbourne, Victoria, Australia). Cell lines were grown at 37° C. in a humidifier atmosphere of 5% CO2. The mAb 806 (IgG2b) was produced after immunization of mice with NR6 mouse fibroblasts expressing the de2-7 EGFR. mAb 806 was selected after rosette assays showed binding to NR6 cells, which overexpressed the de2-7 EGFR (titer of 1:2500). mAb 528, which recognizes both de2-7 and wt EGFR, has been described previously (10) and was produced in the Biological Production Facility (Ludwig Institute for Cancer Research, Melbourne, Victoria, Australia) using a hybridoma obtained from American Type Culture Collection (Rockville, Md.). The DH8.3 mAb, which is specific for the de2-7 EGFR, was kindly provided by Prof. William Gullick (University of Kent and Canterbury, Kent, United Kingdom) (19). The polyclonal antibody sc-03 directed to the COOH-terminal domain of the EGFR was purchased from Santa Cruz Bio-technology (Santa Cruz Biotechnology, Santa Cruz, Calif.).

FACS Analysis of Receptor Expression.

Cultured parental and trans-fected U87MG cell lines were analyzed for wt and de2-7 EGFR expression using the 528, 806, and DH8.3 antibodies. Cells (1 3 10 6) were incubated with 5 mg/ml of the appropriate antibody or an isotype-matched negative control in PBS containing 1% HSA for 30 min at 4° C. After three washes with PBS/1% HSA, cells were incubated an additional 30 min at 4° C. with FITC-coupled goat antimouse antibody (1:100 dilution; Calbiochem, San Diego, Calif.). After three subsequent washes, cells were analyzed on an Epics Elite™ ESP (Beckman Coulter, Hialeah, Fla.) by observing a minimum of 20,000 events and analyzed using EXPO™ (version 2) for Windows.

Scatchard Analysis.

The mAb 806 was labeled with 125I (Amrad, Melbourne, Victoria, Australia) by the Chloramine T method. All binding assays were performed in 1% HSA/PBS on 1−2×106 live U87MG.Δ2-7 or A431 cells for 90 min at 4° C. with gentle rotation. A set concentration of 10 ng/ml 125I-labeled mAb 806 was used in the presence of increasing concentrations of unlabeled antibody. Nonspecific binding was determined in the presence of 10,000-fold excess of unlabeled antibody. After incubation, cells were washed and counted for bound 125I-labeled mAb 806 using a COBRA II gamma counter (Packard Instrument Company, Meriden, Conn.). Scatchard analysis was done after correction for immunoreactivity.

Immunoprecipitation Studies.

Cells were labeled for 16 h with 100 mCi/ml of Tran 35 S-Label (ICN Biomedicals, Irvine, Calif.) in DMEM without methionine/cysteine supplemented with 5% dialyzed FCS. After washing with PBS, cells were placed in lysis buffer (1% Triton X-100, 30 mM HEPES, 150 mM NaCl, 500 mM 4-(2-aminoethyl)benzenesulfonylfluoride, 150 nM aprotinin, 1 mM E-64 protease inhibitor, 0.5 mM EDTA, and 1 mM leupeptin, pH 7.4) for 1 h at 4° C. Lysates were clarified by centrifugation for 10 min at 12,000 3 g and then incubated with 5 mg of appropriate antibody for 30 min at 4° C. before the addition of protein A-Sepharose™ Immunoprecipitates were washed three times with lysis buffer, mixed with SDS sample buffer, separated by gel electrophoresis using a 7.5% gel that was then dried, and exposed to X-ray film.

Xenograft Models.

Consistent with previous reports (23, 25), U87MG cells transfected with de2-7 EGFR grew more rapidly then parental cells and U87MG cells transfected with the wt EGFR. Tumor cells (3×106) in 100 ml of PBS were inoculated s.c. into both flanks of 4-6-week-old, female nude mice (Animal Research Center, Western Australia, Perth, Australia). Therapeutic efficacy of mAb 806 was investigated in both preventative and established tumor models. In the preventative model, five mice with two xenografts each were treated i.p. with either 0.1 or 1 mg of mAb 806 or vehicle (PBS) starting the day before tumor cell inoculation. Treatment was continued for a total of six doses, three times per week for 2 weeks. In the established model, treatment was started when tumors had reached a mean volume of 65 mm 3 (U87MG.Δ2-7), 84 mm 3 (U87MG), 73 mm 3 (U87MG.wtEGFR), or 201 mm 3 (A431 tumors). Tumor volume in mm 3 was determined using the formula (length 3 width 2)/2, where length was the longest axis and width the measurement at right angles to the length (26). Data were expressed as mean tumor volume 6 SE for each treatment group. This research project was approved by the Animal Ethics Committee of the Austin and Repatriation Medical Centre.

Histological Examination of Tumor Xenografts.

Xenografts were excised at the times indicated and bisected. One half was fixed in 10% formalin/PBS before being embedded in paraffin. Four-mm sections were then cut and stained with H&E for routine histological examination. The other half was embedded in Tissue Tek® O.C.T.™ compound (Sakura Finetek, Torrance, Calif.), frozen in liquid nitrogen, and stored at 280° C. Thin (5-mm) cryostat sections were cut and fixed in ice-cold acetone for 10 min, followed by air drying for an additional 10 min. Sections were blocked in protein blocking reagent (Lipshaw Immunon, Pittsburgh, Pa.) for 10 min and then incubated with biotinylated primary antibody (1 mg/ml) for 30 min at room temperature. All antibodies were biotinylated using the ECL™ protein biotinylation module (Amersham, Baulkham Hills, NSW, Australia), as per the manufacturer's instructions. After rinsing with PBS, sections were incubated with a streptavidin-horseradish peroxidase complex for an additional 30 min (Silenus, Melbourne, Victoria, Australia). After a final PBS wash, the sections were exposed to 3-amino-9-ethylcarbazole substrate [0.1 M acetic acid, 0.1 M sodium acetate, 0.02 M 3-amino-9-ethylcarbazole (Sigma Chemical Co., St. Louis, Mo.)] in the presence of hydrogen peroxide for 30 min. Sections were rinsed with water and counterstained with hematoxylin for 5 min and mounted.

Statistical Analysis.

The in vivo tumor measurements in mm 3 are ex-pressed as the mean 6 SE. Differences between treatment groups at given time points were tested for statistical significance using Student's t test.

Results

Binding of Antibodies to Cell Lines.

To determine the specificity of mAb 806, its binding to U87MG, U87MG.Δ2-7, and U87MG.wtEGFR cells was analyzed by FACS. An irrelevant IgG2b (mAb 100-310 directed to the human antigen A33) was included as an isotype control for mAb 806, and the 528 antibody was included because it recognizes both the de2-7 and wt EGFR. Only the 528 antibody was able to stain the parental U87MG cell line (FIG. 29), consistent with previous reports demonstrating that these cells express the wt EGFR (16). mAb 806 had binding levels similar to the control antibody, clearly demonstrating that it is unable to bind the wt EGFR (FIG. 29). Binding of the isotype control antibody to the U87MG.Δ2-7 and U87MG.wtEGFR cell lines was similar to that observed for the U87 MG cells. mAb 806 stained U87 MG.D2-7 and U87 MG. wtEGFR cells, indicating that mAb 806 specifically recognized the de2-7 EGFR and a subset of the overexpressed EGFR (FIG. 29). As expected, the 528 antibody stained both the U87MG.Δ2-7 and U87MG.wtEGFR cell lines (FIG. 29). The intensity of 528 antibody staining on U87MG.wtEGFR cells was much higher than mAb 806, suggesting that mAb 806 only recognizes a portion of the overexpressed EGFR. The mAb 806 reactivity observed with U87MG.wtEGFR cells is similar to that obtained with A431 cells, another cell line that over expresses the wt EGFR.3

A Scatchard analysis was performed using U87MG.Δ2-7 and A431 cells to determine the relative affinity and binding sites for mAb 806 on each cell line. mAb 806 had an affinity for the de2-7 EGFR receptor of 1.1×109M−1 and recognized an average (three separate experiments) of 2.4×105 binding sites/cell. In contrast, the affinity of mAb 806 for the wt EGFR on A431 cells was only 9.5×107 M−1. Interestingly, mAb 806 recognized 2.3×105 binding sites on the surface of A431, which is some 10-fold lower than the reported number of EGFR found in these cells. To confirm the number of EGFR on the surface of our A431 cells, we performed a Scatchard analysis using 125I-labeled 528 antibody. As expected, this antibody bound to approximately 2×106 sites on the surface of A431 cells. Thus, it appears that mAb 806 only binds a portion of the EGFR receptors on the surface of A431 cells. Importantly, 125I-labeled mAb 806 did not bind to the parental U87 MG cells at all, even when the number of cells was increased to 1×107.

Immunoprecipitations.

We further characterized mAb 806 reactivity in the various cell lines by immunoprecipitation after 35 S-labeling using mAb 806, sc-03 (a commercial polyclonal antibody specific for the COOH-terminal domain of the EGFR) and a IgG2b isotype control. The sc-03 antibody immunoprecipitated three bands from U87MG.Δ2-7 cells, a doublet corresponding to the two de2-7 EGFR bands observed in these cells and a higher molecular weight band corresponding to the wt EGFR (FIG. 30). In contrast, although mAb 806 immunoprecipitated the two de2-7 EGFR bands, the wt EGFR was completely absent (FIG. 30). The pattern seen in U87MG.wtEGFR and A431 cells was essentially identical. The sc-03 antibody immunoprecipitated a single band corresponding to the wt EGFR from both cell lines (FIG. 30). The mAb 806 also immunoprecipitated a single band corresponding to the wt EGFR from both U87MG.wtEGFR and A431 cells (FIG. 30). Consistent with the FACS and Scatchard data, the amount of EGFR immunoprecipitated by mAb 806 was substantially less than the total EGFR present on the cell surface. Given that mAb 806 and the sc-03 immunoprecipitated similar amounts of the de2-7 EGFR, this result supports the notion that the mAb 806 antibody only recognizes a portion of the EGFR in cells overexpressing the receptor. Comparisons between mAb 806 and the 528 antibody showed an identical pattern of reactivity (data not shown). An irrelevant IgG2b (an isotype control for mAb 806) did not immunoprecipitate EGFR from any of the cell lines (FIG. 30). Using identical conditions, mAb 806 did not immunoprecipitate the EGFR from the parental U87MG cells (data not shown).

Efficacy of mAb 806 in Preventative Models.

mAb 806 was examined for efficacy against U87MG and U87MG.Δ2-7 tumors in a preventative xenograft model. Antibody or vehicle was administered i.p. the day before tumor inoculation and was given three times per week for 2 weeks (see “Materials and Methods”). At a dose of 1 mg/injection, mAb 806 had no effect on the growth of parental U87MG xenografts that express the wt EGFR (FIG. 9A). In contrast, mAb 806 inhibited significantly the growth of U87MG.Δ2-7 xenografts in a dose-dependent manner (FIG. 9B). Twenty days after tumor inocu-lation, when control animals were sacrificed, the mean tumor volume was 1600±180 mm3 for the control group, a significantly smaller 500±95 mm3 for the 0.1 mg/injection group (P<0.0001) and 200±42 mm3 for the 1 mg/injection group (P<0.0001). Treatment groups were sacrificed at day 24, at which time the mean tumor volumes were 1300±240 mm 3 for the 0.1 mg treated group and 500±100 mm3 for the 1 mg group (P<0.005).

Efficacy of mAb 806 in Established Xenograft Models.

Given the efficacy of mAb 806 in the preventative xenograft model, its ability to inhibit the growth of established tumor xenografts was examined Antibody treatment was as described in the preventative model, except that it commenced when tumors had reached a mean tumor volume of 65 mm3 (10 days after implantation) for the U87MG.Δ2-7 xenografts and 84 mm3 (19 days after implantation) for the parental U87MG xenografts. Once again, mAb 806 had no effect on the growth of parental U87MG xenografts, even at a dose of 1 mg/injection (FIG. 10A). In contrast, mAb 806 significantly inhibited the growth of U87MG.Δ2-7 xenografts in a dose-dependent manner (FIG. 10B). At day 17, 1 day before control animals were sacrificed, the mean tumor volume was 900±200 mm3 for the control group, 400±60 mm3 for the 0.1 mg/injection group (P<0.01), and 220±60 mm3 for the 1 mg/injection group (P<0.002). Treatment of U87MG.Δ2-7 xenografts with an IgG2b isotype control had no effect on tumor growth (data not shown).

To examine whether the growth inhibition observed with mAb 806 was restricted to cells expressing de2-7 EGFR, its efficacy against the U87MG.wtEGFR xenografts was also examined in an established model. These cells serve as a model for tumors containing amplification of the EGFR gene without de2-7 EGFR expression. mAb 806 treatment commenced when tumors had reached a mean tumor volume of 73 mm 3 (22 days after implantation). mAb 806 significantly inhibited the growth of established U87MG.wtEGFR xenografts when compared with control tumors treated with vehicle (FIG. 10C). On the day control animals were sacrificed, the mean tumor volume was 1000±300 mm3 for the control group and 500±80 mm3 for the group treated with 1 mg/injection (P<0.04).

Histological and Immunohistochemical Analysis of Established Tumors.

To evaluate potential histological differences between mAb 806-treated and control U87MG.Δ2-7 and U87MG.wtEGFR xenografts, formalin-fixed, paraffin-embedded sections were stained with H&E (FIG. 31). Areas of necrosis were seen in sections from mAb 806-treated U87MG.Δ2-7 (mAb 806-treated xenografts were collected 24 days after tumor inoculation and vehicle treated xenografts at 18 days), and U87MG.wtEGFR xenografts (mAb 806 xenografts were collected 42 days after tumor inoculation and vehicle treated xenografts at 37 days; FIG. 31). This result was consistently observed in a number of tumor xenografts (n 5 4 for each cell line). However, sections from U87MG.Δ2-7 and U87MG.wtEGFR xenografts treated with vehicle (ii 5 5) did not display the same areas of necrosis seen after mAb 806 treatment (FIG. 31). Vehicle and mAb 806-treated xenografts removed at identical times also showed these differences in tumor necrosis (data not shown). Thus, the increase in necrosis observed was not caused by the longer growth periods used for the mAb 806-treated xenografts. Furthermore, sections from mAb 806-treated U87MG xenografts were also stained with H&E and did not reveal any areas of necrosis (data not shown), further supporting the hypothesis that mAb 806 binding induces decreased cell viability, resulting in increased necrosis within tumor xenografts.

An immunohistochemical analysis of U87MG, U87MG.Δ2-7, and U87MG.wtEGFR xenograft sections was performed to determine the levels of de2-7 and wt EGFR expression after mAb 806 treatment (FIG. 32). As expected, the 528 antibody stained all xenografts sections with no obvious decrease in intensity between treated and control tumors (FIG. 32). Staining of U87MG sections was undetectable with the mAb 806; however, positive staining of U87MG.Δ2-7 and U87MG.wtEGFR xenograft sections was observed (FIG. 32). There was no difference in mAb 806 staining intensity between control and treated U87MG.Δ2-7 and U87MG.wtEGFR xenografts, suggesting that antibody treatment does not lead to the selection of clonal variants lacking mAb 806 reactivity.

Treatment of A431 Xenografts with mAb 806.

To demonstrate that the antitumor effects of mAb 806 were not restricted to U87MG cells, the antibody was administrated to mice containing A431 xenografts. These cells contain an amplified EGFR gene and express approximately 2×106 receptors/cells. We have previously shown that mAb 806 binds ˜10% of these EGFRs and targets A431 xenografts. (3) mAb 806 significantly inhibited the growth of A431 xenografts when examined in the preventative xenograft model described previously (FIG. 11A). At day 13, when control animals were sacrificed, the mean tumor volume was 1400±150 mm3 in the vehicle-treated group and 260±60 mm3 for the 1 mg/injection treatment group (P<0.0001). In a separate experiment, a dose of 0.1 mg of mAb also inhibited significantly (P<0.05) the growth of A431 xenografts in a preventative model (data not shown).

Given the efficacy of mAb 806 in the preventative A431 xenograft model, its ability to inhibit the growth of established tumor xenografts was examined Antibody treatment was as described in the preventative model, except it was not started until tumors had reached a mean tumor volume of 200±20 mm3 mAb 806 significantly inhibited the growth of established A431 xenografts (FIG. 11B). At day 13, the day control animals were sacrificed, the mean tumor volume was 1100±100 mm3 for the control group and 450±70 mm3 for the 1 mg/injection group (P<0.0001).

We have shown previously3 that mAb 806 targets both de2-7 EGFR-transfected U87MG xenografts and A431 xenografts that over express the wt EGFR. mAb 806 did not target parental U87MG cells, which express ˜105 EGFR3 (16). As assessed by FACS, immunohistochemistry, and immunoprecipitation, we now demonstrate that mAb 806 is also able to specifically bind U87MG.wtEGFR cells, which express >106 EGFRs/cell. Thus, the previous observed binding of mAb 806 to A431 cells is not the result of some unusual property of these cells but rather appears to be a more general phenomenon related to over expression of the wt EGFR.

(a) We were unable to detect mAb 806 binding to the parental U87MGcell line, which expresses 1×105 wt EGFRs/cell (16), either by FACS, immunoprecipitation, immunohistochemistry, or with iodinated antibody. Indeed, iodinated mAb 806 did not bind to U87 MG cell pellets containing 1×107 cells, which based on the Scatchard data using 1×106 A431 cells, are conditions that should detect low level antibody binding (i.e., the total number of receptors being similar in both cases).

(b) Scatchard analysis clearly showed that mAb 806 only bound to 10% of the total EGFR on the surface of A431 cells. If mAb 806 simply binds to the wt EGFR with low affinity, then it should have bound to a considerably higher percentage of the receptor.

(c) Comparative immunoprecipitation of the A431 and U87MG. wtEGFR cell lines with mAb 806 and the sc-03 antibody also supported the hypothesis that only a subset of receptors are recognized by mAb 806. Taken together, these results support the notion that mAb 806 recognizes a EGFR subset on the surface of cells overexpressing the EGFR. We are currently analyzing the EGFR immunoprecipitated by mAb 806 to see if it displays altered biochemical properties related to glycosylation or kinase activity.

The xenograft studies with mAb 806 described here demonstrate dose-dependent inhibition of U87MG.Δ2-7 xenograft growth. In contrast, no inhibition of parental U87 MG xenografts was observed, despite the fact that they continue to express the wt EGFR in vivo. mAb 806 not only significantly reduced xenograft volume, it also induced significant necrosis within the tumor. As noted above, other de2-7 EGFR-specific mAbs have been generated (20-22), but this is the first report showing the successful therapeutic use of such an antibody in vivo against a human de2-7 EGFR-expressing glioma xenograft. A recent report demonstrated that the de2-7 EGFR-specific Y10 mAb had in vivo antitumor activity against murine B16 melanoma cells transfected with a murine homologue of the human de2-7 EGFR (33). Y10 mediated in vitro cell lysis (>90%) of B16 melanoma cells expressing the de2-7 EGFR in the absence of complement or effector cells. In contrast to their in vitro observations, the in vivo Y10 antibody efficacy was completely mediated through Fc function when using B16 melanoma cells grown as xenografts in an immunocompetent model. Thus, the direct effects observed in vitro do not seem to be replicated when cells are grown as tumor xenografts.

Overexpression of the EGFR has been reported in a number of different tumors and is observed in most gliomas (4, 14). It has been proposed that the subsequent EGFR over expression mediated by receptor gene amplification may confer a growth advantage by increasing intracellular signaling and cell growth (34). The U87MG cell line was transfected with the wt EGFR to produce a glioma cell that mimics the process of EGFR gene amplification. Treatment of established U87MG.wtEGFR xenografts with mAb 806 resulted in significant growth inhibition. Thus, mAb 806 also mediates in vivo antitumor activity against cells overexpressing the EGFR. Interestingly, mAb 806 inhibition of U87MG.wtEGFR xenografts was less pronounced than that observed with U87MG.Δ2-7 tumors. This probably reflects the fact that mAb 806 has a lower affinity for the overexpressed wt EGFR and only binds a small proportion of receptors expressed on the cell surface. (3) However, it should be noted that despite the small effect on U87MG.wtEGFR xenograft volumes, mAb 806 treatment produced large areas of necrosis within these xenografts. To exclude the possibility that mAb 806 only mediates inhibition of the U87MG-derived cell lines, we tested its efficacy against A431 xenografts. This squamous cell carcinoma-derived cell line contains significant EGFR gene amplification, which is retained both in vitro and in vivo. Treatment of A431 xenografts with mAb 806 produced significant growth inhibition in both a preventative and established model, indicating the antitumor effects of mAb 806 are not restricted to transfected U87MG cell lines.

Complete prevention of A431 xenograft growth by antibody treatment has been reported previously. The wt EGFR mAbs 528, 225, and 425 all prevented the formation of A431 xenografts when administered either on the day or 1 day after tumor inoculation (9, 10). The reason for this difference in efficacy between these wt EGFR anti-bodies and mAb 806 is not known but may be related to the mechanism of cell growth inhibition. The wt EGFR antibodies function by blocking ligand binding to the EGFR, but this is probably not the case with mAb 806 because it only binds a small EGFR subset on the surface of A431 cells. The significant efficacy of mAb 806 against U87MG cells expressing the ligand-independent de2-7 EGFR further supports the notion that this antibody mediates its antitumor activity by a mechanism not involving ligand blockade. Therefore, we are currently investigating the non-immunological and immunological mechanisms that contribute to the antitumor effects of mAb 806. Non-immunological mechanisms may include subtle changes in receptor levels, blockade of signaling, or induction of inappropriate signaling.

Previously, agents such as doxorubicin and cisplatin in conjunction with wt EGFR antibodies have produced enhanced antitumor activity (35, 36). The combination of doxorubicin and mAb 528 resulted in total eradication of established A431 xenografts, whereas treatment with either agent alone caused only temporary in vivo growth inhibition (36). Likewise, the combination of cisplatin and either mAb 528 or 225 also led to the eradication of well-established A431 xenografts, which was not observed when treatment with either agent was used (35). Thus, future studies involving the combination of chemotherapeutic agents with mAb 806 are planned using xenograft models.

Maybe the most important advantage of mAb 806 compared with current EGFR antibodies is that it should be possible to directly conjugate cytotoxic agents to mAb 806. This approach is not feasible with current EGFR-specific antibodies because they target the liver and cytotoxic conjugation would almost certainly induce severe toxicity. Conjugation of cytotoxic agents such as drugs (37) or radioisotopes (38) to antibodies has the potential to improve efficacy and reduce the systemic toxicity of these agents. This study clearly demonstrates that mAb 806 has significant in vivo antitumor activity against de2-7 EGFR-positive xenografts and tumors overexpressing the EGFR. The unique specificity of mAb 806 suggests immunotherapeutic potential in targeting a number of tumor types, particularly head and neck tumors and glioma, without the restrictions associated with normal tissue uptake.

References

  • 1. Scott, A. M., and Welt, S. Antibody-based immunological therapies. Curr. Opin. Immunol., 9: 717-722, 1997.
  • 2. Fan, Z., and Mendelsohn, J. Therapeutic application of anti-growth factor receptor antibodies. Curr. Opin. Oncol., 10: 67-73, 1998.
  • 3. Garcia de Palazzo, I. E., Adams, G. P., Sundareshan, P., Wong, A. J., Testa, J. R., Bigner, D. D., and Weiner, L. M. Expression of mutated epidermal growth factor receptor by non-small cell lung carcinomas. Cancer Res., 53: 3217-3220, 1993.
  • 4. Wikstrand, C. J., Reist, C. J., Archer, G. E., Zalutsky, M. R., and Bigner, D. D. The class III variant of the epidermal growth factor receptor (EGFRvIII): characterization and utilization as an immunotherapeutic target. J. Neurovirol., 4: 148-158, 1998.
  • 5. Korshunov, A., Golanov, A., Sycheva, R., and Pronin, I. Prognostic value of tumor associated antigen immunoreactivity and apoptosis in cerebral glioblastomas: an analysis of 168 cases. J. Clin. Pathol., 52: 574-580, 1999.
  • 6. Grandis, J. R., Melhem, M. F., Gooding, W. E., Day, R., Holst, V. A., Wagener, M. M., Drenning, S. D., and Tweardy, D. J. Levels of TGF-a and EGFR protein in head and neck squamous cell carcinoma and patient survival. J. Natl. Cancer Inst., 90: 824-832, 1998.
  • 7. Fan, Z., Masui, H., Altas, I., and Mendelsohn, J. Blockade of epidermal growth factor receptor function by bivalent and monovalent fragments of 225 anti-epidermal growth factor receptor monoclonal antibodies. Cancer Res., 53: 4322-4328, 1993.
  • 8. Teramoto, T., Onda, M., Tokunaga, A., and Asano, G. Inhibitory effect of anti-epidermal growth factor receptor antibody on a human gastric cancer. Cancer (Phila.), 77: 1639-1645, 1996.
  • 9. Rodeck, U., Herlyn, M., Herlyn, D., Molthoff, C., Atkinson, B., Varello, M., Steplewski, Z., and Koprowski, H. Tumor growth modulation by a monoclonal antibody to the epidermal growth factor receptor immunologically mediated and effector cell-independent effects. Cancer Res., 47: 3692-3696, 1987.
  • 10. Masui, H., Kawamoto, T., Sato, J. D., Wolf, B., Sato, G., and Mendelsohn, J. Growth inhibition of human tumor cells in athymic mice by anti-epidermal growth factor receptor monoclonal antibodies. Cancer Res., 44: 1002-1007, 1984.
  • 11. Faillot, T., Magdelenat, H., Mady, E., Stasiecki, P., Fohanno, D., Gropp, P., Poisson, M., and Delattre, J. Y. A Phase I study of an anti-epidermal growth factor receptor monoclonal antibody for the treatment of malignant gliomas. Neurosurgery, 39: 478-483, 1996.
  • 12. Divgi, C. R., Welt, S., Kris, M., Real, F. X., Yeh, S. D., Gralla, R., Merchant, B., Schweighart, S., Unger, M., Larson, S. M., et al. Phase I and imaging trial of indium 11-labeled anti-epidermal growth factor receptor monoclonal antibody 225 in patients with squamous cell lung carcinoma. J. Natl. Cancer Inst., 83: 97-104, 1991.
  • 13. Baselga, J., Pfister, D., Cooper, M. R., Cohen, R., Burtness, B., Bos, M., D'Andrea, G., Seidman, A., Norton, L., Gunnett, K., Falcey, J., Anderson, V., Waksal, H., and Mendelsohn, J. Phase I studies of anti-epidermal growth factor receptor chimeric antibody C225 alone and in combination with cisplatin. J. Clin. Oncol., 18: 904, 2000.
  • 14. Voldborg, B. R., Damstrup, L., Spang-Thomsen, M., and Poulsen, H. S. Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials. Ann. Oncol., 8: 1197-1206, 1997.
  • 15. Sugawa, N., Ekstrand, A. J., James, C. D., and Collins, V. P. Identical splicing of aberrant epidermal growth factor receptor transcripts from amplified rearranged genes in human glioblastomas. Proc. Natl. Acad. Sci. USA, 87: 8602-8606, 1990.
  • 16. Nishikawa, R., Ji, X. D., Harmon, R. C., Lazar, C. S., Gill, G. N., Cavenee, W. K., and Huang, H. J. A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc. Natl. Acad. Sci. USA, 91: 7727-7731, 1994.
  • 17. Tang, C. K., Gong, X. Q., Moscatello, D. K., Wong, A. J., and Lippman, M. E. Epidermal growth factor receptor vIII enhances tumorigenicity in human breast cancer. Cancer Res., 60: 3081-3087, 2000.
  • 18. Olapade-Olaopa, E. O., Moscatello, D. K., MacKay, E. H., Horsburgh, T., Sandhu, D. P., Terry, T. R., Wong, A. J., and Habib, F. K. Evidence for the differential expression of a variant EGF receptor protein in human prostate cancer. Br. J. Cancer, 82: 186-194, 2000.
  • 19. Moscatello, D. K., Holgado-Madruga, M., Godwin, A. K., Ramirez, G., Gunn, G., Zoltick, P. W., Biegel, J. A., Hayes, R. L., and Wong, A. J. Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors. Cancer Res., 55: 5536-5539, 1995.
  • 20. Wikstrand, C. J., Hale, L. P., Batra, S. K., Hill, M. L., Humphrey, P. A., Kurpad, S. N., McLendon, R. E., Moscatello, D., Pegram, C. N., Reist, C. J., et al. Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas. Cancer Res., 55: 3140-3148, 1995.
  • 21. Hills, D., Rowlinson-Busza, G., and Gullick, W. J. Specific targeting of a mutant, activated FGF receptor found in glioblastoma using a monoclonal antibody. Int. J. Cancer, 63: 537-543, 1995.
  • 22. Okamoto, S., Yoshikawa, K., Obata, Y., Shibuya, M., Aoki, S., Yoshida, J., and Takahashi, T. Monoclonal antibody against the fusion junction of a deletion-mutant epidermal growth factor receptor. Br. J. Cancer, 73: 1366-1372, 1996.
  • 23. Nagane, M., Coufal, F., Lin, H., Bogler, O., Cavenee, W. K., and Huang, H. J. A common mutant epidermal growth factor receptor confers enhanced tumorigenicity on human glioblastoma cells by increasing proliferation and reducing apoptosis. Cancer Res., 56: 5079-5086, 1996.
  • 24. Sato, J. D., Le, A. D., and Kawamoto, T. Derivation and assay of biological effects of monoclonal antibodies to epidermal growth factor receptors. Methods Enzymol., 146: 63-81, 1987.
  • 25. Huang, H. S., Nagane, M., Klingbeil, C. K., Lin, H., Nishikawa, R., Ji, X. D., Huang, C. M., Gill, G. N., Wiley, H. S., and Cavenee, W. K. The enhanced tumorigenic activity of a mutant epidermal growth factor receptor common in human cancers is mediated by threshold levels of constitutive tyrosine phosphorylation and unattenuated signaling. J. Biol. Chem., 272: 2927-2935, 1997.
  • 26. Clarke, K., Lee, F. T., Brechbiel, M. W., Smyth, F. E., Old, L. J., and Scott, A. M. Therapeutic efficacy of anti-Lewis(y) humanized 3S193 radioimmunotherapy in a breast cancer model: enhanced activity when combined with Taxol chemotherapy. Clin. Cancer Res., 6: 3621-3628, 2000.
  • 27. Atlas, I., Mendelsohn, J., Baselga, J., Fair, W. R., Masui, H., and Kumar, R. Growth regulation of human renal carcinoma cells: role of transforming growth factor a. Cancer Res., 52: 3335-3339, 1992.
  • 28. Perez-Soler, R., Donato, N.J., Shin, D. M., Rosenblum, M. G., Zhang, H. Z., Tornos, C., Brewer, H., Chan, J. C., Lee, J. S., Hong, W. K., et al. Tumor epidermal growth factor receptor studies in patients with non-small-cell lung cancer or head and neck cancer treated with monoclonal antibody RG 83852. J. Clin. Oncol., 12: 730-739, 1994.
  • 29. Wersall, P., Ohlsson, I., Biberfeld, P., Collins, V. P., von Krusenstjerna, S., Larsson, S., Mellstedt, H., and Boethius, J. Intratumoral infusion of the monoclonal antibody, mAb 425, against the epidermal-growth-factor receptor in patients with advanced malignant glioma. Cancer Immunol. Immunother., 44: 157-164, 1997.
  • 30. Brady, L. W., Miyamoto, C., Woo, D. V., Rackover, M., Emrich, J., Bender, H., Dadparvar, S., Steplewski, Z., Koprowski, H., Black, P., et al. Malignant astrocytomas treated with iodine-125 labeled monoclonal antibody 425 against epidermal growth factor receptor: a Phase II trial. Int. J. Radiat. Oncol. Biol. Phys., 22: 225-230, 1992.
  • 31. Reist, C. J., Archer, G. E., Kurpad, S, N., Wikstrand, C. J., Vaidyanathan, G., Willingham, M. C., Moscatello, D. K., Wong, A. J., Bigner, D. D., and Zalutsky, M. R. Tumor-specific anti-epidermal growth factor receptor variant III monoclonal antibodies: use of the tyramine-cellobiose radioiodination method enhances cellular retention and uptake in tumor xenografts. Cancer Res., 55: 4375-4382, 1995.
  • 32. Reist, C. J., Archer, G. E., Wikstrand, C. J., Bigner, D. D., and Zalutsky, M. R. Improved targeting of an anti-epidermal growth factor receptor variant III monoclonal antibody in tumor xenografts after labeling using N-succinimidyl 5-iodo-3-pyridinecarboxylate. Cancer Res., 57: 1510-1515, 1997.
  • 33. Sampson, J. H., Crotty, L. E., Lee, S., Archer, G. E., Ashley, D. M., Wikstrand, C. J., Hale, L. P., Small, C., Dranoff, G., Friedman, A. H., Friedman, H. S., and Bigner, D. D. Unarmed, tumor-specific monoclonal antibody effectively treats brain tumors.

Proc. Natl. Acad. Sci. USA, 97: 7503-7508, 2000.

  • 34. Filmus, J., Trent, J. M., Pollak, M. N., and Buick, R. N. Epidermal growth factor receptor gene-amplified MDA-468 breast cancer cell line and its nonamplified variants. Mol. Cell. Biol., 7: 251-257, 1987.
  • 35. Fan, Z., Baselga, J., Masui, H., and Mendelsohn, J. Antitumor effect of anti-epidermal growth factor receptor monoclonal antibodies plus cis-diamminedichloroplatinum on well established A431 cell xenografts. Cancer Res., 53: 4637-4642, 1993.
  • 36. Baselga, J., Norton, L., Masui, H., Pandiella, A., Coplan, K., Miller, W. H., Jr., and Mendelsohn, J. Antitumor effects of doxorubicin in combination with anti-epidermal growth factor receptor monoclonal antibodies. J. Natl. Cancer Inst., 85: 1327-1333, 1993.
  • 37. Trail, P. A., and Bianchi, A. B. Monoclonal antibody drug conjugates in the treatment of cancer. Curr. Opin. Immunol., 11: 584-588, 1999.
  • 38. DeNardo, S. J., Kroger, L. A., and DeNardo, G. L. A new era for radiolabeled antibodies in cancer? Curr. Opin. Immunol., 11: 563-569, 1999.

EXAMPLE 21 Construction, Expression and Analysis of Chimeric 806 Antibody

Chimeric antibodies are a class of molecules in which heavy and light chain variable regions of for instance, a mouse, rat or other species are joined onto human heavy and light chain regions. Chimeric antibodies are produced recombinantly. One advantage of chimeric antibodies is that they can reduce xenoantigenic effects, the inherent immunogenicity of non-human antibodies (for instance, mouse, rat or other species). In addition, recombinantly prepared chimeric antibodies can often be produced in large quantities, particularly when utilizing high level expression vectors.

For high level production, the most widely used mammalian expression system is one which utilizes the gene amplification procedure offered by dehydrofolate reductase deficient (“dhfr−”) Chinese hamster ovary cells. The system is well known to the skilled artisan. The system is based upon the dehydrofolate reductase “dhfr” gene, which encodes the DHFR enzyme, which catalyzes conversion of dehydrofolate to tetrahydrofolate. In order to achieve high production, dhfr-CHO cells are transfected with an expression vector containing a functional DHFR gene, together with a gene that encodes a desired protein. In this case, the desired protein is recombinant antibody heavy chain and/or light chain.

By increasing the amount of the competitive DHFR inhibitor methotrexate (MTX), the recombinant cells develop resistance by amplifying the dhfr gene. In standard cases, the amplification unit employed is much larger than the size of the dhfr gene, and as a result the antibody heavy chain is co-amplified.

When large scale production of the protein, such as the antibody chain, is desired, both the expression level, and the stability of the cells being employed, are critical. In long term culture, recombinant CHO cell populations lose homogeneity with respect to their specific antibody productivity during amplification, even though they derive from a single, parental clone.

Bicistronic expression vectors were prepared for use in recombinant expression of the chimeric antibodies. These bicistronic expression vectors, employ an “internal ribosomal entry site” or “IRES.” In these constructs for production of chimericanti-EGFR, the immunoglobulin chains and selectable markers cDNAs are linked via an IRES. IRES are cis-acting elements that recruit the small ribosomal subunits to an internal initiator codon in the mRNA with the help of cellular trans-acting factors. IRES facilitate the expression of two or more proteins from a polycistronic transcription unit in eukaryotic cells. The use of bicistronic expression vectors in which the selectable marker gene is translated in a cap dependent manner, and the gene of interest in an IRES dependent manner has been applied to a variety of experimental methods. IRES elements have been successfully incorporated into vectors for cellular transformation, production of transgenic animals, recombinant protein production, gene therapy, gene trapping, and gene targeting.

Synopsis of Chimeric Antibody 806 (ch806) Construction

The chimeric 806 antibody was generated by cloning the VH and VL of the 806 antibody from the parental murine hybridoma using standard molecular biology techniques. The VH and VL were then cloned into the pREN mammalian expression vectors, the construction of which are set forth in Table 6 and Table 7 and transfected into CHO (DHFR−/−ve) cells for amplification and expression. Briefly, following trypsinization 4×106 CHO cells were co-transferred with 10 |g of each of the LC and HC expression vectors using electroporation under standard conditions. Following a 10 min rest period at room temperature, the cells were added to 15 ml medium (10% fetal calf serum, hypoxanthine/thymidine supplement with additives) and transferred to 15×10 cm cell culture petri dishes. The plates were then placed into the incubator under normal conditions for 2 days. At this point, the addition of gentamycin, 5 nM methotrexate, the replacement of fetal calf serum with dialyzed fetal calf serum and the removal of hypoxanthine/thymidine, initiated the selection for clones that were successfully transfected with both the LC and HC from the medium. At day 17 following transfection, individual clones growing under selection were picked and screened for expression of the chimeric 806 antibody. An ELISA was utilized for screening and consisted of coating an ELISA plate with denatured soluble EGF receptor (denatured EGFR is known to allow 806 binding). This assay allows for the screening of production levels by individual clones and also for the functionality of the antibody being screened. All clones were shown to be producing functional ch806 and the best producer was taken and expanded for amplification. To amplify the level of ch806 being produced, the highest producing clone was subjected to reselection under a higher methotrexate concentration (100 nM vs 5 nM). This was undertaken using the aforementioned procedures.

Clones growing at 100 nM MTX were then passed onto the Biological Production Facility, Ludwig Institute, Melbourne, Australia for measurement of production levels, weaning off serum, cell banking. The cell line has been shown to stably produce ˜10 mg/liter in roller bottles.

The nucleic acid sequence of the pREN ch806 LC neo vector is provided in SEQ ID NO:7 and the corresponding amino acid sequence is defined in SEQ ID NO:9. The nucleic acid sequence of the pREN ch806 HC DHFR vector is provided in SEQ ID NO:8 and the corresponding amino acid sequence is set forth in SEQ ID NO:10.

FIG. 33 depicts the vectors pREN-HC and pREN-LC, which employ an IRES. The pREN bicistronic vector system is described and disclosed in co-pending U.S. Ser. No. 60/355,838 filed Feb. 13, 2002, which is incorporated herein by reference in its entirety.

Ch806 was assessed by FACS analysis to demonstrate that the chimeric 806 displays identical binding specificity to that of the murine parental antibody. Analysis was performed using wild type cells (U87MG parental cells), cells overexpressing the EGF receptor (A431 cells and UA87 wt EGFR cells) and UA87 Δ2-7 cells (data not shown). Similar binding specificity of Mab806 and ch806 was obtained using cells overexpressing EGFR and cells expressing the de2-7 EGFR. No binding was observed in wild type cells. Scatchard analysis revealed a binding affinity for radiolabeled ch806 of 6.4×109 M−1 using U87MG.de2-7 cells (data not shown).

Biodistribution analysis of the ch806 antibody was performed in BALB/c nude mice bearing U87MG-de2-7 xenograft tumors and the results are shown in FIG. 34. Mice were injected with 5 μg of radiolabelled antibody and were sacrificed in groups of four per time point at 8, 24, 48 and 74 hours. Organs were collected, weighed and radioactivity measured in a gamma counter. 125I-labelled ch806 displays reduced targeting to the tumor compared to 111In-labelled ch806, which has high tumor uptake and cumulative tumor retention over the 74 hour time period. At 74 hours, the 111In-labelled antibody displays approximately 30% ID/gram tissue and a tumor to blood ratio of 4.0 (FIG. 35). The 111In-labelled ch806 shows some nonspecific retention in the liver, spleen and kidneys. This is common for the use of this isotope and decreases with time, which supports that this binding is non-specific to ch806 and due to 111In binding.

Chimeric antibody ch806 was assessed for therapeutic efficacy in an established tumor model. 3×106 U87MG.Δ2-7 cells in 100 μl of PBS were inoculated s.c. into both flanks of 4-6 week old female nude mice (Animal Research Center, Western Australia, Australia). The mAb806 was included as a positive control. The results are depicted in FIG. 36. Treatment was started when tumors had reached a mean volume of 50 mm3 and consisted of 1 mg of ch806 or mAb806 given i.p. for a total of 5 injections on the days indicated. Tumor volume in mm3 was determined using the formula (length×width2)/2, where length was the longest axis and width the measurement at right angles to the length. Data was expressed as mean tumor volume+/−S.E. for each treatment group. The ch806 and mAb806 displayed nearly identical anti-tumor activity against U87MG.Δ2-7 xenografts.

Analysis of Ch806 Immune Effector Function

Materials and Methods

Antibodies and Cell Lines:

Murine anti-de2-7 EGFR monoclonal mAb 806, chimeric antibody ch806 (IgG1) and control isotype matched chimeric anti-G250 monoclonal antibody cG250 were prepared by the Biological Production Facility, Ludwig Institute for Cancer Research, Melbourne, Australia. Both complement-dependant cytotoxicity (CDC) and antibody-dependent cellular-cytotoxicity (ADCC) assays utilised U87MG.de2-7 and A431 cells as target cells. The previously described U87MG.de2-7 cell line is a human astrocytoma cell line infected with a retrovirus containing the de2-7EGFR (Nishikawa, R. et al. (1994) Proc Natl Acad Sci USA. 91: 7727-31). Human squamous carcinoma A431 cells were purchased from the American Type Culture Collection (Manassas, Va.). All cell lines were cultured in DMEM/F-12 with GLUTAMAX™ (Life Technologies, Melbourne, Australia) supplemented with 10% heat-inactivated FCS(CSL, Melbourne, Australia), 100 units/ml penicillin and 100 ng/ml streptomycin. To maintain selection for retrovirally transfected U87MG.de2-7 cells, 400 ng/ml G418 was included in the media.

Preparation of human peripheral blood mononuclear cells (PBMC) Effector Cells: PBMCs were isolated from healthy volunteer donor blood. Heparinised whole blood was fractionated by density centrifugation on Ficoll-Hypaque (ICN Biomedical Inc., Ohio, USA). PBMC fractions was collected and washed three times with RPML+ 1640 supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM L-glutamine, containing 5% heat-inactivated FCS.

Preparation of Target Cells:

CDC and ADCC assays were performed by a modification of a previously published method ((Nelson, D. L. et al. (1991) In: J. E. Colignan, A. M. Kruisbeek, D. D. Margulies, E. M. Shevach, and W. Strober (eds.), Current Protocols in Immunology, pp. 7.27.1. New York: Greene Publishing Wiley Interscience). Briefly, 5×106 target U87MG.de2-7 and A431 cells were labeled with 50 μCi 51Cr (Geneworks, Adelaide, Australia) per 1×106 cells and incubated for 2 hr at 37° C. The cells were then washed three time with PBS (0.05M, pH 7.4) and a fourth wash with culture medium. Aliquots (1×104 cells/50 ul) of the labeled cells were added to each well of 96-well microtitre plates (NUNC, Roskilde, Denmark).

CDC Assay:

To 50 μl labeled target cells, 50 μl ch806 or isotype control antibody cG250 were added in triplicate over the concentration range 0.00315-10 μg/ml, and incubated on ice 5 min. Fifty μl of freshly prepared healthy donor complement (serum) was then added to yield a 1:3 final dilution of the serum. The microtitre plates were incubated for 4 hr at 37° C. Following centrifugation, the released 51Cr in the supernatant was counted (COBRA II™ automated Gamma Counter, Can berra Packard, Melbourne, Australia). Percentage specific lysis was calculated from the experimental 51Cr release, the total (50 μl target cells+100 μl 10% TWEEN™ 20 and spontaneous (50 μl target cells+100 μl medium) release.

ADCC Assay:

Ch806-mediated ADCC effected by healthy donor PBMCs was measured by two 4-hr 51Cr release assays. In the first assay, labelled target cells were plated with the effector cells in 96-well “U” bottom microplates (NUNC, Roskilde, Denmark) at effector/target (E:T) cell ratios of 50:1. For ADCC activity measurements, 0.00315-10 μg/mg (final concentration) test and control antibodies were added in triplicate to each well. In the second ADCC assay, the ADCC activity of ch806 was compared with the parental murine mAb 806 over a range of Effector: Target cell ratios with the test antibody concentration constant at 1 ug/ml. In both assays, micotitre plates were incubated at 37° C. for 4 hours, then 50 ul supernatant was harvested from each well and released 51Cr was determined by gamma counting (COBRA II™ automated Gamma Counter, Can berra Packard, Melbourne, Australia). Controls included in the assays corrected for spontaneous release (medium alone) and total release (10% TWEEN™ 20/PBS). Appropriate controls with the same subclass antibody were run in parallel.

The percentage cell lysis (cytotoxicity) was calculated according to the formula:

Percentage ⁢ ⁢ Cytotoxicity = Sample ⁢ ⁢ Counts - Spontaneous ⁢ ⁢ Release Total ⁢ ⁢ Release - Spontaneous ⁢ ⁢ Release × 100

The percent (%) cytotoxicity was plotted versus concentration of antibody (μg/ml).

Results

The results of the CDC analyses are presented in FIG. 37. Minimal CDC activity was observed in the presence of up to 10 μg/ml ch806 with CDC comparable to that observed with isotype control cG250.

Ch806 mediated ADCC on target U87MG.de2-7 and A431 cells at E:T ratio of 50:1 is presented in FIG. 38. Effective ch806 specific cytotoxicity was displayed against target U87MG.de2-7 cells, but minimal ADCC was mediated by ch806 on A431 cells. The levels of cytotoxicity achieved reflect the number of ch806 binding sites on the two cell populations. Target U87MG.de2-7 cells express ˜1×106 de2-7EGFR which are specifically recognised by ch806, while only a subset of the 1×106 wild-type EGFR molecules expressed on A431 cells are recognised by ch806 (see above Examples).

Further ADCC analyses were performed to compare the ADCC mediated by 1 ug/ml ch806 on target U87MG.de2-7 cells with that effected by 1 μg/ml parental murine mAb 806. Results are presented in FIG. 39. Chimerisation of mAb 806 has effected marked improvement of the ADCC achieved by the parental murine mAb with greater than 30% cytotoxicity effected at E:T ratios 25:1 and 50:1.

The lack of parental murine mAb 806 immune effector function has been markedly improved upon chimerisation. Ch806 mediates good ADCC, but minimal CDC activity.

EXAMPLE 22 Generation of Anti-Idiotype Antibodies to Chimeric Antibody ch806

To assist the clinical evaluation of mAb806 or ch806, laboratory assays are required to monitor the serum pharmacokinetics of the antibodies and quantitate any immune responses to the mouse-human chimeric antibody. Mouse monoclonal anti-idiotypic antibodies (anti-ids) were generated and characterised for suitability as ELISA reagents for measuring ch806 in patient sera samples and use as positive controls in human anti-chimeric antibody immune response analyses. These anti-idiotype antibodies may also be useful as therapeutic or prophylactic vaccines, generating a natural anti-EGFR antibody response in patients.

Methods for generating anti-idiotype antibodies are well known in the art (Bhattacharya-Chatterjee, Chatterjee et al. 2001, Uemura et al. 1994, Steffens, Boerman et al. 1997, Safa and Foon 2001, Brown and Ling 1988).

Mouse monoclonal anti-idiotypic antibodies (anti-ids) were, briefly, generated as follows. Splenocytes from mice immunized with ch806 were fused with SP2/0-AG14 plasmacytoma cells and antibody producing hybridomas were selected through ELISA for specific binding to ch806 and competitive binding for antigen (FIG. 40). Twenty-five hybridomas were initially selected and four, designated LMH-11, -12-13 and -14, secreted antibodies that demonstrated specific binding to ch806, mAb 806 and were able to neutralise ch806 or mAb 806 antigen binding activity (FIG. 41). The recognition of the ch806/mAb 806 idiotope or CDR region was demonstrated by lack of cross-reactivity with purified polyclonal human IgG.

In the absence of readily available recombinant antigen de2-7 EGFR to assist with the determination of ch806 in serum samples, the ability of the novel anti-idiotype ch806 antibodies to concurrently bind 806 variable regions was exploited in the development of a sensitive, specific ELISA for measuring ch806 in clinical samples (FIG. 42).

Using LMH-12 for capture and Biotinylated-LMH-12 for detection, the validated ELISA demonstrated highly reproducible binding curves for measuring ch806 (2 μg/ml-1.6 ng/ml) in sera with a 3 ng/ml limit of detection. (n=12; 1-100 ng/ml, Coefficient of Variation <25%; 100 ng/ml-5 ug/ml, Coefficient of Variation <15%). No background binding was evident with the three healthy donor sera tested and negligible binding was observed with isotype control hu3S193. The hybridoma produces high levels of antibody LMH-12, and larger scale production is planned to enable the measurement of ch806 and quantitation of any immune responses in clinical samples. (Brown and Ling 1988)

Results

Mice Immunization and Hybridoma Clone Selection

Immunoreactivity of pre- and post-immunization sera samples indicated the development of high titer mouse anti-ch806 and anti-huIgG mAbs. Twenty-five hybridomas producing antibodies that bound ch806, but not huIgG, were initially selected. The binding characteristics of some of these hybridomas are shown in FIGS. 42A and 42B. Four of these anti-ch806 hybridomas with high affinity binding (clones 3E3, 5B8, 9D6 and 4D8) were subsequently pursued for clonal expansion from single cells by limiting dilution and designated Ludwig Institute for Cancer Research Melbourne Hybridoma (LMH)-11, -12, -13, and -14, respectively (FIG. 42).

Binding and Blocking Activities of Selected Anti-Idiotype Antibodies

The ability of anti-ch806 antibodies to concurrently bind two ch806 antibodies is a desirable feature for their use as reagents in an ELISA for determining serum ch806 levels. Clonal hybridomas, LMH-11, -12, -13, and -14 demonstrated concurrent binding (data not shown).

After clonal expansion, the hybridoma culture supernatants were examined by ELISA for the ability to neutralise ch806 or mAb 806 antigen binding activity with sEGFR621. Results demonstrated the antagonist activity of anti-idiotype mAbs LMH-11, -12, -13, and -14 with the blocking in solution of both ch806 and murine mAb 806 binding to plates coated with sEGFR (FIG. 41 for LMH-11, -12, -13). Following larger scale culture in roller bottles the binding specificity's of the established clonal hybridomas, LMH-11, -12, -13, and -14 were verified by ELISA. LMH-11 through -14 antibodies were identified as isotype IgG1κ by mouse monoclonal antibody isotyping kit.

ch806 in Clinical Serum Samples: Pharmacokinetic ELISA Assay Development

To assist with the determination of ch806 in serum samples, the ability of the anti-idiotype ch806 antibodies to concurrently bind the 806 variable region was exploited in the development of a sensitive and specific ELISA assay for ch806 in clinical samples. The three purified clones LMH-11, -12, and -13 (FIGS. 49B and 49C, respectively) were compared for their ability to capture and then detect bound ch806 in sera. Results indicated using LMH-12 (10 μg/ml) for capture and biotinylated-LMH-12 for detection yielded the highest sensitivity for ch806 in serum (3 ng/ml) with negligible background binding.

Having established the optimal pharmacokinetic ELISA conditions using 1 μg/ml anti-idiotype LMH-12 and 1 μg/ml biotinylated LMH-12 for capture and detection, respectively, validation of the method was performed. Three separate ELISAs were performed in quadruplicate to measure ch806 in donor serum from three healthy donors or 1% BSA/media with isotype control hu3S193. Results of the validation are presented in FIG. 43 and demonstrate highly reproducible binding curves for measuring ch806 (2 μg/ml-1.6 ng/ml) in sera with a 3 ng/ml limit of detection. (n=12; 1-100 ng/ml, Coefficient of Variation <25%; 100 ng/ml-5 μg/ml, Coefficient of Variation <15%). No background binding was evident with any of the three sera tested and negligible binding was observed with isotype control hu3S193.

References

  • Brown, G. and N. Ling (1988). Murine Monoclonal Antibodies. Antibodies, Volume I. A Practical Approach. D. Catty. Oxford, England, IRL Press: 81-104.
  • Bhattacharya-Chatterjee, M., S. K. Chatterjee, et al. (2001). “The anti-idiotype vaccines for immunotherapy.” Curr Opin Mol Ther 3(1): 63-9.
  • Domagala, T., N. Konstantopoulos, et al. (2000). “Stoichiometry, kinetic and binding analysis of the interaction between epidermal growth factor (EGF) and the extracellular domain of the EGF receptor.” Growth Factors 18(1): 11-29.
  • Safa, M. M. and K. A. Foon (2001). “Adjuvant immunotherapy for melanoma and colorectal cancers.” Semin Oncol 28(1): 68-92.
  • Uemura, H., E. Okajima, et al. (1994). “Internal image anti-idiotype antibodies related to renal-cell carcinoma-associated antigen G250.” Int J Cancer 56(4): 609-14.

EXAMPLE 23 Assessment of Carbohydrate Structures and Antibody Recognition

Experiments were undertaken to further assess the role of carbohydrate structures in the binding and recognition of the EGFR, both amplified and de2-7 EGFR, by the mAb806 antibody.

To determine if carbohydrate structures are directly involved in the mAb 806 epitope, the recombinant sEGFR expressed in CHO cells was treated with PNGase F to remove N-linked glycosylation. Following treatment, the protein was run on SDS-PAGE, transferred to membrane and immunoblotted with mAb 806 (FIG. 44). As expected, the deglycosylated sEGFR ran faster on SDS-PAGE, indicating that the carbohydrates had been successfully removed. The mAb 806 antibody clearly bound the deglycosylated material demonstrating the antibody epitope is peptide in nature and not solely a glycosylation epitope.

Lysates, prepared from cell lines metabolically labelled with 35S, were immunoprecipitated with different antibodies directed to the EGFR (FIG. 45). As expected, the 528 antibody immunoprecipitated 3 bands from U87MG.Δ2-7 cells, an upper band corresponding to the wild type (wt) EGFR and two lower bands corresponding to the de2-7 EGFR. These two de2-7 EGFR bands have been reported previously and are assumed to represent differential glycosylation (Chu et al. (1997) Biochem. J. June 15; 324 (Pt 3):885-861). In contrast, mAb 806 only immunoprecipitated the two de2-7 EGFR bands, with the wt receptor being completely absent even after over-exposure (data not shown). Interestingly, mAb806 showed increased relative reactivity with the lower de2-7 EGFR band but decreased reactivity with the upper band when compared to the 528 antibody. The SC-03 antibody, a commercial rabbit polyclonal antibody directed to C-terminal domain of the EGFR, immunoprecipitated the three EGFR bands as seen with the 528 antibody although the total amount of receptor immunoprecipitated by this antibody was considerably less. No bands were observed when using an irrelevant IgG2b antibody as a control for mAb 806.

The 528 antibody immunoprecipitated a single band from U87MG.wtEGFR cells corresponding to the wt receptor (FIG. 45). MAb 806 also immunoprecipitated a single band from these cells, however, this EGFR band clearly migrated faster than the 528 reactive receptor. The SC-03 antibody immunoprecipitated both EGFR reactive bands from U87MG.wtEGFR cells, further confirming that the mAb 806 and 528 recognize different forms of the EGFR in whole cell lysates from these cells.

As observed with U87MG.wtEGFR cells, the 528 antibody immunoprecipitated a single EGFR band from A431 cells (FIG. 45). The 528 reactive EGFR band is very broad on these low percentage gels (6%) and probably reflects the diversity of receptor glycosylation. A single EGFR band was also seen following immunoprecipitation with mAb 806. While this EGFR band did not migrate considerably faster than the 528 overall broad reactive band, it was located at the leading edge of the broad 528 band in a reproducible fashion. Unlike U87MG.Δ2-7 cell lysates, the total amount of EGFR immunoprecipitated by mAb 806 from A431 lysates was considerably less than with the 528 antibody, a result consistent with our Scatchard data showing mAb 806 only recognizes a portion of the EGFR on the surface of these cells (see Example 4 above) Immunoprecipitation with SC-03 resulted in a single broad EGFR band as for the 528 antibody. Similar results were obtained with HN5 cells (data not shown). Taken together, this data indicates that mAb 806 preferentially reacts with faster migrating species of the EGFR, which may represent differentially glycosylated forms of the receptor.

In order to determine at what stage of receptor processing mAb 806 reactivity appeared a pulse/chase experiment was conducted. A431 and U87MG.Δ2-7 cells were pulsed for 5 min with 35S methionine/cysteine, then incubated at 37° C. for various times before immunoprecipitation with mAb 806 or 528 (FIG. 46). The immunoprecipitation pattern in A431 cells with the 528 antibody was typical for a conformational dependent antibody specific for the EGFR. A small amount of receptor was immunoprecipitated at 0 min (i e after 5 min pulse) with the amount of labelled EGFR increasing at each time point. There was also a concurrent increase in the molecular weight of the receptor with time. In contrast, the mAb 806 reactive EGFR material was present at high levels at 0 min, peaked at 20 min and then reduced at each further time point. Thus, it appears that mAb 806 preferentially recognizes a form of the EGFR found at an early stage of processing.

The antibody reactivity observed in pulse-labelled U87MG.Δ2-7 cells was more complicated. Immunoprecipitation with the 528 antibody at 0 min revealed that a small amount of the lower de2-7 EGFR band was labelled (FIG. 46). The amount of 528 reactive de2-7 EGFR lower band increased with time, peaking at 60 min and declining slowly at 2 and 4 h. No significant amount of the labelled upper band of de2-7 EGFR was detected until 60 min, after which the level continued to increase until the end of the time course. This clearly indicates that the upper de2-7 EGFR is a more mature form of the receptor. MAb 806 reactivity also varied during the time course study, however mAb 806 preferentially precipitated the lower band of the de2-7 EGFR. Indeed, there were no significant levels of mAb 806 upper band seen until 4 h after labelling.

The above experiments suggest that mab 806 preferentially reacts with a more immature glycosylation form of the de2-7 and wt EGFR. This possibility was tested by immunoprecipitating the EGFR from different cells lines labelled overnight with 35S methionine/cysteine and then subjecting the resultant precipitates to Endoglycosidase H (Endo H) digestion. This enzyme preferentially removes high mannose type carbohydrates (i.e. immature glycosylation) from proteins while leaving complex carbohydrates (i.e. matureglycosylation) intact. Immunoprecipitation and digestion with Endo H of labelled U87MG.Δ2-7 cell lysates with 528, mAb 806 and SC-03 gave similar results (FIG. 47). As predicted, the lower de2-7 EGFR band was fully sensitive to Endo H digestion, migrating faster on SDS-PAGE after Endo H digestion, demonstrating that this band represents the high mannose form of the de2-7 EGFR. The upper de2-7 EGFR band was essentially resistant to Endo H digestion, showing only a very slight difference in migration after Endo H digestion, indicating that the majority of the carbohydrate structures are of the complex type. The small but reproducible decrease in the molecular weight of the upper band following enzyme digestion suggests that while the carbohydrates on the upper de2-7 EGFR band are predominantly of the complex type, it does possess some high mannose structures. Interestingly, these cells also express low amounts of endogenous wt EGFR that is clearly visible following 528 immunoprecipitation. There was also a small but noticeable reduction in molecular weight of the wt receptor following Endo H digestion, indicating that it also contains high mannose structures.

The sensitivity of the immunoprecipitated wt EGFR to Endo H digestion was similar in both U87MG.wtEGFR and A431 cells (FIG. 47). The bulk of the material precipitated by the 528 antibody was resistant to the Endo H enzyme although a small amount of the material was of the high mannose form. Once again there was a small decrease in the molecular weight of the wt EGFR following Endo H digestion suggesting that it does contain some high mannose structures. The results using the SC-03 antibody were similar to the 528 antibody. In contrast, the majority of the EGFR precipitated by mAb 806 was sensitive to Endo H in both U87MG.wtEGFR and A431 cells, confirming that mAb 806 preferentially recognizes the high mannose form of the EGFR. Similar results were obtained with HN-5 cells, wherein the majority of the material precipitated by mAb 806 was sensitive to Endo H digestion, while the majority of the material precipitated by mAb528 and SC-03 was resistant to Endo H digestion (data not shown).

Cell surface iodination of the A431 cell line, was performed with 125I followed by immunoprecipitation with the 806 antibody. The protocol for surface iodination was as follows: The cell lysis, immunoprecipitation, Endo H digestion, SDS PAGE and autoradiography are as described above herein. For labeling, cells were grown in media with 10% FCS, detached with EDTA, washed twice with PBS then resuspended in 400 ul of PBS (approx 2-3×106 cells). To this was added 15 ul of 125I (100 mCi/ml stock), 100 ul bovine lactoperoxidase (1 mg/ml) stock, 10 ul H2O2 (0.1% stock) and this was incubated for 5 min. A further 10 ul of H2O2 was then added and the incubation continued for a further 3 min. Cells were then washed again 3 times with PBS and lysed in 1% TRITON™. Cell surface iodination of the A431 cell line with lactoperoxidase, followed by immunoprecipitation with the 806 antibody, showed that, similar to the whole cell lysates described above, the predominant form of the EGFR recognized by 806 bound on the cell surface of A431 cells was sensitive to EndoH digestion (FIG. 48). This confirms that the form of EGFR bound by 806 on the cell surface of A431 cells is an EndoH sensitive form and thus is the high mannose type.

This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety.

TABLE 6  pREN ch806 LC Neo Vector Xho I 1 CTCGAGAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTC 51 ATTAGGCACCCCAGGCTTTACACTTTATGCTCCCGGCTCGTATGTTGTGT                                   EcoRI     EF1α promoter 101 GGAGATTGTGAGCGGATAACAATTTCACACAGAATTCGTGAGGCTCCGGT 151 GCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGG 201 GGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAA 251 ACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGG 301 GGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAA 351 CGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGC 401 CTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACG 451 CCCCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTG 501 GGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCT 551 TGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTG 601 GCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAA 651 ATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTA 701 AATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGG 751 CGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCC 801 TGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGG 851 CCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGC 901 GGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGC 951 TTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGA 1001 GAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTC 1051 AGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACC 1101 TCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAG 1151 GGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGT 1201 TAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTG 1251 AGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTT                     MluI       HindIII   PmeI 1301 TTTCTTCCATTTCAGGTGTACGCGTCTCGGGAAGCTTTAGTTTAAACGCC 1351 GCCACCATGGTGTCCACAGCTCAGTTCCTTGCATTCTTGTTGCTTTGGTTT         M  V  S  T  A  Q  F  L  A  F  L  L  L  W  F 1401 CCAGGTGCAAGATGTGACATCCTGATGACCCAATCTCCATCCTCCATGTCT P  G  A  R  C  D  I  L  M  T  Q  S  P  S  S  M  S 1451 GTATCTCTGGGAGACACAGTCAGCATCACTTGCCATTCAAGTCAGGACATT           V S L G D T V S I T C H S S Q D I 1501 AACAGTAATATAGGGTGGTTGCAGCAGAGACCAGGGAAATCATTTAAGGGC  N  S  N  I  G  W  L  Q  Q  R  P  G  K  S  F  K  G 1551 CTGATCTATCATGGAACCAACTTGGACGATGAAGTTCCATCAAGGTTCAGT           L I Y H G T N L D D E V P S R F S 1601 GGCAGTGGATCTGGAGCCGATTATTCTCTCACCATCAGCAGCCTGGAATCT           G S G S G A D Y S L T I S S L E S 1651 GAAGATTTTGCAGACTATTACTGTGTACAGCATGCTCAGTTTCCGTGGACG  E  D  F  A  D  Y  Y  C  V  Q  H  A  Q  F  P  W  T                                         BamHI 1701 TTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGTGAGTGGATCCATCTGGG  F  G  G  G  T  K  L  E  I  K  R 1751 ATAAGCATGCTGTTTTCTGTCTGTCCCTAACATGCCCTGTGATTATGCGCAAA 1801 CAACACACCCAAGGGCAGAACTTTGTTACTTAAACACCATCCTGTTTGCTTCTT 1851 TCCTCAGGAACTGTGGCTGCACCA        T  V  A  A  P 1876 TCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGC  S  V  F  I  F  P  P  S  D  E  Q  L  K  S  G  T  A 1926 CTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC   S  V  V  C  L  L  N  N  F  Y  P  R  E  A  K  V  Q 1976 AGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC    W  K  V  D  N  A  L  Q  S  G  N  S  Q  E  S  V 2026 ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC  T  E  Q  D  S  K  S  T  Y  Y  S  L  S  S  T  L  T 2076 GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCA   L  S  K  A  D  Y  E  K  H  K  V  Y  A  C  E  V  T 2126 CCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG    H  Q  G  L  S  S  P  V  T  K  S  F  N  R  G  E        Nhe/Xba 2176 TGTTGAGCTAGAACTAACTAACTAAGCTAGCAACGGTTTCCCTCTAGCGG  C  * 2226 GATCAATTCCGCCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAA 2276 TAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTC 2326 TTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCA 2376 TTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAAT 2426 GTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTC 2476 TGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCC 2526 TCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAA 2576 CCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCT 2626 CTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCC 2676 ATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACGTGTGTT 2751 TAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTT 2801 TTCCTTTGAAAAACACGATAATACCATGGTTGAACAAGATGGATTGCACG 2851 CAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCA 2901 CAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCA 2951 GGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATG 3001 AACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTT 3051 CCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCT 3101 GCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTC 3151 CTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACG 3201 CTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGA 3251 GCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGG 3301 ACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAG 3351 GCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTG 3401 CTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACT 3451 GTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACC 3501 CGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGT 3551 GCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCC                     blunt end SalI/SalI 3601 TTCTTGACGAGTTCTTCTGAGTCGATCGACCTGGCGTAATAGCGAAGAGG 3651 CCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGG 3701 GACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCG 3751 CAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTT 3801 TCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTA 3851 AATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGA 3901 CCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCT 3951 GATAGACGGTTTTTCGCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTG 4001 GACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTTA 4051 TAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTA 4101 ACAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGT 4151 GGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATATTTGTTTATTTTTC 4201 TAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAAT 4251 GCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTG 4301 TCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTACTGTTTTTGCTCAC 4351 CCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACG 4401 AGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTT 4451 TTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTA 4501 TGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCG 4551 CCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAG 4601 AAAAGCATATTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCC 4651 ATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGG 4701 AGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAA 4751 CTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGAC 4801 GAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACT 4851 ATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACT 4901 GGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCG 4951 GCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCG 5001 CGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAG 5051 TTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAG 5101 ATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA 5151 AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTA 5201 AAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCT 5251 TAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAA 5301 AGGATGTTCTTGAGATCCTTTTTTTCTGCACGTAATCTGCTGCTTGCAAA 5351 CAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTAC 5401 CAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAAT 5451 ACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGT 5501 AGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTG 5551 CCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTA 5601 CCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCC 5651 CAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGC 5701 TATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCG 5751 GTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGG 5801 AAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTG 5851 AGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAAC 5901 GCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGC 5951 TCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA 6001 CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGC 6051 AGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCC 6101 TCTCCCCGCGCGTTGGCCGATTCATTAATGCAGGTATCACGAGGCCCTTT 6151 CGTCTTCAC

The above nucleic acid sequence corresponds to SEQ ID NO:7. The above amino acid sequence, including any underlined amino acid sequence corresponds to the sequence of SEQ ID NO:9.

TABLE 7  pREN 806 HC DHFR Vector Xho I 1 CTCGAGAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTC 52 ATTAGGCACCCCAGGCTTTACACTTTATGCTCCCGGCTCGTATGTTGTGT                                   EcoRI     EF1α promoter 102 GGAGATTGTGAGCGGATAACAATTTCACACAGAATTCGTGAGGCTCCGGT 152 GCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGG 202 GGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAA 252 ACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGG 302 GGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAA 352 CGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGC 402 CTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACG 452 CCCCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTG 502 GGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCT 552 TGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTG 602 GCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAA 652 ATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTA 702 AATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGG 752 CGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCC 802 TGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGG 852 CCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGC 902 GGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGC 952 TTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGA 1002 GAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTC 1052 AGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACC 1102 TCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAG 1152 GGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGT 1202 TAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTG 1251 AGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTT                    MluI        HindIII    PmeI 1302 TTTCTTCCATTTCAGGTGTACGCGTCTCGGGAAGCTTTAGTTTAAACGCC 1352 GCCACCATGAGAGTGCTGATTCTTTTGTGGCTGTTCACAGCCTTTCCTGGT       M  R  V  L  I  L  L  W  L  F  T  A  F  P  G 1401 GTCCTGTCTGATGTGCAGCTTCAGGAGTCGGGACCTAGCCTGGTGAAACCT       V L S D V Q L Q E S G P S L V K P 1451 TCTCAGACTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGT  S  Q  T  L  S  L  T  C  T  V  G  Y  I  T  S  T  S 1501 GATTTTGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAGCTGGAGTGG  D  F  A  W  N  W  I  R  Q  F  P  G  N  K  L  E  W 1551 ATGGGCTACATAAGTTATAGTGGTAACACTAGGTACAACCCATCTCTCAAA  M  G  Y  I  S  Y  S  G  N  T  R  Y  N  P  S  L  K 1601 AGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAATTCTTCCTGCAG  S  R  I  S  I  T  R  D  T  S  K  N  Q  F  F  L 1651 TTGAATTCTGTGACTATTGAGGACACAGCCACATATTACTGTGTAACGGCG               1701 GGACGCGGGTTTCCTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA  G  R  G  F  P  Y  W  G  Q  G  T  L  V  T  V  S  A         BamHI 1751 CAGTGAGTGGATCCTCTGCGCCTGGGCCCAGCTCTGTC 1801 CCACACCGCGGTCACATGGCACCACCTCTCTTGCAGCCTCCACCAAGGGC                                       S  T  K  G 1851 CCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCAC p  s  v  f  p  l  a  p  s  s  k  s  t  s  g  g  t 1901 AGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG  A  A  L  G  C  L  V  K  D  Y  F  P  E  P  V  T  V 1951 TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT s  w  n  s  g  a  l  t  s  g  v  h  t  f  p  a 2001 GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCC v  l  q  s  s  g  l  y  s  l  s  s  v  y  s  v  p 2051 CTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGC  S  S  S  L  G  T  Q  T  Y  I  C  N  V  N  H  K  P 2101 CCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAA   S  N  T  K  V  D  K  K  V  E  P  K  S  C  D  K 2151 ACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTC T  H  T  C  P  P  C  P  A  P  E  L  L  G  G  P  S 2201 AGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA  V  F  L  F  P  P  K  P  K  D  T  L  M  I  S  R  T 2251 CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG   P  E  V  T  C  V  V  V  D  V  S  H  E  D  P  E 2301 GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAACGCCAAGAC V  K  F  N  W  Y  V  D  G  V  E  V  H  N  A  K  T 2351 AAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCC  K  P  R  E  E  Q  Y  N  S  T  Y  R  V  V  S  V  L 2401 TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG   T  V  L  H  Q  D  W  L  N  G  K  E  Y  K  C  K 2451 GTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGC V  S  N  K  A  L  P  A  P  I  E  K  T  I  S  K  A 2501 CAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGG  K  G  Q  P  R  E  P  Q  V  Y  T  L  P  P  S  R  E 2551 AGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC   E  M  T  K  N  Q  V  S  L  T  C  L  V  K  G  F 2601 TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA Y  P  S  D  I  A  V  E  W  E  S  N  G  Q  P  E  N 2651 CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC  N  Y  K  T  T  P  P  V  L  D  S  D  G  S  F  F  L 2701 TCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTC   Y  S  K  L  T  V  D  K  S  R  W  Q  Q  G  N  V 2751 TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAA F  S  C  S  V  M  H  E  A  L  H  N  H  Y  T  Q  K                                         Nhe/Xba 2801 GAGCCTCTCCCTGTCTCCGGGTAAATGAGCTAGAAACTAACTAAGCTAGC  S  L  S  L  S  P  G  K  * 2851 AACGGTTTCCCTCTAGCGGGATCAATTCCGCCCCCCCCCCCTAACGTTAC 2901 TGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTAT 2951 TTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGC 3001 CCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGG 3051 AATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTT 3101 CTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCC 3151 CCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATAC 3201 ACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTG 3251 TGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAG 3301 GATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTG 3351 CACATGCTTTACGTGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCC 3401 GAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATAATACCATGGTT 3451 CGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATTGGCAA 3501 GAACGGAGACCTACCCTGGCCTCCGCTCAGGAACGAGTTCAAGTACTTCC 3551 AAAGAATGACCACAACCTCTTCAGTGGAAGGTAAACAGAATCTGGTGATT 3601 ATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAA 3651 GGACAGAATTAATGGTTCGATATAGTTCTCAGTAGAGAACTCAAAGAACC 3701 ACCACGAGGAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCCTTAAGAC 3751 TTATTGAACAACCGGAATTGGCAAGTAAAGTAGACATGGTTTGGATAGTC 3801 GGAGGCAGTTCTGTTTACCAGGAAGCCATGAATCAACCAGGCCACCTCAG 3851 ACTCTTTGTGACAAGGATCATGCAGGAATTTGAAAGTGACACGTTTTTCC 3901 CAGAAATTGATTTGGGGAAATATAAACTTCTCCCAGAATACCCAGGCGTC 3951 CTCTCTGAGGTCCAGGAGGAAAAAGGCATCAAGTATAAGTTTGAAGTCTA 4001 CGAGAAGAAAGACTAACAGGAAGATGCTTTCAAGTTCTCTGCTCCCCTCC Blunt end SalI/SalI 4051 TAAAGCTATGCATTTTTATAAGACCATGGGACTTTTGCTGGTCGATCGAC 4101 CTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGC 4151 GCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCG 4201 GCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCT 4251 AGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCG 4301 GCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTT 4351 AGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTC 4401 ACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCTTTGACGTTGGA 4451 GTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCA 4501 ACCCTATCTCGGTCTATTTATAAGGGATTTTGCCGATTTCGGCCTATTGG 4551 TTAAAAAATGAGCTGATTTAACAAAATTTAACGCGAATTTTAACAAAATA 4601 TTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACC 4651 CCTATATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATG 4701 AGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTAT 4751 GAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTT 4801 GCCTTACTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCT 4851 GAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAG 4901 CGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGA 4951 GCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCC 5001 GGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGT 5051 TGAGTACTCACCAGTCACAGAAAAGCATATTACGGATGGCATGACAGTAA 5101 GAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAAC 5151 TTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCA 5201 CAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGA 5251 ATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATG 5301 GCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTC 5351 CCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCAC 5401 TTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGA 5451 GCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGG 5501 TAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTA 5551 TGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAG 5601 CATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTT 5651 AAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATA 5701 ATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCA 5751 GACCCCGTAGAAAAGATCAAAGGATGTTCTTGAGATCCTTTTTTTCTGCA 5801 CGTAATCTGCTGCTTGCAAACAAAAAACCACCGCTACCAGCGGTGGTTTG 5851 TTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCA 5901 GCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGC 5951 CACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAAT 6001 CCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGT 6051 TGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG 6101 GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACT 6151 GAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGA 6201 GAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGC 6251 ACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGG 6301 GTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGG 6351 GGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTG 6401 GCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGA 6451 TTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCC 6501 GCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAG 6551 CGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG 6601 CAGGTATCACGAGGCCCTTTCGTCTTCAC

The above nucleic acid sequence corresponds to SEQ ID NO:8. The above amino acid sequence, including any underlined amino acid sequence corresponds to the sequence of SEQ ID NO:10.

Claims

1. An isolated antibody produced by hybridoma cell line 806 (ATCC Accession No. PTA-3858).

2. The hybridoma cell line 806 deposited under ATCC Accession No. PTA-3858.

Referenced Cited
U.S. Patent Documents
4137230 January 30, 1979 Hashimoto et al.
4151042 April 24, 1979 Higashide et al.
4169888 October 2, 1979 Hanka et al.
4190580 February 26, 1980 Hashimoto et al.
4225494 September 30, 1980 Higashide et al.
4248870 February 3, 1981 Miyashita et al.
4256746 March 17, 1981 Miyashita et al.
4260608 April 7, 1981 Miyashita et al.
4263294 April 21, 1981 Miyashita et al.
4264596 April 28, 1981 Miyashita et al.
4265814 May 5, 1981 Hashimoto et al.
4294757 October 13, 1981 Asai
4307016 December 22, 1981 Asai et al.
4308268 December 29, 1981 Miyashita et al.
4308269 December 29, 1981 Miyashita et al.
4309428 January 5, 1982 Miyashita et al.
4313946 February 2, 1982 Powell et al.
4317821 March 2, 1982 Miyashita et al.
4322348 March 30, 1982 Asai et al.
4331598 May 25, 1982 Hasegawa et al.
4342566 August 3, 1982 Theofilopoulos et al.
4360462 November 23, 1982 Higashide et al.
4361650 November 30, 1982 Asai et al.
4362663 December 7, 1982 Kida et al.
4364866 December 21, 1982 Asai et al.
4371533 February 1, 1983 Akimoto et al.
4413132 November 1, 1983 Wierenga
4418064 November 29, 1983 Powell et al.
4671958 June 9, 1987 Rodwell et al.
4762707 August 9, 1988 Jansen et al.
4816567 March 28, 1989 Cabilly et al.
4867973 September 19, 1989 Goers et al.
4933294 June 12, 1990 Waterfield et al.
4937183 June 26, 1990 Ultee et al.
4943533 July 24, 1990 Mendelsohn et al.
4952394 August 28, 1990 Senter
4997913 March 5, 1991 Hellstrom et al.
5013547 May 7, 1991 Sweet et al.
5028697 July 2, 1991 Johnson et al.
5034223 July 23, 1991 Abrams et al.
5047324 September 10, 1991 Fredrickson
5087616 February 11, 1992 Myers et al.
5106951 April 21, 1992 Morgan, Jr. et al.
5122368 June 16, 1992 Greenfield et al.
5130116 July 14, 1992 Woo et al.
5141736 August 25, 1992 Iwasa et al.
5164311 November 17, 1992 Gupta
5171563 December 15, 1992 Abrams et al.
5208020 May 4, 1993 Chari et al.
5212290 May 18, 1993 Vogelstein et al.
5217713 June 8, 1993 Iwasa et al.
5225539 July 6, 1993 Winter
5306809 April 26, 1994 Boon et al.
5332837 July 26, 1994 Kelly et al.
5401828 March 28, 1995 Vogelstein et al.
5416064 May 16, 1995 Chari et al.
5457105 October 10, 1995 Barker et al.
5459061 October 17, 1995 Sato et al.
5475092 December 12, 1995 Chari et al.
5541339 July 30, 1996 Kelly et al.
5556623 September 17, 1996 Barton et al.
5558864 September 24, 1996 Bendig et al.
5563250 October 8, 1996 Hylarides et al.
5585499 December 17, 1996 Chari et al.
5606017 February 25, 1997 Willner et al.
5612474 March 18, 1997 Patel
5622929 April 22, 1997 Willner et al.
5635483 June 3, 1997 Pettit et al.
5635603 June 3, 1997 Hansen et al.
5639641 June 17, 1997 Pedersen et al.
5643573 July 1, 1997 Barton et al.
5665358 September 9, 1997 Barton et al.
5674977 October 7, 1997 Gariepy
5677171 October 14, 1997 Hudziak et al.
5708146 January 13, 1998 Willner et al.
5708156 January 13, 1998 Ilekis et al.
5720954 February 24, 1998 Hudziak et al.
5739350 April 14, 1998 Kelly et al.
5760041 June 2, 1998 Wissner et al.
5770195 June 23, 1998 Hudziak et al.
5780588 July 14, 1998 Pettit et al.
5795965 August 18, 1998 Tsuchiya et al.
5807715 September 15, 1998 Morrison et al.
5814317 September 29, 1998 Vogelstein et al.
5824805 October 20, 1998 King et al.
5844093 December 1, 1998 Kettleborough et al.
5846545 December 8, 1998 Chari et al.
5851526 December 22, 1998 Welt et al.
5869045 February 9, 1999 Hellstrom et al.
5869619 February 9, 1999 Studnicka
5880270 March 9, 1999 Berninger et al.
5891996 April 6, 1999 Mateo de Acosta del Rio et al.
5911995 June 15, 1999 Uckun
5942602 August 24, 1999 Wels et al.
5980896 November 9, 1999 Hellstrom et al.
6010902 January 4, 2000 Ledbetter et al.
6060608 May 9, 2000 Boger
6214345 April 10, 2001 Firestone et al.
6217866 April 17, 2001 Schlessinger et al.
6224868 May 1, 2001 Wong et al.
6235883 May 22, 2001 Jakobovits et al.
6281354 August 28, 2001 Boger
6306393 October 23, 2001 Goldenberg et al.
6331175 December 18, 2001 Goldenberg
6331415 December 18, 2001 Cabilly et al.
6333410 December 25, 2001 Chari et al.
6340701 January 22, 2002 Chari et al.
6355683 March 12, 2002 Baell et al.
6372738 April 16, 2002 Chari et al.
6395272 May 28, 2002 Deo et al.
6436931 August 20, 2002 Chari et al.
6441163 August 27, 2002 Chari et al.
6506883 January 14, 2003 Meteo de Acosta del Rio et al.
6512101 January 28, 2003 King et al.
6534660 March 18, 2003 Yongxin et al.
6548530 April 15, 2003 Boger
6570024 May 27, 2003 Eldridge et al.
6586618 July 1, 2003 Zhao et al.
6596757 July 22, 2003 Chari et al.
6630579 October 7, 2003 Chari et al.
6660742 December 9, 2003 Lee
6675105 January 6, 2004 Hogarth et al.
6699715 March 2, 2004 Ledbetter et al.
6706708 March 16, 2004 Chari et al.
6716821 April 6, 2004 Zhao et al.
6756397 June 29, 2004 Zhao et al.
6759509 July 6, 2004 King et al.
6790954 September 14, 2004 Chung et al.
6797492 September 28, 2004 Daugherty et al.
6835753 December 28, 2004 Baell et al.
6849625 February 1, 2005 Lambert et al.
6884869 April 26, 2005 Senter et al.
6884874 April 26, 2005 Eldridge et al.
6913748 July 5, 2005 Widdison
6941229 September 6, 2005 Elleman et al.
6946543 September 20, 2005 Ward et al.
6989452 January 24, 2006 Ng et al.
7008942 March 7, 2006 Chari et al.
7049316 May 23, 2006 Zhao et al.
7060808 June 13, 2006 Goldstein et al.
7091186 August 15, 2006 Senter et al.
7097840 August 29, 2006 Erickson et al.
7098308 August 29, 2006 Senter et al.
7129261 October 31, 2006 Ng et al.
7129332 October 31, 2006 Pastan et al.
7132511 November 7, 2006 Carr et al.
7132554 November 7, 2006 Rose
7192750 March 20, 2007 Chung et al.
7214685 May 8, 2007 Tietze et al.
7217819 May 15, 2007 Chari et al.
7223837 May 29, 2007 De Groot et al.
7226592 June 5, 2007 Kreysch
7247301 July 24, 2007 van de Winkel et al.
7256257 August 14, 2007 Doronina et al.
7276497 October 2, 2007 Chari et al.
7276499 October 2, 2007 Chari et al.
7276585 October 2, 2007 Lazar et al.
7301019 November 27, 2007 Widdison et al.
7303749 December 4, 2007 Chari
7329760 February 12, 2008 Zhao et al.
7368565 May 6, 2008 Chari et al.
7374762 May 20, 2008 Amphlett et al.
7375078 May 20, 2008 Feng
7388026 June 17, 2008 Zhao et al.
7390898 June 24, 2008 Baloglu et al.
7396810 July 8, 2008 Clinton
7411063 August 12, 2008 Widdison et al.
7414073 August 19, 2008 Baloglu et al.
7423116 September 9, 2008 Doronina et al.
7432088 October 7, 2008 Kuo et al.
7449559 November 11, 2008 Ward et al.
7473796 January 6, 2009 Chari et al.
7476669 January 13, 2009 Chari et al.
7494649 February 24, 2009 Amphlett et al.
7495114 February 24, 2009 Baloglu et al.
7498298 March 3, 2009 Doronina et al.
7498302 March 3, 2009 Ng et al.
7501120 March 10, 2009 Amphlett et al.
7514080 April 7, 2009 Amphlett et al.
7517903 April 14, 2009 Chen et al.
7521541 April 21, 2009 Eigenbrot et al.
7528130 May 5, 2009 Chari et al.
7550609 June 23, 2009 Chari et al.
7553809 June 30, 2009 Baell et al.
7553816 June 30, 2009 Senter et al.
7575748 August 18, 2009 Erickson et al.
7585857 September 8, 2009 Chari et al.
7589180 September 15, 2009 Old et al.
7595378 September 29, 2009 van de Winkel et al.
7598290 October 6, 2009 Miller et al.
7598375 October 6, 2009 Ho et al.
7601354 October 13, 2009 Chari
7625558 December 1, 2009 Greene et al.
7628986 December 8, 2009 Weber et al.
7635570 December 22, 2009 Siena et al.
7651687 January 26, 2010 Buck et al.
7655660 February 2, 2010 Zhao et al.
7655661 February 2, 2010 Zhao et al.
7659241 February 9, 2010 Senter et al.
7667054 February 23, 2010 Miller et al.
7691962 April 6, 2010 Boyd et al.
7723484 May 25, 2010 Beidler et al.
7736644 June 15, 2010 Weber et al.
7745394 June 29, 2010 Doronina et al.
7750116 July 6, 2010 Doronina et al.
7754681 July 13, 2010 Feng
7767792 August 3, 2010 Johns et al.
7790164 September 7, 2010 Cao
7807798 October 5, 2010 Jakobovits et al.
20010005747 June 28, 2001 Ball et al.
20010036923 November 1, 2001 Chari et al.
20010046686 November 29, 2001 Wong et al.
20010048922 December 6, 2001 Romet-Lemonne et al.
20010055595 December 27, 2001 Goldenberg
20020001587 January 3, 2002 Erickson et al.
20020004587 January 10, 2002 Miller et al.
20020006379 January 17, 2002 Hansen et al.
20020012663 January 31, 2002 Waksal
20020013485 January 31, 2002 Chari et al.
20020049335 April 25, 2002 Boger
20020062009 May 23, 2002 Taylor
20020064785 May 30, 2002 Mass
20020082424 June 27, 2002 Boger
20020156274 October 24, 2002 Terfloth
20020173629 November 21, 2002 Jakobovits et al.
20030009152 January 9, 2003 O'hara et al.
20030050331 March 13, 2003 Ng et al.
20030054497 March 20, 2003 Co et al.
20030055226 March 20, 2003 Chari et al.
20030073731 April 17, 2003 Lee
20030073852 April 17, 2003 Ng et al.
20030083263 May 1, 2003 Doronina et al.
20030091561 May 15, 2003 van de Winkel et al.
20030096743 May 22, 2003 Senter et al.
20030109682 June 12, 2003 Santi et al.
20030130189 July 10, 2003 Senter et al.
20030190363 October 9, 2003 O'connor et al.
20030194403 October 16, 2003 van de Winkel et al.
20030195365 October 16, 2003 Zhao et al.
20030199519 October 23, 2003 Zhao et al.
20030211097 November 13, 2003 Pastan et al.
20030211112 November 13, 2003 Debinski
20030215387 November 20, 2003 Harrison
20030224001 December 4, 2003 Goldstein et al.
20040006212 January 8, 2004 Goldstein et al.
20040033543 February 19, 2004 Schwab et al.
20040086943 May 6, 2004 Andres et al.
20040109867 June 10, 2004 Yongxin et al.
20040131611 July 8, 2004 Oliver et al.
20040144948 July 29, 2004 Akhavan-Tafti et al.
20040147428 July 29, 2004 Pluenneke
20040157782 August 12, 2004 Doronina et al.
20040202666 October 14, 2004 Griffiths
20040235074 November 25, 2004 Siegall et al.
20040235840 November 25, 2004 Chari et al.
20040248196 December 9, 2004 Adams et al.
20040253645 December 16, 2004 Daugherty et al.
20050009751 January 13, 2005 Senter et al.
20050014700 January 20, 2005 Boger
20050026987 February 3, 2005 Boger
20050031627 February 10, 2005 Mazzola et al.
20050032860 February 10, 2005 Boger
20050053608 March 10, 2005 Weber et al.
20050059087 March 17, 2005 Weber et al.
20050064492 March 24, 2005 DeSauvage et al.
20050100546 May 12, 2005 Jakobovits et al.
20050106644 May 19, 2005 Cairns et al.
20050107595 May 19, 2005 Cairns et al.
20050113308 May 26, 2005 Senter et al.
20050113571 May 26, 2005 Terfloth
20050142133 June 30, 2005 Lazar et al.
20050152913 July 14, 2005 Eldridge
20050169933 August 4, 2005 Steeves et al.
20050214310 September 29, 2005 Toki et al.
20050227324 October 13, 2005 Huang et al.
20050238640 October 27, 2005 Sliwkowski
20050238649 October 27, 2005 Doronina et al.
20050255555 November 17, 2005 Johns et al.
20050256030 November 17, 2005 Feng
20050271671 December 8, 2005 Griffiths
20050272083 December 8, 2005 Seshagiri
20050272798 December 8, 2005 Ng et al.
20050276812 December 15, 2005 Ebens et al.
20060004081 January 5, 2006 Chen et al.
20060009462 January 12, 2006 Yongxin et al.
20060024317 February 2, 2006 Boyd et al.
20060029574 February 9, 2006 Albitar et al.
20060074008 April 6, 2006 Senter et al.
20060084141 April 20, 2006 Floss et al.
20060088523 April 27, 2006 Andya et al.
20060116422 June 1, 2006 De Groot et al.
20060121044 June 8, 2006 Amler et al.
20060147959 July 6, 2006 Bell et al.
20060154334 July 13, 2006 Tarnowski et al.
20060165685 July 27, 2006 Kreysch
20060182750 August 17, 2006 Chari et al.
20060183887 August 17, 2006 Jakobovits et al.
20060229253 October 12, 2006 Doronina et al.
20060234343 October 19, 2006 Ward et al.
20060247295 November 2, 2006 Gangwar et al.
20070031402 February 8, 2007 Zhang et al.
20070037972 February 15, 2007 Ho et al.
20070048314 March 1, 2007 Dai et al.
20070071675 March 29, 2007 Wu et al.
20070092940 April 26, 2007 Eigenbrot et al.
20070112188 May 17, 2007 Widdison et al.
20070116707 May 24, 2007 Goldstein et al.
20070134243 June 14, 2007 Gazzard et al.
20070135346 June 14, 2007 Zhao et al.
20070202101 August 30, 2007 Rosen et al.
20070264266 November 15, 2007 Chari et al.
20070269447 November 22, 2007 Chari et al.
20070270585 November 22, 2007 Chari et al.
20080008704 January 10, 2008 Rubin
20080025983 January 31, 2008 Adams et al.
20080114153 May 15, 2008 Steeves et al.
20080145374 June 19, 2008 Steeves et al.
20080171040 July 17, 2008 Ebens et al.
20080171856 July 17, 2008 Steeves et al.
20080171865 July 17, 2008 Steeves et al.
20080226657 September 18, 2008 Doronina et al.
20080226659 September 18, 2008 Erickson et al.
20080241128 October 2, 2008 Jeffrey
20080248051 October 9, 2008 Doronina et al.
20080248053 October 9, 2008 Doronina et al.
20080249085 October 9, 2008 Cassady et al.
20080260685 October 23, 2008 Zhao et al.
20080267960 October 30, 2008 Drachman et al.
20080279868 November 13, 2008 Boyd et al.
20080281102 November 13, 2008 Gangwar et al.
20080293800 November 27, 2008 Gangwar et al.
20080300192 December 4, 2008 Doronina et al.
20080305044 December 11, 2008 McDonagh et al.
20080311136 December 18, 2008 Beusker et al.
20090010945 January 8, 2009 Alley et al.
20090018086 January 15, 2009 Doronina et al.
20090028821 January 29, 2009 Zhao et al.
20090031668 February 5, 2009 Llorente Gonzalez et al.
20090041791 February 12, 2009 Feng
20090047296 February 19, 2009 Doronina et al.
20090053240 February 26, 2009 Lazar et al.
20090111756 April 30, 2009 Doronina et al.
20090117134 May 7, 2009 Ward et al.
20090137782 May 28, 2009 Old et al.
20090142361 June 4, 2009 Amphlett et al.
20090155282 June 18, 2009 Weber et al.
20090156790 June 18, 2009 Weber et al.
20090175865 July 9, 2009 Eigenbrot et al.
20090175887 July 9, 2009 Weber et al.
20090175888 July 9, 2009 Ng et al.
20090202536 August 13, 2009 Ebens, Jr. et al.
20090214541 August 27, 2009 Gillies et al.
20090220510 September 3, 2009 Old et al.
20090240038 September 24, 2009 Weber et al.
20090269343 October 29, 2009 Bigner et al.
20090274713 November 5, 2009 Chari et al.
20090280503 November 12, 2009 Fiore
20090281158 November 12, 2009 Zhao et al.
20090297509 December 3, 2009 Waksal et al.
20090304693 December 10, 2009 Ghayur et al.
20090306101 December 10, 2009 Solca et al.
20090318668 December 24, 2009 Beusker et al.
20090324621 December 31, 2009 Senter et al.
20100008929 January 14, 2010 Van De Winkel et al.
20100056762 March 4, 2010 Old
20100092475 April 15, 2010 Johns et al.
20100166744 July 1, 2010 Wong
20100196265 August 5, 2010 Adams et al.
20100203007 August 12, 2010 Li et al.
20100322937 December 23, 2010 Johns et al.
20110008766 January 13, 2011 Ghayur et al.
20110076232 March 31, 2011 Old et al.
20110150759 June 23, 2011 Johns et al.
20110313230 December 22, 2011 Johns et al.
20120183471 July 19, 2012 Old et al.
20130095112 April 18, 2013 Overgaard
Foreign Patent Documents
120694 October 1984 EP
125023 November 1984 EP
519596 December 1992 EP
0586002 March 1994 EP
0699755 March 1996 EP
1510221 March 2005 EP
1392359 October 2009 EP
2163256 March 2010 EP
8503357 August 1985 WO
9103489 March 1991 WO
9116350 October 1991 WO
9215683 September 1992 WO
9311161 June 1993 WO
9413804 June 1994 WO
WO-95/20045 July 1995 WO
9525167 September 1995 WO
9616988 June 1996 WO
96040210 December 1996 WO
9944645 September 1999 WO
0211677 February 2002 WO
02092771 November 2002 WO
03014149 February 2003 WO
03014159 February 2003 WO
2004003019 January 2004 WO
2004056847 July 2004 WO
2004085474 October 2004 WO
2005081854 September 2005 WO
2005094357 October 2005 WO
2007089149 August 2007 WO
2007103288 September 2007 WO
2008033495 March 2008 WO
2008091701 July 2008 WO
2008154927 December 2008 WO
2009017394 February 2009 WO
2009023265 February 2009 WO
2008115404 October 2009 WO
2010096434 August 2010 WO
2011035465 March 2011 WO
2011041319 April 2011 WO
WO-2012/135360 August 2012 WO
Other references
  • Riemer et al. (Mol. Immunol. 2005; 42: 1121-1124).
  • Jiang et al. (J. Biol. Chem. Feb. 11, 2005; 280 (6): 4656-4662).
  • Rudikoff et al (Proc. Natl. Acad. Sci. USA. 1982; 79: 1979-1983).
  • Greenspan et al. (Nature Biotechnology. 1999; 7: 936-937).
  • Abbruzzese J.L., et al., “Phase II Study of Anti-Epidermal Growth Factor Receptor (EGFR) Antibody Cetuximab (IMC-C225) in Combination with Gemcitabine in Patients with Advanced Pancreatic Cancer,” Proceedings of American Society of Clinical Oncology, 2001, vol. 20, pp. 130A.
  • Aboud-Pirak E., et al., “Efficacy of Antibodies to Epidermal Growth Factor Receptor Against KB Carcinoma in Vitro and in Nude Mice,” Journal of the National Cancer Institute, 1988, vol. 80 (20), pp. 1605-1611.
  • Aboud-Pirak E., et al., “Inhibition of Human Tumor Growth in Nude Mice by a Conjugate of Doxorubicin with Monoclonal Antibodies to Epidermal Growth Factor Receptor,” Proceedings of the National Academy of Sciences, 1989, vol. 86 (10), pp. 3778-3781.
  • Aboud-Pirak, et al., “Efficacy of Antibodies to Epidermal Growth Factor Receptor Against Kb Carcinoma In Vitro and in Nude Mice,” Chemical Abstracts, 1989, vol. 110 (9), pp. 69068k.
  • Adams G.P., et al., “Monoclonal Antibody Therapy of Cancer,” Nature Biotechnology, 2005, vol. 23 (9), pp. 1147-1157.
  • Aden D.P., et al., “Cell Surface Antigens Coded for by the Human Chromosome 7,” Immunogenetics, 1976, vol. 3 (1), pp. 209-221.
  • Aghajanian, et al., “A Phase I Study of Cetuximab/ Paclitaxellcarboplatin for the Initial Treatment of Advanced Stage Ovarian, Primary Peritoneal, and Fallopian Tube Cancer,” Journal of Clinical Oncology, ASCO Annual Meeting Proceedings, 2005, Abstract 5047, pp. 16S.
  • Agosti R.M., et al., “Expression of the Epidermal Growth Factor Receptor in Astrocytic Tumours is Specifically Associated with Glioblastoma Multiforme,” Virchows Archiv. A, Pathological Anatomy and Histopathology, 1992, vol. 420 (4), pp. 321-325.
  • Agulnik M., et al., “Predictive and Pharmacodynamic Biomarker Studies in Tumor and Skin Tissue Samples of Patients with Recurrent or Metastatic Squamous Cell Carcinoma of the Head and Neck Treated with Erlotinib,” Journal of Clinical Oncology, 2007, vol. 25 (16), pp. 2184-2190.
  • Agus D.B., et al., “Phase I Clinical Study of Pertuzumab, A Novel Her Dimerization Inhibitor, In Patients with Advanced Cancer,” Journal of Clinical Oncology, 2005, vol. 23 (11), pp. 2534-2543.
  • Agus D.B., et al., “Targeting Ligand-Activated ErbB2 Signaling Inhibits Breast and Prostate Tumor Growth,” Cancer Cell, 2002, vol. 2 (2), pp. 127-137.
  • Akiyama T., et al., “Genistein, a Specific Inhibitor of Tyrosine-specific Protein Kinases,” Journal of Biological Chemistry, 1987, vol. 262 (12), pp. 5592-5595.
  • Albanell J., et al., “Activated Extracellular Signal-Regulated Kinases: Association with Epidermal Growth Factor Receptor/Transforming Growth Factor Alpha Expression in Head and Neck Squamous Carcinoma and Inhibition by Anti-Epidermal Growth Factor Receptor Treatments,” Cancer Research, 2001, vol. 61 (17), pp. 6500-6510.
  • Albanell J., et al., “Pharmacodynamic Studies of the Epidermal Growth Factor Receptor Inhibitor Zd1839 in Skin from Cancer Patients: Histopathologic and Molecular Consequences of Receptor Inhibition,” Journal of Clinical Oncology, 2002, vol. 20 (1), pp. 110-124.
  • Albanell J., et al., “Pharmacodynamic Studies With the Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor ZD1839,” Seminars in Oncology , 2001, vol. 28 (5) (suppl. 16), pp. 56-66.
  • Aldape K.D., et al., “Immunohistochemical Detection of Egfrviii in High Malignancy Grade Astrocytomas and Evaluation of Prognostic Significance,” Journal of Neuropathology & Experimental Neurology, 2004, vol. 63 (7), pp. 700-707.
  • Alimirah F., et al., “Du-145 and Pc-3 Human Prostate Cancer Cell Lines Express Androgen Receptor: Implications for the Androgen Receptor Functions and Regulation,” FEBS Letters, 2006, vol. 580 (9), pp. 2294-2300.
  • Alroyl., et al., “The Erbb Signaling Network in Embryogenesis and Oncogenesis: Signal Diversification Through Combinatorial Ligand-Receptor Interactions,” FEBS Letters, 1997, vol. 410 (1), pp. 83-36.
  • Anderson N.G., et al., “ZD1839 (Iressa), a Novel Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor, Potently Inhibits the Growth of EGFR-Positive Cancer Cell Lines with or without erbB2 Overexpression,” International Journal of Cancer, 2001, vol. 94 (6), pp. 774-782.
  • Andrews P.A., et al., “Cellular Pharmacology of Cisplatin: Perspectives on Mechanisms of Acquired Resistance,” Cancer Cells, 1990, vol. 2 (2), pp. 35-43.
  • Ang K.K., et al., “Impact of epidermal growth factor receptor expression on survival and pattern of relapse in patients with advanced head and neck carcinoma,” Cancer Research, 2002, vol. 62 (24), pp. 7350-7356.
  • Anido J., et al., “ZD1839, a Specific Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor, Induces the Formation of Inactive EGFR/HER2 and EGFR/HER3 Heterodimers and Prevents Heregulin Signaling in HER2-overexpressing Breast Cancer Cells,” Clinical Cancer Research , 2003, vol. 9 (4), pp. 1274-1283.
  • Annual Report 1999-2000, Ludwig Institute for Cancer Research.
  • Annual Report 2002-2003, Ludwig Institute for Cancer Research, 2003, pp. 8, 84-86 and 99-100.
  • Annual Research Highlights Report 2005, Ludwig Institute for Cancer Research, pp. 3.
  • Annual Research Highlights Report 2006, Ludwig Institute for Cancer Research, 2007, pp. 1-56.
  • Annual Research Report 2002-2003, Ludwig Institute for Cancer Research, 2003. pp. 1-7.
  • Annual Search Report 2003, Ludwig Institute for Cancer Research, 2003, pp. 81-83, 93 and 152.
  • Annual Search Report 2004, Ludwig Institute for Cancer Research, 2005, pp. 12, 79, 83-84, 98, 204 and 240.
  • Annual Search Report 2005, Ludwig Institute for Cancer Research, pp. 3, 27, 29-31 and 36.
  • Archer G.E., et al., “Regional Treatment of Epidermal Growth Factor Receptor VIII-expressing Neoplastic Meningitis with a Single-Chain Immunotoxin, MR-1,” Clinical Cancer Research , 1999, vol. 5 (9), pp. 2646-2652.
  • Arteaga C.L., “The Epidermal Growth Factor Receptor: From Mutant Oncogene in Nonhuman Cancers to Therapeutic Target in Human Neoplasia,” Journal of Clinical Oncology, 2001, vol. 19 (suppl. 18), pp. 32S-42S.
  • Arteaga C.L., “Epidermal Growth Factor Receptor Dependence in Human Tumors: More Than Just Expression,” The Oncologist, 2002, vol. 7 (suppl. 4), pp. 31-39.
  • Arteaga C.L., “ErbB-Targeted Therapeutic Approaches in Human Cancer,” Experimental Cell Research, 2003, vol. 284 (1), pp. 122-130.
  • Arteaga C.L., et al. , “Unliganded Epidermal Growth Factor Receptor Dimerization Induced by Direct Interaction of Quinazolines with the ATP Binding Site,” Journal of Biological Chemistry, 1997, vol. 272 (37), pp. 23247-23254.
  • Arteaga C.L., et al., “Overview of Rationale and Clinical Trials With Signal Transduction Inhibitors in Lung Cancer,” Seminars in Oncology , 2002, vol. 29 (1) (suppl. 4), pp. 15-26.
  • Arteaga C.L., et al., “Tyrosine Kinase Inhibitors:Why Does the Current Process of Clinical Development Not Apply to Them”, Cancer Cell, 2004, vol. 5 (6), pp. 525-531.
  • Arteaga C.L., et al., “Tyrosine kinase inhibitors-ZD1839 (Iressa),” Current Opinion in Oncology, 2001, vol. 13 (6), pp. 491-498.
  • Arteaga C.L., “Overview of Epidermal Growth Factor Receptor Biology and its Role as a Therapeutic Target in Human Neoplasia,” Seminars in Oncology, 2002, vol. 29 (5 Suppl. 14), pp. 3-9.
  • Arteaga, et al., “Antibodies Against P185her2 Enhance Etoposide-Induced Cytotoxicity Against Human Breast Carcinoma Cells,” Proceedings of the American Society of Clinical Oncology, 1993, vol. 75 (Abstract 101), pp. 12.
  • Ashley D.M., et al., “Monoclonal Antibodies to Growth Factors and Growth Factor Receptors: Their Diagnostic and Therapeutic Potential in Brain Tumors,” Journal of Neuro-oncology, 1997, vol. 35 (3), pp. 259-273.
  • Atlas I., et al., “Growth Regulation of Human Renal Carcinoma Cells: Role of Transforming Growth Factor,” Journal of Cancer Research, 1992, vol. 52 (12), pp. 3335-3339.
  • Aug K.K., et al., “Epidermal Growth Factor Receptor and Response of Head-and-neck carcinoma to therapy,” 2004, vol. 58 (3), pp. 959-965.
  • Aujame L., et al., “High Affinity Human Antibodies by Phage Display,” Human Antibodies, 1997, vol. 8 (4), pp. 155-168.
  • Austin C.D., et al., “Endocytosis and Sorting of Erbb2 and the Site of Action of Cancer Therapeutics Trastuzumab and Geldanamycin,” Molecular Biology of the Cell, 2004, vol. 15 (12), pp. 5268-5282.
  • Avital S., et al., “Radioimmunoguided Surgery for Recurrent Colorectal Cancer Manifested by Isolated CEA Elevation,” Cancer, 2000, vol. 89 (8), pp. 1692-1698.
  • Azzazy H.M., et al., “Phage Display Technology: Clinical Applications and Recent Innovations,” Clinical Biochemistry, 2002, vol. 35 (6), pp. 425-445.
  • Baerga-Ortiz A., et al., “Epitope Mapping of a Monoclonal Antibody against Human Thrombin by H/D-exchange Mass Spectrometry Reveals Selection of a Diverse Sequence in a Highly Conserved Protein,” Protein Science, 2002, vol. 11 (6), pp. 1300-1308.
  • Bailey, et al., “Evaluation of Epidermal Growth Factor Receptor (Egfr) as a Predictive Marker in Patients With Non-Small-Cell Lung Cancer (Nsclc) Receiving First- Line Gefitinib Combined With Platinum-Based Chemotherapy,” Journal of Clinical Oncology, ASCO Annual Meeting Proceedings, Post-Meeting Edition, 2004, Abstract 7013, pp. 22 (14S).
  • Balaban N., et al., “The Effect of Ionizing Radiation on Signal Transduction: Antibodies to EGF Receptor Sensitize A431 Cells to Radiation,” Biochimica Biophysica Acta. , 1996, vol. 1314 (1-2), pp. 147-156.
  • Baly, et al., “Development and Characterization of a Rhumab Her2 Antibody Adcc Assay for Clinical Evaluation of Cytotoxic Potency,” Proceedings of the American Association for Cancer Research, 1997, vol. 27-28 (Abstract 181), pp. 38.
  • Bandyopadhyay D., et al., “Physical Interaction between Epidermal Growth Factor Receptor and DNA-dependent Protein Kinase in Mammalian Cells,” Journal of Biological Chemistry, 1998, vol. 273 (3), pp. 1568-1573.
  • Barendswaard E.C., et al., “Rapid and Specific Targeting of Monoclonal Antibody A33 to a Colon Cancer Xenograft in Nude Mice,” International Journal of Oncology, 1998, vol. 12 (1), pp. 45-53.
  • Barnette M.S., et al., “Association of the Anti-Inflammatory Activity of Phosphodiesterase 4 (PDE4) Inhibitors with either Inhibition of PDE4 Catalytic Activity or Competition for (3H)Rolipram Binding,” Biochemical Pharmacology, 1996, vol. 51 (7), pp. 949-956.
  • Baselga, et al., “Antitumor Activity of Paclitaxel in Combination With Anti-Growth Factor Receptor Monoclonal Antibodies in Breast Cancer Xenografts,” Proceedings of the American Association for Cancer Research, 1994, vol. 380 (Abstract 2262), pp. 35.
  • Baselga, et al., “Cetuximab (C225) Plus Cisplatin/Carboplatin is Active in Patients (Pts) With Recurrent/Metastatic Squamous Cell Carcinoma of the Head and Neck (Scchn) Progressing on a Same Dose and Schedule Platinum-Based Regimen,” Proceedings of the American Society of Clinical Oncology, 2002, Abstract 900, pp. 21.
  • Baselga, et al., “Mechanism of Action of Anti-Her2 Monoclonal Antibodies,” Annals of Oncology, 2001, vol. 12 (Suppl. 1), pp. S35-S41.
  • Baselga, et al., “Phase I Study of Aee788, a Novel Multitargeted Inhibitor of Erbb and Vegf Receptor Family Tyrosine Kinases (A Pharmacokinetic (Pk)-Pharmacodynamic (Pd) Study to Identify the Optimal Therapeutic Dose Regimen),” Journal of Clinical Oncology, 2005, Abstract 3028, pp. 23.
  • Baselga J., “Clinical Trials of Herceptin(TrastuzumAb),” European Journal of Cancer, 2001, vol. 37 (Suppl. 1), pp. S18-S24.
  • Baselga J., “Combining the Anti-EGFR Agent Gefitinib With Chemotherapy in Non-Small-Cell Lung Cancer: How Do We Go From Intact to Impact,” Journal of Clinical Oncology, 2004, vol. 22 (5), pp. 759-761.
  • Baselga J., et al. , “Continuous Administration of Z01839 (Iressa), a Novel Oral Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor (EGFR-TKI), in Patients with Five Selected Tumor Types: Evidence of Activity and Good Tolerability.,” Proceedings of American Society of Clinical Oncology, 2000, vol. 19, pp. 177A.
  • Baselga J., et al., “Antitumor Effects of Doxorubicin in Combination with Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies,” Journal of the National Cancer Institute , 1993, vol. 85, pp. 1327-1333.
  • Baselga J., et al., “Critical Update and Emerging Trends in Epidermal Growth Factor Receptor Targeting in Cancer,” Journal of Clinical Oncology, 2005, vol. 23 (11), pp. 2445-2459.
  • Baselga J., et al., “Mechanism of Action of Trastuzumab and Scientific Update,” Seminars in Oncology , 2001, vol. 28 (5) (suppl. 16), pp. 4-11.
  • Baselga J., et al., “Phase I Safety, Pharmacokinetic, and Pharmacodynamic Trial of ZD1839, a Selective Oral Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, in Patients With Five Selected Solid Tumor Types,” Journal of Clinical Oncology, 2002, vol. 20 (21), pp. 4292-4302.
  • Baselga J., et al., “Phase I Studies of Anti-Epidermal Growth Factor Receptor Chimeric Antibody C225 alone and in Combination with Cisplatin,” Journal of Clinical Oncology, 2000, vol. 18 (4), pp. 904-914.
  • Baselga J., et al., “Phase II and Tumor Pharmacodynamic Study of Gefitinib in Patients With Advanced Breast Cancer,” Journal of Clinical Oncology, 2005, vol. 23 (23), pp. 5323-5333.
  • Baselga J., et al., “Phase II Multicenter Study of the Antiepidermal Growth Factor Receptor Monoclonal Antibody Cetuximab in Combination With Platinum-Based Chemotherapy in Patients With Platinum-Refractory Metastatic and/or Recurrent Squamous Cell Carcinoma of the Head and Neck,” Journal of Clinical Oncology, 2005, vol. 23 (24), pp. 5568-5577.
  • Baselga J., et al., “Phase II Study of Efficacy, Safety, and Pharmacokinetics of Trastuzumab Monotherapy Administered on a 3-Weekly Schedule,” Journal of Clinical Oncology, 2005, vol. 23 (10), pp. 2162-2171.
  • Baselga J., et al., “Phase II Study of Weekly Intravenous Recombinant Humanized Anti-P185her2 Monoclonal Antibody in Patients with Her2/Neu-Overexpressing Metastatic Breast Cancer,” Journal of Clinical Oncology, 1996, vol. 14 (3), pp. 737-744.
  • Baselga J., et al., “Receptor Blockade with Monoclonal Antibodies as Anti-Cancer Therapy,” Pharmacology and Therapeutics , 1994, vol. 64 (1), pp. 127-154.
  • Baselga J., et al., “Recombinant Humanized Anti-HER2 Antibody (Herceptin) Enhances the Antitumor Activity of Paclitaxel and Doxorubicin Against HER2/neu Overexpressing Human Breast Cancer Xenografts,” Journal of Cancer Research, 1998, vol. 58 (13), pp. 2825-2831.
  • Baselga J., et al., “ZD1839 (Iressa)1, 2 as an Anticancer Agent,” Drugs, 2000, vol. 60 (1), pp. 33-42.
  • Baselga J., “Herceptin Alone or in Combination with Chemotherapy in the Treatment of HER2-Positive Metastatic Breast Cancer: Pivotal Trials,” Oncology, 2001, vol. 61 (suppl. 2), pp. 14-21.
  • Baselga J., “Targeting the Epidermal Growth Factor Receptor: A Clinical Reality,” Journal of Clinical Oncology, 2001, vol. 19 (suppl. 18), pp. 41S-44S.
  • Baselga J., “Targeting Tyrosine Kinases in Cancer: The Second Wave,” Science, 2006, vol. 312 (5777), pp. 1175-1178.
  • Baselga J., “The EGFR as a Target for Anticancer Therapy—focus on Cetuximab,” European Journal of Cancer, 2001, vol. 37 (Suppl. 4), pp. S16-S22.
  • Baselga J., “Why the Epidermal Growth Factor Receptor” The Rationale for Cancer Therapy, The Oncologist, 2002, vol. 7 (4), pp. 2-8.
  • Batra S.K., et al., “Epidermal Growth Factor Ligand-Independent, Unregulated, Cell-Transforming Potential of a Naturally Occurring Human Mutant EGFRvIII Gene,” Cell Growth and Differentiation, 1995, vol. 6 (10), pp. 1251-1259.
  • Beckmann M.W., et al., “Expression Analyses of Epidermal Growth Factor Receptor and Her-2/Neu: No Advantage of Prediction of Recurrence or Survival in Breast Cancer Patients,” Oncology, 1996, vol. 53 (6), pp. 441-447.
  • Beers R., et al., “Immunotoxins with Increased Activity against Epidermal Growth Factor Receptor vIII-expressing Cells Produced by Antibody Phage Display,” Clinical Cancer Research , 2000, vol. 6 (7), pp. 2835-2843.
  • Behr T.M., et al., “Radioimmunotherapy of Small Volume Disease of Colorectal Cancer Metastatic to the Liver: Preclinical Evaluation in Comparison to Standard Chemotherapy and Initial Results of a Phase I Clinical Study,” Clinical Cancer Research, 1999, vol. 5 (Suppl. 10), pp. 3232s-3242s.
  • Behr T.M., et al., “Radioimmunotherapy of Small-Volume Disease of Metastatic Colorectal Cancer,” Cancer, 2002, vol. 94 (4), pp. 1373-1381.
  • Bell D.W., et al., “Epidermal Growth Factor Receptor Mutations and Gene Amplification in Non-Small-Cell Lung Cancer: Molecular Analysis of the Ideal/Intact Gefitinib Trials,” Journal of Clinical Oncology, 2005, vol. 23 (31), pp. 8081-8092.
  • Bell D.W., et al., “Inherited Susceptibility to Lung Cancer may be Associated with the T790M Drug Resistance Mutation in EGFR,” Nature Genetics, 2005, vol. 37 (12), pp. 1315-1316.
  • Bender H., et al., “Immunotherapy of Human Glioma Xenografts With Unlabeled, 131i-, or 125i-Labeled Monoclonal Antibody 425 to Epidermal Growth Factor Receptor,” Cancer Research, 1992, vol. 52 (1), pp. 121-126.
  • Benichou S., et al., “Random Fragment Libraries Using Yeast Expression Plasmid,” Methods in Molecular Biology, 1996, vol. 66, pp. 241-255.
  • Bequinot, et al., “Down-Regulation of the Epidermal Growth Factor Receptor in Kb Cells is Due to Receptor Internalization and Subsequent Degradation in Lysosomes,” Chemical Abstracts, 1984, Abstract 1592k, pp. 141 (101).
  • Berkers J.A., et al., “The Effects of Receptor Density and Cell Shape on Epidermal Growth Factor Binding,” Journal of Receptor Research, 1992, vol. 12 (1), pp. 71-100.
  • Bernier J., “Centuximab in the Treatment of the Head and Neck Cancer,” Expert Review of Anticancer, 2006, vol. 6 (11), pp. 1539-1552.
  • Bertics P.J., et al., “Alteration of Epidermal Growth Factor Receptor Activity by Mutation of Its Primary Carboxyl-Terminal Site of Tyrosine Self-Phosphorylation,” Journal of Biological Chemistry, 1988, vol. 263 (8), pp. 3610-3617.
  • Bertics P.J., et al., “Self-Phosphorylation Enhances the Protein-Tyrosine Kinase Activity of the Epidermal Growth Factor Receptor,” Journal of Biological Chemistry, 1985, vol. 260 (27), pp. 14642-14647.
  • Bhattacharya-Chatterjee M., et al., “The Anti-Idiotype Vaccines for Immunotherapy,” Current Opinion in Molecular Therapeutics, 2001, vol. 3 (1), pp. 63-69.
  • Bianco C., et al., “Antitumor Activity of Combined Treatment of Human Cancer Cells with Ionizing Radiation and Anti-Epidermal Growth Factor Receptor Monoclonal Antibody C225 Plus Type I Protein Kinase a Antisense Oligonucleotide,” Clinical Cancer Research , 2000, vol. 6 (11), pp. 4343-4350.
  • Bianco R., et al., “Loss of PTEN/MMAC1/TEP in EGF Receptor-expressing Tumor Cells Counteracts the Antitumor Action of EGFR Tyrosine Inase Inhibitors,” Oncogene, 2003, vol. 22 (18), pp. 2812-2822.
  • Bier H., et al., “Anti-(Epidermal Growth Factor) Receptor Monoclonal Antibodies for the Induction of Antibody-Dependent Cell-Mediated Cytotoxicity Against Squamous Cell Carcinoma Lines of the Head and Neck,” Cancer Immunology, Immunotherapy, 1998, vol. 46 (3), pp. 167-173.
  • Bier H., et al., “Clinical Trial with Escalating Doses of the Aantiepidermal Growth Factor Receptor Humanized Monoclonal Antibody EMD 72 000 in Patients with Advanced Squamous Cell Carcinoma of the Larynx and Hypopharynx,” Cancer Chemotherapy and Pharmacology, 2001, vol. 47 (6), pp. 519-524.
  • Bier H., et al., “Dose-Dependent Access of Murine Anti-Epidermal Growth Factor Receptor Monoclonal Antibody to Tumor Cells in Patients With Advanced Laryngeal and Hypopharyngeal Carcinoma,” European archives of oto-rhino-laryngology, 1995, vol. 252 (7), pp. 433-439.
  • Biernat W., et al., “Predominant Expression of Mutant Egfr (Egfrviii) is Rare in Primary Glioblastomas,” Brain Pathology, 2004, vol. 14 (2), pp. 131-136.
  • Bigner S.H., et al., “Characterization of the Epidermal Growth Factor Receptor in Human Glioma Cell Lines and Xenografts,” Cancer Research, 1990, vol. 50 (24), pp. 8017-8022.
  • Bindon C.I., et al., “Importance of Antigen Specificity for Complement-Mediated Lysis by Monoclonal Antibodies,” European Journal of Immunology, 1988, vol. 18 (10), pp. 1507-1514.
  • Bird R.E., et al., “Single-Chain Antigen-Binding Proteins,” Science, 1988, vol. 242 (4877), pp. 423-426.
  • Biscardi J.S., et al., “C-Src, Receptor Tyrosine Kinases, and Human Cancer,” Advanced Cancer Research, 1999, vol. 76, pp. 61-119.
  • Bishop J.M., “The Molecular Genetics of Cancer,” Science, 1987, vol. 235 (4786), pp. 305-311.
  • Bishop P.C., et al., “Dierential Sensitivity of Cancer Cells to Inhibitors of the Epidermal Growth Factor Receptor Family,” Oncogene, 2002, vol. 21 (1), pp. 119-127.
  • Blagosklonny M.V., et al., “Why Iressa Failed: Toward Novel Use of Kinase Inhibitors (Outlook).,” Cancer Biology and Therapy, 2003, vol. 2 (2), pp. 137-140.
  • Bleeker W.K., et al., “Dual Mode of Action of a Human Anti-Epidermal Growth Factor Receptor Monoclonal Antibody for Cancer Therapy,” Journal of Immunology, 2004, vol. 173 (7), pp. 4699-4707.
  • Blume-Jensen P., et al., “Oncogenic Kinase Signalling,” Nature, 2001, vol. 411 (6835), pp. 355-365.
  • Boder E.T., et al., “Directed Evolution of Antibody Fragments with Monovalent Femtomolar Antigen-Binding Affinity,” Proceedings of the National Academy of Sciences, 2000, vol. 97 (20), pp. 10701-10705.
  • Boder E.T., et al., “Yeast Surface Display for Directed Evolution of Protein Expression, Affinity, and Stability,” Methods in Enzymology, 2000, vol. 328, pp. 430-444.
  • Boder E.T., et al., “Yeast Surface Display for Screening Combinatorial Polypeptide Libraries,” Nature Biotechnology, 1997, vol. 15 (6), pp. 553-557.
  • Bogan A.A., et al., “Anatomy of Hot Spots in Protein Interfaces,” Journal of Molecular Biology, 1998, vol. 280 (1), pp. 1-9.
  • Boger, “Design, Synthesis, and Evaluation of DNA Minor Groove Binding Agents: the Duocarmycins,” Pure and Applied Chemistry, 1994, vol. 66 (4), pp. 837-844.
  • Boghaert E.R., et al., “Antibody-Targeted Chemotherapy with the Calicheamicin Conjugate hu3S193-N-Acetyl” Calicheamicin Dimethyl Hydrazide Targets Lewisy and Eliminates Lewisy-Positive Human Carcinoma Cells and Xenografts , Clinical Cancer Research , 2004, vol. 10 (13), pp. 4538-4549.
  • Bonner, et al., “Cetuximab Improves Locoregional Control and Survival of Locoregionally Advanced Head and Neck Cancer: Independent Review of Mature Data With a Median Follow-Up of 45 Months,” Presented at the Annual Aacr-Nci-Eortc International Conference on Molecular Targets and Cancer Therapeutics: Discovery, Biology, and Clinical Applications; Nov. 2005, philadelphia; pa. (2011), Abstract B106.
  • Bonner, et al., “Cetuximab Prolongs Survival in Patients With Locoregionally Advanced Squamous Cell Carcinoma of Head and Neck (A Phase Iii Study of High Dose Radiation Therapy With or Without Cetuximab),” Journal of Clinical Oncology, 2004 ASCO Annual Meeting Proceedings (Post-Meeting Edition), 2004, Abstract 5507, pp. 22 (14S).
  • Bonner, et al., “Continued Response Following Treatment With Imc-C225, An Egfr Moab Combined With Rt in Advanced Head and Neck Malignancies,” Proceedings of the Annual Meeting of the American Society of Clinical Oncology, 2000, vol. 4A (Abstract 5F), pp. 10.
  • Bonner J.A., et al., “Enhanced Apoptosis With Combination C225/Radiation Treatment Serves as the Impetus for Clinical Investigation in Head and Neck Cancers,” Journal of Clinical Oncology, 2000, vol. 18 (suppl. 21), pp. 47S-53S.
  • Bonner J.A., et al., “Radiotherapy Plus Cetuximab for Squamous-Cell Carcinoma of the Head and Neck,” The New England Journal of Medicine, 2006, vol. 354 (6), pp. 567-578.
  • Boonstra J., et al., “The Epidermal Growth Factor,” Cell Biology International, 1995, vol. 19 (5), pp. 413-430.
  • Bos, et al., “Phase I Studies of Anti-Epidermal Growth Factor Receptor Chimeric Monoclonal Antibody C225 in Patients With Egfr Overexpressing Tumors,” American Society of Clinical Oncolog, 1996, vol. 443 (Abstract 1381), pp. 15.
  • Bos M., et al., “PD153035 a Tyrosine Kinase Inhibitor Prevents Epidermal Growth Factor Receptor Activation and Inhibits Growth of Cancer Cells in a Receptor Number Dependent Manner,” Clinical Cancer Research, 1997, vol. 3 (11), pp. 2099-2106.
  • Boschelli, “Small Molecule Inhibitors of Receptor Tyrosine Kinases,” Drugs of the Future, 1999, vol. 24 (5), pp. 515-537.
  • Bouyain S., et al., “The Extracellular Region of ErbB4 Adopts a Tethered Conformation in the Absence of Ligand,” Proceedings of the National Academy of Sciences, 2005, vol. 102 (42), pp. 15024-15029.
  • Boyer C.M., et al., “Relative Cytotoxic Activity of Immunotoxins Reactive with Different Epitopes on the Extracellular Domain of the c-ErbB-2 (HER-2/neu) Gene Product p185,” International Journal of Cancer, 1999, vol. 82, pp. 525-531.
  • Brady L.W., et al., “Malignant Astrocytomas Treated With Iodine-125 Labeled Monoclonal Antibody 425 Against Epidermal Growth Factor Receptor: A Phase II Trial,” International Journal of Radiation Oncology Biology Physics, 1991, vol. 22 (1), pp. 225-230.
  • Brock C.S., et al., “Review Current Perspectives in Gliomas,” Medical Oncology, 1997, vol. 14 (2), pp. 103-120.
  • Brown D., et al., “Antiepidermal Growth Factor Receptor Antibodies Augment Cytotoxicity of Chemotherapeutic Agents on Squamous Cell Carcinoma Cell Lines,” Otolaryngology Head and Neck Surgery, 2000, vol. 122 (1), pp. 75-83.
  • Brown G., et al., “Murine Monoclonal Antibodies” in: Antibodies: A Practical Approach, vol. 1, Catty D., ed., IRL Press, 1988, pp. 81-104.
  • Bruggemann M., et al., “The Immunogenicity of Chimeric Antibodies,” The Journal of Experimental Medicine, 1989, vol. 170 (6), pp. 2153-2157.
  • Bruns C.J., et al., “Blockade of the Epidermal Growth Factor Receptor Signaling by a Novel Tyrosine Kinase Inhibitor Leads to Apoptosis of Endothelial Cells and Therapy of Human Pancreatic Cells and Therapy of Human Pancreatic,” Clinical Cancer Research , 2000, vol. 60 (11), pp. 2926-2935.
  • Bruns C.J., et al., “Epidermal Growth Factor Receptor Blockade with C225 Plus Gemcitabine Results in Regression of Human Pancreatic Carcinoma Growing Orthotopically in Nude Mice by Antiangiogenic Mechanisms,” Clinical Cancer Research , 2000, vol. 6 (5), pp. 1936-1948.
  • Bucci B., et al., “Egf-R Expression in Ductal Breast Cancer: Proliferation and Prognostic Implications,” Anticancer Research, 1997, vol. 17 (1B), pp. 769-774.
  • Bucholtz J.D., “Radiolabeled Antibody Therapy,” Seminars in Oncology Nursing, 1987, vol. 3 (1), pp. 67-73.
  • Buchsbaum D.J., et al., “Experimental Radioimmunotherapy,” Medical Physics, 1993, vol. 20 (2 Pt 2), pp. 551-567.
  • Budillon, et al., “Zd1839, an Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, Upgregulates P27kip1 Inducing Gi Arrest and Enhancing the Antitumor Effect of Interferon,” Proceedings of the Annual Meeting of the American Association for Cancer Research, 2000, vol. 773 (Abstract 4910).
  • Burgess A.W., “EGFR Family: Structure Physiology Signalling and Therapeutic Targets,” Growth Factors, 2008, vol. 26 (5), pp. 263-274.
  • Burgess A.W., et al., “An Open-and-Shut Case” Recent Insights into the Activation of EGF/ErbB Receptors, Molecular Cell, 2003, vol. 12 (3), pp. 541-552.
  • Burgess A.W., et al., “Murine Epidermal Growth Factor: Heterogeneity on High Resolution Ion-exchange Chromatography,” The EMBO Journal, 1983, vol. 2 (11), pp. 2065-2069.
  • Burris H.A. 3rd., et al., “Phase I Safety, Pharmacokinetics, and Clinical Activity Study of Lapatinib (Gw572016), a Reversible Dual Inhibitor of Epidermal Growth Factor Receptor Tyrosine Kinases, in Heavily Pretreated Patients with Metastatic Carcinomas,” Journal of Clinical Oncology, 2005, vol. 23 (23), pp. 5305-5313.
  • Burstein, et al., “A Phase II, Open-Label, Multicenter Study of Lapatinib in Two Cohorts of Patients with Advanced or Metastatic Breast Cancer Who have Progressed While Receiving Trastuzumab-Containing Regimens,” Annals of Oncology, 2004, vol. 27 (Abstract 1040), 15 (Suppl. 3).
  • Burstein H.J., et al., “Trastuzumab and Vinorelbine as First-Line Therapy for Her2-Overexpressing Metastatic Breast Cancer: Multicenter Phase II Trial with Clinical Outcomes, Analysis of Serum Tumor Markers as Predictive Factors, and Cardiac Surveillance Algorithm,” Journal of Clinical Oncology, 2003, vol. 21 (15), pp. 2889-2895.
  • Burtness B., et al., “Phase III Randomized Trial of Cisplatin Plus Placebo Compared with Cisplatin Plus Cetuximab in Metastatic/Recurrent Head and Neck Cancer: an Eastern Cooperative Oncology Group Study,” Journal of Clinical Oncology, 2005, vol. 23 (34), pp. 8646-8654.
  • Burtness B.A., et al., “Phase III Trial Comparing Cisplatin (C) + Placebo (P) to C + Anti-Epidermal Growth Factor Antibody (EGF-R) C225 in Patients (pts) with Metastatic/Recurrent Head & Neck Cancer (HNC),” Proceedings of American Society of Clinical Oncology, 2002, vol. 21, pp. 226A.
  • Busam K.J., et al., “Cutaneous Side-Effects in Cancer Patients Treated with the Antiepidermal Growth Factor Receptor Antibody C225,” British Journal of Dermatology, 2001, vol. 144 (6), pp. 1169-1176.
  • Buss J.E., et al., “Altered Epidermal Growth Factor (EGF)-Stimulated Protein Kinase Activity in Variant A431 cells with Altered Growth Responses to EGF,” Proceedings of the National Academy of Sciences, 1982, vol. 79 (8), pp. 2574-2578.
  • Cadena D.L., et al., “The Intracellular Tyrosine Kinase Domain of the Epidermal Growth Factor Receptor Undergoes a Conformational Change Upon Autophosphorylation,” Journal of Biological Chemistry, 1994, vol. 269 (1), pp. 260-265.
  • Cadena, et al., “Receptor Protein Tyrosine Kinases” In: Protein Phosphorylation, chapter 9, marks, eds, vch publishers, inc., new york, 1996, pp. 265-284.
  • Cai W., et al., “Quantitative Pet of EGFR Expression in Xenograft-Bearing Mice Using 64cu-Labeled Cetuximab, a Chimeric Anti-EGFR Monoclonal Antibody,” European Journal of Nuclear Medicine and Molecular Imaging, 2007, vol. 34 (6), pp. 850-858.
  • Caldas C., et al., “Humanization of the Anti-CD18 Antibody 6.7: an Unexpected Effect of a Framework Residue in Binding to Antigen,” Molecular Immunology, 2003, vol. 39 (15), pp. 941-952.
  • Callaghan T., et al., “A Complete Description of the Egf-Receptor Exon Structure: Implication in Oncogenic Activation and Domain Evolution,” Oncogene, 1993, vol. 8 (11), pp. 2939-2948.
  • Campos-Gonzalez R., et al., “Immunodetection of the Ligand-Activated Receptor for Epidermal Growth Factor,” Growth Factors, 1991, vol. 4 (4), pp. 305-316.
  • Cappuzzo F., et al., “Epidermal Growth Factor Receptor Gene and Protein and Gefitinib Sensitivity in Non-Small-Cell Lung Cancer,” Journal of the National Cancer Institute, 2005, vol. 97 (9), pp. 643-655.
  • Cappuzzo F., et al., “Increased Her2 Gene Copy Number is Associated with Response to Gefitinib Therapy in Epidermal Growth Factor Receptor-Positive Non-Small-Cell Lung Cancer Patients,” Journal of Clinical Oncology, 2005, vol. 23 (22), pp. 5007-5018.
  • Carlin C.R., et al., “S6 is theHhuman Receptor for Epidermal Growth Factor (EGF). (Abstract) Cytogenet,” Cell Genet, 1982, vol. 32, pp. 256.
  • Carlin C.R., et al., “Identity of Human Epidermal Growth Factor (EGF) Receptor with Glycoprotein SA-7: Evidence for Differential Phosphorylation of the Two Components of the EGF Receptor from A431 Cells,” Proceedings of the National Academy of Sciences , 1982, vol. 79 (16), pp. 5026-5030.
  • Carpenter G., “Properties of the Receptor for Epidermal Growth Factor,” Cell, 1984, vol. 37, pp. 357-358.
  • Carpenter G., “Receptors for Epidermal Growth Factor and Other Polypeptide Mitogens,” Annual Review of Biochemistry, 1987, vol. 56, pp. 881-914.
  • Carteni G., et al., “Panitumumab a Novel Drug in Cancer Treatment,” Annals of Oncology, 2007, vol. 18 Suppl. 6, pp. vi16-vi21.
  • Carter P., et al., “Humanization of an Anti-p185 HER2 Antibody for Human Cancer Therapy ,” Proceedings of the National Academy of Sciences of the United States of America, 1992, vol. 89, pp. 4285-4289.
  • Carter P., et al., “Identification and Validation of Cell Surface Antigens for Antibody Targeting in Oncology,” Endocrine-related Cancer, 2004, vol. 11 (4), pp. 659-687.
  • Carter P., “Improving the Efficacy of Antibody-based Cancer Therapies ,” Nature Reviews, 2001, vol. 1 (2), pp. 118-129.
  • Carter T.H., et al., “Tissue-Specific Transformation by Oncogenic Mutants of Epidermal Growth Factor Receptor,” Critical Reviews in Oncogenesis, 1994, vol. 5 (4), pp. 389-428.
  • Cartron G., et al., “Therapeutic Activity of Humanized Anti-CD20 Monoclonal Antibody and Polymorphism in IgG Fc Receptor Fc gRIIIa Gene,” Blood, 2002, vol. 99 (3), pp. 754-758.
  • Casado, et al., “A Phase I/11a Pharmacokinetic (Pk) and Serial Skin and Tumor Pharmacodynamic (Pd) Study of the EGFR Irreversible Tyrosine Kinase Inhibitor Ekb-569 in Combination with 5-Fluorouracil (5fu), Leucovorin (Lv) and Irinotecan (Cpt-11) (Folfiri Regimen) in Patients (Pts) with Advanced Colorectal Cancer (Acc),” Journal of Clinical Oncology, 2004, vol. 255s (Abstract 3543), pp. 22.
  • Casset, F., et al., “A Peptie Mimetic of an Anti-CD4 Monoclonal Antibody by Rational Design,” Biochemical and Biophysical Research Communications, 2003, vol. 307 (1), pp. 198-205.
  • Catimel B., et al., “Purification and Characterization of a Novel Restricted Antigen Expressed by Normal and Transformed Human Colonic Epithelium,” Journal of Biological Chemistry, 1996, vol. 271 (41), pp. 25664-25670.
  • Chaffanet M., et al., “EGF Receptor Amplification and Expression in Human Brain Tumours,” European Journal of Cancer, 1992, vol. 28 (1), pp. 11-17.
  • Chakravarthi A., et al., “Insulin like Growth Factor Receptor I Mediates Resistance to Anti-Epidermal Growth Factor Receptor Therapy in Primary Human Glioblastoma Cells through Continued Activation of Phosphoinositide 3-Kinase Signaling,” Cancer Research, 2002, vol. 62 (1), pp. 200-207.
  • Chan, et al., “EGFR Tyrosine Kinase Inhibition Decreases Epithelial Proliferation in Dcis of the Breast, Whereas C-Erbb2 Blockade Does Not,” Proceedings of the Annual Meeting of the American Association for Cancer Research, 2000, vol. 482 (Abstract 3074), pp. 41.
  • Chang C.P., et al., “Ligand-induced Internalization of the Epidermal Growth Factor Receptor is Mediated by Multiple Endocytic Codes Analogous to the Tyrosine Motif Found in Constitutively Internalized Receptors,” The Journal of Biological Chemistry, 1993, vol. 268 (26), pp. 19312-19320.
  • Chantry A., “The Kinase Domain and Membrane Localization Determine Intracellular Interactions between Epidermal Growth Factor Receptors,” Journal of Biological Chemistry, 1995, vol. 270 (7), pp. 3068-3073.
  • Chao G., “Characterizing and Engineering Antibodies Against the Epidermal Growth Factor Receptor (Project Report),” in: Doctor of Philosophy in Chemical Enginering, 2008.
  • Chao G., “Characterizing and f“ngineel”ing antibodies against the epiderinal grow1h factor receptor (Project Report),” Chemical Engineering, 2002.
  • Chao G., et al., “Fine Epitope Mapping of Anti-Epidermal Growth Factor Receptor Antibodies through Random Mutagenesis and Yeast Surface Display,” Journal of Molecular Biology, 2004, vol. 342 (2), pp. 539-550.
  • Chau N.G., et al., “The Association between EGFR variant III, HPV, p16, c-MET, EGFR Gene Copy Number and Response to EGFR Inhibitors in Patients with Recurrent or Metastatic Squamous Cell Carcinoma of the Head and Neck,” Head and Neck Oncology, 2011, vol. 3, pp. 11.
  • Chen B., et al., “Mice Mutant for Egfr and Shp2 have Defective Cardiac Semilunar Valvulogenesis,” Nature Genetics, 2000, vol. 24 (3), pp. 296-299.
  • Cherk M.H., et al., “Lack of Correlation of Hypoxic Cell Fraction and Angiogenesis with Glucose Metabolic Rate in Non-Small Cell Lung Cancer Assessed by 18 F-Fluoromisonidazole and 18 F-FDG PET,” Journal of Nuclear Medicine, 2006, vol. 47 (12), pp. 1921-1926.
  • Chien N.C., et al., “Significant Structural and Functional Change of an Antigen-Binding Site by a Distant Amino Acid Substitution: Proposal of a Structural Mechanism,” The Proceedings of the National Academy of Sciences, 1989, vol. 86 (14), pp. 5532-5534.
  • Ching K.Z., et al., “Expression of mRNA for Epidermal Growth Factor, Transforming Growth Factor-Alpha and their Receptor in Human Prostate Tissue and Cell Lines,” Molecular and Cellular Biochemistry, 1993, vol. 126 (2), pp. 151-158.
  • Chinkers M., et al., “Rapid Induction of Morphological changes in Human Carcinoma Cells A-431 by Epidermal Growth Factor,” 1979, vol. 83 (1), pp. 260-265.
  • Cho H.S., et al., “Structure of the Extracellular Region of HER2 alone and in Complex with the Herceptin Fab,” Nature, 2003, vol. 421 (6924), pp. 756-760.
  • Cho H.S., et al., “Structure of the Extracellular Region of HER3 Reveals an Interdomain Tether,” Science, 2002, vol. 297 (5585), pp. 1330-1333.
  • Chong G., et al., “Phase I Trial of 131 I-huA33 in Patients with Advanced Colorectal Carcinoma,” Clinical Cancer Research , 2005, vol. 11 (13), pp. 4818-4826.
  • Chopra, “1111n-Labeled Chimeric Monoclonal Antibody, ch806, Targeting the Epidermal Growth Factor Receptor Deletion Variant De2-7 (Egfrviii),” Molecular Imaging and Contrast Agent Database (Micad) [Internet]. Bethesda (MD), National Center for Biotechnology Information (US), 2010, pp. 1-5.
  • Chopra, “1111n-Labeled CHX-/V-CTPA Conjugated Monoclonal Antibody (MAB) 806 Targeting the Epidermal Growth Factor Receptor Deletion Variant De2-7 (EGFRvIII),” Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD), National Center for Biotechnology Information (US), 2010, pp. 1-5.
  • Chopra, “1241-Labeled Residulizing Ligand IMP-R4 Conjugated Chimeric Monoclonal Antibody ch806 Targeting the epidermal growth factor receptor deletion variant de2-7 (EGFRvIII),” Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD), National Center for Biotechnology Information (US), 2010, pp. 1-5.
  • Chopra, “1251-Labeled Monoclonal Antibody (MAB) 806 Targeting the Epidermal Growth Factor Receptor Deletion Variant de2-7 (EGFRvIII),” Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD), National Center for Biotechnology Information (US), 2010, pp. 1-4.
  • Christensen, et al., “Immunohistochemical Detection of Epidermal Growth Factor Receptor in Laryngeal Squamous Cell Carcinomas,” Acta Otolarvngol, 1992, vol. 112 (4), pp. 734-738.
  • Christensen J.G., et al., “High Levels of Her 2 Expression Alter the Ability of Epidermal Growth Factor Receptor (EGFR) Family Tyrosine Kinase inhibitors to Inhibit EGFR Phosphorylation in Vivo,” Clinical Cancer Research, 2001, vol. 7 (12), pp. 4230-4238.
  • Christmann A., et al., “Epitope Mapping and Affinity Purification of Monospecific Antibodies by Escherichia coli Cell Surface display of Gene-derived Random Peptide Libraries,” Journal of Immunological Methods, 2001, vol. 257 (1-2), pp. 163-173.
  • Chu C.T., et al., “Receptor Dimerization is not a Factor in the Signalling Activity of a Transforming Variant Epidermal Growth Factor Receptor (EGFRvIII),” Biochemical Journal, 1997, vol. 324 (Pt 3), pp. 855-861.
  • Chung C.H., et al., “Increased Epidermal Growth Factor Receptor Gene Copy Number is Associated with Poor Prognosis in Head and Neck Squamous Cell Carcinomas,” Journal of Clinical Oncology, 2006, vol. 24 (25), pp. 4170-4176.
  • Chung K.Y., et al., “Cetuximab Shows Activity in Colorectal Cancer Patients With Tumors That Do Not Express the Epidermal Growth Factor Receptor by Immunohistochemistry,” Journal of Clinicak Oncology, 2005, vol. 23 (9), pp. 1803-1810.
  • Ciardiello, et al., “Potentiation of Cytotoxic Drugs Activity in Human Cancer Cells by Zd-1839 (Iressa), and EGFR-Selective Tyrosine Kinase Inhibitor,” Proceedings of the American Association for Cancer Research, 2000, vol. 482 (Abstract 3075), pp. 41.
  • Ciardiello F., et al., “A Novel Approach in the Treatment of Cancer: Targeting the Epidermal Growth Factor Receptor,” Clinical Cancer Research , 2001, vol. 7 (10), pp. 2958-2970.
  • Ciardiello F., et al., “Antiangiogenic and Antitumor Activity of Anti-Epidermal Growth Factor Receptor C225 Monoclonal Antibody in Combination with Vascular Endothelial Growth Factor Antisense Oligonucleotide in Human GEO Colon Cancer Cells,” Clinical Cancer Research , 2000, vol. 6 (9), pp. 3739-3747.
  • Ciardiello F., et al., “Antitumor Activity of Combined Blockade of Epidermal Growth Factor Receptor and Protein Kinase A,” Journal of National Cancer Institute, 1996, vol. 88 (23), pp. 1770-1776.
  • Ciardiello F., et al., “Antitumor Activity of Sequential Treatment with Topotecan and Anti-Epidermal Growth Factor Receptor Monoclonal Antibody C225,” Clinical Cancer Research , 1999, vol. 5 (4), pp. 909-916.
  • Ciardiello F., et al., “Antitumor Effect and Potentiation of Cytotoxic Drugs Activity in Human Cancer Cells by ZD-1839 (Iressa), an Epidermal Growth Factor Receptor-selective Tyrosine Kinase Inhibitor,” Clinical Cancer Research , 2000, vol. 6 (5), pp. 2053-2063.
  • Ciardiello F., et al., “Cooperative Antiproliferative Effects of 8-Chloro-Cyclic AMP and 528 Anti-Epidermal Growth Factor Receptor Monoclonal Antibody on Human Cancer Cells,” Clinical Cancer Research , 1995, vol. 1 (2), pp. 161-167.
  • Ciardiello F., et al., “Cooperative Inhibition of Renal Cancer Growth by Anti-Epidermal Growth Factor Receptor Antibody and Protein Kinase a Antisense Oligonucleotide,” Journal of National Cancer Institute, 1998, vol. 90 (14), pp. 1087-1094.
  • Ciardiello F., et al., “Epidermal Growth Factor Receptor (EGFR) as a Target in Cancer Therapy: Understanding the Role of Receptor Expression and Other Molecular Determinants That Could Influence the Response to Anti-EGFR Drugs,” European Journal of Cancer, 2003, vol. 39 (10), pp. 1348-1354.
  • Ciardiello F., et al., “Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors as Anticancer Agents,” Drugs, 2000, vol. 60 (1), pp. 25-32.
  • Ciardiello F., et al., “Inhibition of Growth Factor Production and Angiogenesis in Human Cancer Cells by ZD1839 (Iressa), a Selective Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor,” Clinical Cancer Research , 2001, vol. 7 (5), pp. 1459-1465.
  • Ciesielski M.J., et al., “Oncogenic Epidermal Growth Factor Receptor Mutants with Tandem Duplication: Gene Structure and Effects on Receptor Function,” Oncogene, 2000, vol. 19 (6), pp. 810-820.
  • Clark M., “Antibody Humanization: A Case of the ‘Emperor's New Clothes’”, Immunology Today, 2000, vol. 21 (8), pp. 397-402.
  • Clarke K., et al., “In Vivo Biodistribution of a Humanized Anti-Lewis Y Monoclonal Antibody (hu3S193) in MCF-7 xenografted BALB/c Nude Mice,” Cancer Research, 2000, vol. 60 (17), pp. 4804-4811.
  • Clarke K., et al., “Mutant Epidermal Growth Factor Receptor Enhances Induction of Vascular Endothelial Growth Factor by Hypoxia and Insulin-Like Growth Factor-1 via a PI3 Kinase Dependent Pathway,” British Journal of Cancer, 2001, vol. 84 (10), pp. 1322-1329.
  • Clarke K., et al., “Therapeutic Efficacy of Anti-Lewis (Y) Humanized 3S 193 Radioimmunotherapy in a Breast Cancer Model: Enhanced Activity When Combined with Taxol Chemotherapy,” Clinical Cancer Research, 2000, vol. 6 (9), pp. 3621-3628.
  • Clayton A.H., et al., “Ligand-Induced Dimer-Tetramer Transition During the Activation of the Cell Surface Epidermal Growth Factor Receptor-A Multidimensional Microscopy Analysis,” Journal of Biological Chemistry, 2005, vol. 280 (34), pp. 30392-30399.
  • Clayton A.H., et al., “Unligated Epidermal Growth Factor Receptor Forms Higher Order Oligomers within Microclusters on A431 Cells That Are Sensitive to Tyrosine Kinase Inhibitor Binding,” Biochemistry, 2007, vol. 46 (15), pp. 4589-4597.
  • Clynes R.A., et al., “Inhibitory Fc Receptors Modulate in Vivo Cytoxicity Against Tumor Targets,” Nature Medicine, 2000, vol. 6 (4), pp. 443-446.
  • Co M.S., et al., “Humanized Antibodies for Therapy,” Nature, 1991, vol. 351, pp. 501-502.
  • Cobleigh M.A., et al., “Multinational Study of the Efficacy and Safety of Humanized Anti-HER2 Monoclonal Antibody in Women Who Have HER2-Overexpressing Metastatic Breast Cancer That Has Progressed After Chemotherapy for Metastatic Disease,” Journal of Clincal Oncology, 1999, vol. 17 (9), pp. 2639-2648.
  • Cochran J.R., et al., “Domain-Level Antibody Epitope Mapping through Yeast Surface Display of Epidermal Growth Factor Receptor Fragments,” Journal of Immunological Methods, 2004, vol. 287 (1-2), pp. 147-158.
  • Cohen E.E.W., et al., “Phase II Study of Z01839 (Iressa) in Recurrent or Metastatic Squamous Cell Carcinoma of the Head and Neck,” Proceedings of American Society of Clinical Oncology, 2002, vol. 21, pp. 225A.
  • Cohen, et al., “Safety Profile of the Monoclonal Antibody (Moab) Imc-C255, an Anti- Epidermal Growth Factor Receptor (EGFR) Used in the Treatment of EGFR-Positive Tumors,” Proceedings of the American Society of Clinical Oncology, 2000, vol. 474a (Abstract 1862), pp. 19.
  • Cohen M.H., et al., “United States Food and Drug Administration Drug Approval Summary: Gefitinib (ZD1839; Iressa) Tablets,” Clinical Cancer Research , 2004, vol. 10, pp. 1212-1218.
  • Cohen S., et al., “Epidermal Growth Factor-Receptor-Protein Kinase Interactions,” Journal of Biological Chemistry, 1980, vol. 225 (10), pp. 4834-4842.
  • Cokgor I., et al., “Phase I Trial Results of Iodine-131-Labeled Antitenascin Monoclonal Antibody 81C6 Treatment of Patients With Newly Diagnosed Malignant Gliomas,” Journal of Clinical Oncology, 2000, vol. 18 (22), pp. 3862-3872.
  • Colapinto E.V., et al., “Comparative Localization of Murine Monoclonal Antibody Me1-14 F(Ab')2 Fragment and Whole Igg2a in Human Glioma Xenografts,” Cancer Research, 1988, vol. 48 (20), pp. 5701-5707.
  • Collins V.P., et al., “Amplified Genes in Human Gliomas,” Cancer Biology, 1993, vol. 4 (1), pp. 27-32.
  • Collins V.P., “Gene Amplification in Human Gliomas,” Glia, 1995, vol. 15 (3), pp. 289-296.
  • Cortez C., et al., “Influence of Size, Surface, Cell Line, and Kinetic Properties on the Specific Binding of A33 Antigen-Targeted Multilayered Particles and Capsules to Colorectal Cancer Cells,” ACS Nano , 2007, vol. 1 (2), pp. 93-102.
  • Cortez C., et al., “Targeting and Uptake of Multilayered Particles to Colorectal Cancer Cells,” Advanced Materials, 2006, vol. 18 (15), pp. 1998-2003.
  • Corti A., et al., “Idiotope Determining Regions of a Mouse Monoclonal Antibody and its Humanized Versions. Identification of Framework Residues that Affect Idiotype Expression,” Journal of Molecular Biology , 1994, vol. 235 (1), pp. 53-60.
  • Cowley G.P., et al., “Increased EGF Receptors on Human Squamous Carcinoma Cell Lines,” British Journal of Cancer, 1986, vol. 53 (2), pp. 223-229.
  • Cragg M.S., et al., “Signaling Antibodies in Cancer Therapy,” Current Opinion in Immunology, 1999, vol. 11 (5), pp. 541-547.
  • Crawford, et al., “Abx-EGF in Combination with Paclitaxel and Carboplatin for Advanced Non-Small Cell Lung Cancer (Nsclc),” Journal of Clinical Oncology, ASCO Annual Meeting Proceedings (Post-Meeting Edition), 2004, vol. 22 (14S), pp. 7083.
  • Crombet T., et al., “Phase I Clinical Evaluation of a Neutralizing Monoclonal Antibody Against Epidermal Growth Factor Receptor in Advanced Brain Tumor Patients: Preliminary Study,” Hybridoma, 2001, vol. 20 (2), pp. 131-136.
  • Crombet T., et al., “Use of the Anti-EGFR Antibody h-R3 in Combination with Radiotherapy in the Treatment of Advanced Head and Neck Cancer,” Proceedings of American Society of Clinical Oncology, 2002, vol. 21, pp. 14A.
  • Crombet T., et al., “Use of the Humanized Anti-Epidermal Growth Factor Receptor Monoclonal Antibody H-R3 in Combination with Radiotherapy in the Treatment of Locally Advanced Head and Neck Cancer Patients,” Journal of Clinical Oncology, 2004, vol. 22 (9), pp. 1646-1654.
  • Cunningham B.C., et al. , “High-Resolution Epitope Mapping of hGH-Receptor Interactions by Alanine-Scanning Mutagenesis,” Science, 1989, vol. 244 (4908), pp. 1081-1085.
  • Cunningham D., et al., “Cetuximab Monotherapy and Cetuximab Plus Irinotecan in Irinotecan-Refractory Metastatic Colorectal Cancer,” New England Journal of Medicine, 2004, vol. 351 (4), pp. 337-345.
  • Cvrljevic A.N., et al., “Activation of Src Induces Mitochondrial Localisation of De2-7EGFR (EGFRviii) in Glioma Cells: Implications for Glucose Metabolism,” Journal of Cell Science, 2011, vol. 124 (Pt 17), pp. 2938-2950.
  • Dadparvar S., et al., “Indium-111-Labeled Anti-Egfr-425 Scintigraphy in the Detection of Malignant Gliomas,” Cancer, 1994, vol. 73 (Suppl. 3), pp. 884-889.
  • Daley G.Q., et al., “Transformation of an Interleukin 3-Dependent Hematopoietic Cell Line by the Chronic Myelogenous Leukemia-Specific P210bcr/abl Protein,” Proceedings of the National Academy of Sciences, 1988, vol. 85 (23), pp. 9312-9316.
  • Damjanov I., et al., “Immunohistochemical Localization of the Epidermal Growth Factor Receptor in Normal Human Tissues,” Laboratory Investigation, 1986, vol. 55 (5), pp. 588-592.
  • Damle N.K., “Antibody-Drug Conjugates Ace the Tolerability Test,” Nature Biotechnology, 2008, vol. 26 (8), pp. 884-885.
  • Damstrup L., et al., “Epidermal Growth Factor Receptor Mutation Type III Transfected into a Small Cell Lung Cancer Cell Line is Predominantly Localized at the Cell Surface and Enhances the Malignant Phenotype,” International Journal of Cancer, 2002, vol. 97 (1), pp. 7-14.
  • Damstrup L., et al., “In Vitro Invasion of Small-Cell Lung Cancer Cell Lines Correlates with Expression of Epidermal Growth Factor Receptor,” British Journal of Cancer, 1998, vol. 78 (5), pp. 631-640.
  • Daugherty B.L., et al., “Polymerase Chain Reaction Facilitates the Cloning, CDR-Grafting, and Rapid Expression of a Murine Monoclonal Antibody Directed Against the CD18 Component of Leukocyte Integrins,” Nucleic Acids Research, 1991, vol. 19 (9), pp. 2471-2476.
  • Davies J., et al., “Affinity Improvement of Single Antibody VH Domains: Residues in all Three Hypervariable Regions Affect Antigen Binding,” Immunotechnology, 1996, vol. 2 (3), pp. 169-179.
  • Davies R.L., et al., “Genetic Analysis of Epidermal Growth Factor Action: Assignment of Human Epidermal Growth Factor Receptor Gene to Chromosome 7,” Proceedings of the National Academy of Sciences , 1980, vol. 77 (7), pp. 4188-4192.
  • Davies SP., et al., “Specificity and Mechanism of Action of Some Commonly used Protein Kinase Inhibitors,” Biochemical Journal, 2000, vol. 351 (Pt 1), pp. 95-105.
  • Davis C.G., et al., “Transgenic Mice as a Source of Fully Human Antibodies for the Treatment of Cancer ,” Cancer and Metastasis Reviews, 1999, vol. 18 (4), pp. 421-425.
  • Dawson N.A., et al., “A Phase II Trial of Gefitinib (Iressa, Zd1839) in Stage IV and Recurrent Renal Cell Carcinoma,” Clinical Cancer Research, 2004, vol. 10 (23), pp. 7812-7819.
  • Dazzi H., et al., “Expression of Epidermal Growth Factor Receptor (Egf-R) in Non-Small Cell Lung Cancer use of Archival Tissue and Correlation of Egf-R with Histology, Tumour Size, Node Status and Survival,” British Journal of Cancer, 1989, vol. 59 (5), pp. 746-749.
  • De Bono J.S., et al., “The ErbB Receptor Family: A Therapeutic Target for Cancer,” Trends in Molecular Medicine, 2002, vol. 8 (4), pp. S19-S26.
  • De Jong J.S., et al., “Expression of Growth Factors, Growth-Inhibiting Factors, and their Receptors in Invasive Breast Cancer. II: Correlations with Proliferation and Angiogenesis,” The Journal of pathology, 1998, vol. 184 (1), pp. 53-57.
  • De Larco J.E., et al., “Sarcoma Growth Factor from Mouse Sarcoma Virus-Transformed Cells. Purification by Binding and Elution from Epidermal Growth Factor Receptor-Rich Cells,” Journal of Biological Chemistry, 1980, vol. 225 (8), pp. 3685-3690.
  • De Larco J.E., et al., “Epithelioid and Fibroblastic Rat Kidney Cell Clones: Epidermal Growth Factor (EGF) Receptors and the Effect of Mouse Sarcoma Virus Transformation,” Journal of Cellular Physiology, 1978, vol. 94 (3), pp. 335-342.
  • De Nardo J., et al., “A New Era for Radiolabeled Antibodies in Cancer”, Current Opinion Immunol, 1999, vol. 11 (5), pp. 563-569.
  • De Pascalis R., et al., “Grafting of “Abbreviated” Complementarity-Determining Regions Containing Specificity-Determining Residues Essential for Ligand Contact to Engineer a Less Immunogenic Humanized Monoclonal Antibody,” Journal of Immunological Methods, 2002, vol. 169 (6), pp. 3076-3084.
  • De Santes K., et al., “Radiolabeled Antibody Targeting of the Her-2/Neu Oncoprotein,” Cancer Research, 1992, vol. 52 (7), pp. 1916-1923.
  • Dechant, et al., “Effect of combinations of EGFR-R antibodies on complement- dependent tumor cell lysis,” Journal of Clinical Oncology, 2008, vol. 26 (15S), pp. 14005.
  • Decker S.J., “Aspects of the Metabolism of the Epidermal Growth Factor Receptor in A431 Human Epidermoid Carcinoma Cells,” Molecular and Cellular Biology, 1984, vol. 4 (4), pp. 571-575.
  • Decker S.J., “Transmembrane Signaling by Epidermal Growth Factor Receptors Lacking Autophosphorylation Sites,” Journal of Biological Chemistry, 1993, vol. 268 (13), pp. 9176-9179.
  • Deen D.F., “Brain Tumor Working Group Report on the 9th International Conference on Brain Tumor Research and Therapy. Organ System Program, National Cancer Institute,” Journal of Neuro-oncolog, 1993, vol. 16 (3), pp. 243-272.
  • Dehm S.M., et al., “SRC Gene Expression in Human Cancer: The Role of Transcriptional Activation,” Biochemistry and Cell Biology, 2004, vol. 82 (2), pp. 263-274.
  • Denardo G.L., et al., “Strategies for Developing Effective Radioimmunotherapy for Solid Tumors,” Clinical Cancer Research, 1999, vol. 5 (Suppl. 10), pp. 3219s-3223s.
  • Dewitt A.E., et al., “Quantitative Analysis of the EGF Receptor Autocrine System Reveals Cryptic Regulation of Cell Response by Ligand Capture,” Journal of Cell Science, 2001, vol. 114 (Pt 12), pp. 2301-2313.
  • Di Fiore P.P., et al., “Overexpression of the Human EGF Receptor Confers an Egf-Dependent Transformed Phenotype to Nih 3t3 Cells,” Cell, 1987, vol. 51 (6), pp. 1063-1070.
  • Di Lorenzo G., et al., “Expression of Epidermal Growth Factor Receptor Correlates With Disease Relapse and Progression to Androgen-Independence in Human Prostate Cancer,” Clinical Cancer Research, 2002, vol. 8 (11), pp. 3438-3444.
  • Dicosimo, et al., “Schedule-Dependent Effects of the Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor Gefitinib in Combination with the Mammalian Target of Rapamycin (Mtor) Inhibitor Everolimus (Rad001),” Proceedings of the American Society of Clinical Oncology, 2004, vol. 213s (Abstract 3074).
  • Diedrich U., et al., “Distribution of Epidermal Growth Factor Receptor Gene Amplification in Brain Tumours and Correlation to Prognosis,” Journal of Neurology, 1995, vol. 242 (10), pp. 683-688.
  • Discafani C.M., et al., “Irreversible Inhibition of Epidermal Growth Factor Receptor Tyrosine Kinase with in Vivo Activity by N-[4-[(3-Bromophenyl)Amino]-6-Quinazolinyl]-2-Butynamide (Cl-387,785),” Biochemical Pharmacology, 1999, vol. 57 (8), pp. 917-925.
  • Dittadi R., et al., “Epidermal Growth Factor Receptor in Lung Malignancies Comparison Between Cancer and Normal Tissue,” British Journal of Cancer, 1991, vol. 64 (4), pp. 741-744.
  • Divgi C.R., et al., “Phase I and Imaging Trial of Indium 111-Labeled Anti-Epidermal Growth Factor Receptor Monoclonal Antibody 225 in Patients with Squamous Cell Lung Carcinoma,” Journal of the National Cancer Institute, 1991, vol. 83 (2), pp. 97-104.
  • Domagala T., et al., “Stoichiometry, Kinetic and Binding Analysis of the Interaction Between Epidermal Growth Factor (EGF) and the Extracellular Domain of the EGF Receptor,” Growth Factors, 2000, vol. 18 (1), pp. 11-29.
  • Doronina S.O., et al., “Enhanced Activity of Monomethylauristatin F Through Monoclonal Antibody Delivery: Effects of Linker Technology on Efficacy and Toxicity,” Bioconjugate chemistry, 2006, vol. 17 (1), pp. 114-124.
  • Downward J., et al., “Close Similarity of Epidermal Growth Factor Receptor and V-Erb-B Oncogene Protein Sequences,” Nature, 1984, vol. 307 (5951), pp. 521-527.
  • Eberhard D.A., et al., “Mutations in the Epidermal Growth Factor Receptor and in Kras Are Predictive and Prognostic Indicators in Patients with Non-Small-Cell Lung Cancer Treated with Chemotherapy Alone and in Combination with Erlotinib,” Journal of Clinical Oncology, 2005, vol. 23 (25), pp. 5900-5909.
  • Egloff A.M., et al., “Targeting Epidermal Growth Factor Receptor and Src Pathways in Head and Neck Cancer,” Seminars in Oncology, 2008, vol. 35 (3), pp. 286-297.
  • Ekstrand A.J., et al., “Altered Subcellular Location of an Activated and Tumour-Associated Epidermal Growth Factor Receptor,” Oncogene, 1995, vol. 10 (7), pp. 1455-1460.
  • Ekstrand A.J., et al., “Amplified and Rearranged Epidermal Growth Factor Receptor Genes in Human Glioblastomas Reveal Deletions of Sequences Encoding Portions of the N- and/or C-terminal Tails,” Proceedings of the National Academy of Sciences, 1992, vol. 89 (10), pp. 4309-4313.
  • Ekstrand A.J., et al., “Functional Characterization of an Egf Receptor with a Truncated Extracellular Domain Expressed in Glioblastomas with Egfr Gene Amplification,” Oncogene , 1994, vol. 9 (8), pp. 2313-2320.
  • Ekstrand A.J., et al., “Genes for Epidermal Growth Factor Receptor, Transforming Growth Factor Alpha, and Epidermal Growth Factor and Their Expression in Human Gliomas in Vivo,” Cancer Research, 1991, vol. 51 (8), pp. 2164-2172.
  • Elleman T.C., et al., “Identification of a Determinant of Epidermal Growth Factor Receptor Ligand-Binding Specificity using a Truncated, High-Affinity form of the Ectodomain,” Biochemistry, 2001, vol. 40 (30), pp. 8930-8939.
  • Eller J.L., et al., “Activity of Anti-Epidermal Growth Factor Receptor Monoclonal Antibody C225 Against Glioblastoma Multiforme, Discussion: 1013-1014,” Neurosurgery, 2002, vol. 51 (4), pp. 1005-1013.
  • Ellgaard L., et al. , “Quality Control in the Endoplasmic Reticulum,” Nature Reviews. Molecular Cell Biology, 2003, vol. 4 (3), pp. 181-191.
  • Ellis A.G., et al., “Preclinical Analysis of the Analinoquinazoline Ag1478, A Specific Small Molecule Inhibitor of EGF Receptor Tyrosine Kinase,” Biochemical Pharmacology, 2006, vol. 71, pp. 1442-1434.
  • Emsley P., et al., “Coot: Model-Building Tools for Molecular Graphics,” Acta Crystallographica, 2004, vol. D60, pp. 2126-2132.
  • Ennis B.W., et al., “Anti-Epidermal Growth Factor Receptor Antibodies Inhibit the Autocrine-Stimulated Growth of Mda-468 Human Breast Cancer Cells,” Molecular Endocrinology, 1989, vol. 3 (11), pp. 1830-1838.
  • Ennis B.W., et al., “The EGF Receptor System as a Target for Antitumor Therapy,” Cancer Investigation, 1991, vol. 9 (5), pp. 553-562.
  • Ennis, “Monoclonal Anti-EGF Receptor Antibodies Inhibit the Growth of Malignant and Nonmalignant Human Mammary Epithelial Cells,” Journal of Cellular Biochemistry, 1989, vol. 104 (Abstract E207) (Suppl. 13B).
  • Epenetos A.A., et al., “Antibody Guided Irradiation of Brain Glioma by Arterial Infusion of Radioactive Monoclonal Antibody against Epidermal Growth Factor Receptor and Blood Group A Antigen,” British Medical Journal, 1985, vol. 290 (6480), pp. 1463-1466.
  • Epenetos A.A., et al., “Long Term Survival of Patients with Advanced Ovarian Cancer Treated with Intraperitoneal Radioimmunotherapy,” International Journal of Gynecological Cancer, 2000, vol. 10 (51), pp. 44-46.
  • Erickson H.K., et al., “Antibody-Maytansinoid Conjugates Are Activated in Targeted Cancer Cells by Lysosomal Degradation and Linker-Dependent Intracellular Processing,” Cancer Research, 2006, vol. 66 (8), pp. 4426-4433.
  • Eriksen, et al., “Phase I Trial of Chimerized Anti-Epidermal Growth Factor Receptor (Anti-EGFR) Antibody in Combination with Either Once-Daily or Twice-Daily Irradiation for Locally Advanced Head and Neck Malignancies,” Proceedings of the American Society of Clinical Oncology, 1999, vol. 388a (Abstract 1501), pp. 18.
  • Eriksen, et al., “The EGFR VIII Variant in Squamous Cell Carcinomas of the Head and Neck: Expression and Correlation with Clinico-Pathological Parameters in 675 Patients From the Randomised Dahanca 6/7 Study,” ECCO 15—34th ESMO Multidisciplinary Congress (Berlin), 2009, vol. 472 (Abstract P-8507).
  • Extended European Search Report for Application No. EP08726810, mailed on Jan. 28, 2011, 7 pages.
  • Faillot T., et al., “A phase I study of an anti-epidermal growth factor receptor monoclonal antibody for the treatment of malignant gliomas,” Neurosurgery, 1996, vol. 39 (3), pp. 478-483.
  • Fairlie W.D., et al., “A Fusion Protein System for the Recombinant Production of Short Disulfide-Containing Peptides,” Protein expression and purification, 2002, vol. 26 (1), pp. 171-178.
  • Fan, et al., “Blockade of Epidermal Growth Factor Receptor by Anti-EGFR Monoclonal Antibody 225 Causes Gi Arrest of A431 Cells with Induction of P27KIPI,” Proceedings of the American Association for Cancer Research, 1999, vol. 10 (Abstract 69), pp. 37.
  • Fan Z., et al., “Antibody-Induced Epidermal Growth Factor Receptor Dimerization Mediates Inhibition of Autocrine Proliferation of A431 Squamous Carcinoma Cells,” Journal of Biological Chemistry, 1994, vol. 269 (44), pp. 27595-27602.
  • Fan Z., et al., “Antitumor Effect of Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies Plus cis-Diamminedichloroplatinum on well Established A431 Cell Xenografts,” Cancer Research, 1993, vol. 53, pp. 4637-4642.
  • Fan Z., et al., “Blockade of Epidermal Growth Factor Receptor Function by Bivalent and Monovalent Fragments of225 Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies,” Journal of Cancer Research, 1993, vol. 53 (18), pp. 4322-4328.
  • Fan Z., et al., “Therapeutic Application of Anti-Growth Factor Receptor Antibodies,” Current Opinion in Oncology, 1998, vol. 10 (1), pp. 67-73.
  • Fantl W.J., et al., “Signalling by Receptor Tyrosine Kinases,” Annual Review of Biochemistry, 1993, vol. 62, pp. 453-481.
  • Farrugia W., et al., “A Possible Role for Metallic Ions in the Carbohydrate Cluster Recognition Displayed by a Lewis Y Specific Antibody,” Plos One, 2009, vol. 4 (11), pp. e7777.
  • Feldhaus M.J., et al., “Flow-Cytometric Isolation of Human Antibodies from a Nonimmune Saccharomyces Cerevisiae Surface Display Library,” Nature Biotechnology, 2003, vol. 21 (2), pp. 163-170.
  • Feldkamp M.M., et al., “Expression of Activated-Epidermal Growth Factor Receptors, Ras-Guanosine Triphosphate, and Mitogenactivated Protein Kinase in Human Glioblastoma Multiforme Specimens,” Neurosurgery, 1999, vol. 45 (6), pp. 1442-1453.
  • Fendly B.M., et al., “Characterization of Murine Monoclonal Antibodies Reactive to Either the Human Epidermal Growth Factor Receptor or Her2/Neu Gene Product,” Cancer Research, 1990, vol. 50 (5), pp. 1550-1558.
  • Fenstermaker R.A., et al., “Deletion and Tandem Duplication of Exons 2-7 in the Epidermal Growth Factor Receptor Gene of a Human Malignant Glioma,” Oncogene, 2000, vol. 19 (39), pp. 4542-4548.
  • Ferguson K.M., et al., “EGF Activates its Receptor by Removing Interactions that Autoinhibit Ectodomain Dimerization,” Molecular Cell, 2003, vol. 11 (2), pp. 507-517.
  • Ferguson K.M., “Structure-Based View of Epidermal Growth Factor Receptor Regulation,” Annual Review of Biophysics, 2008, vol. 37, pp. 353-373.
  • Fernandes H., et al., “Glycosylation-Induced Conformational Modification Positively Regulates Receptor-Receptor Association: A Study with an Aberrant Epidermal Growth Factor Receptor (EGFRvIII/DeltaEGFR) Expressed in Cancer Cells,” Journal of Biological Chemistry, 2001, vol. 276 (7), pp. 5375-5383.
  • Ferrara N., et al., “Discovery and Development of Bevacizumab, an Anti-VEGF Antibody for Treating Cancer,” Nature Reviews Drug Discovery, 2004, vol. 3 (5), pp. 391-400.
  • Ferry D., et al., “Intermittent Oral Zd1839 (Iressa), a Novel Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor (Egfr-Tki), Shows Evidence of Good Tolerability and Activity: Final Results from a Phase I Study,” Proceedings of American Society of Clinical Oncology , 2000, vol. 19, pp. 3A.
  • Figlin R.A., et al., “ABX-EGF, a fully human anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) in patients with advanced cancer: phase 1 clinical results,” Proceedings of American Society of Clinical Oncology, 2002, vol. 21, pp. 10A.
  • Filmus J., et al., “Amplified, Overexpressed and Rearranged Epidermal Growth Factor Receptor Gene in a Human Astrocytoma Cell Line,” Biochemical and Biophysical Research Communications, 1985, vol. 131 (1), pp. 207-215.
  • Filmus J., et al., “Epidermal Growth Factor Receptor Gene-Amplified MDA-468 Breast Cancer Cell Line and Its Nonamplified Variants,” Molecular and Cellular Biology, 1987, vol. 7 (1), pp. 251-257.
  • Filmus J., et al., “MDA-468, A Human Breast Cancer Cell Line with a High Number of Epidermal Growth Factor (EGF) Receptors, Has an Amplified EGF Receptor Gene and is Growth Inhibited by EGF,” Biochemical and Biophysical Research Communications, 1985, vol. 128 (2), pp. 898-905.
  • Finkler N., et al., “Phase 2 Evaluation of OSI-774, a Potent Oral Antagonist of the EGFR-TK in Patients with Advanced Ovarian Carcinoma,” Proceedings of American Society of Clinical Oncology, 2001, vol. 20, pp. 208A.
  • Fischer-Colbrie J., et al., “EGFR and Steroid Receptors in Ovarian Carcinoma: Comparison with Prognostic Parameters and Outcome of Patients,” Anticancer Research, 1997, vol. 17 (1B), pp. 613-619.
  • Fisher G.H., et al., “Induction and Apoptotic Regression of Lung Adenocarcinomas by Regulation of a K-Ras Transgene in the Presence and Absence of Tumor Suppressor Genes,” Genes & Development, 2001, vol. 15 (24), pp. 3249-3262.
  • Flynn J.M., et al., “Campath-1h Monoclonal Antibody Therapy,” Current Opinion in Oncology, 2000, vol. 12 (6), pp. 574-581.
  • Fong C.J., et al., “Epidermal Growth Factor Receptor Monoclonal Antibody Inhibits Constitutive Receptor Phosphorylation, Reduces Autonomous Growth, and Sensitizes Androgen-Independent Prostatic Carcinoma Cells to Tumor Necrosis Factor Alpha,” Cancer Research, 1992, vol. 52 (21), pp. 5887-5892.
  • Foo S.S., et al., “Functional Imaging of Intratumoral Hypoxia,” Molecular Imaging and Biology, 2004, vol. 6 (5), pp. 291-305.
  • Forastiere A., et al., “Head and Neck Cancer,” The New England Journal of Medicine, 2001, vol. 345 (26), pp. 1890-1900.
  • Ford A.C., et al., “Targeting Epidermal Growth Factor Receptor in Head and Neck Cancer,” Head & Neck, 2003, vol. 25 (1), pp. 67-73.
  • Ford, et al., “Pharmacogenomic Approaches for Identifying Markers Predictive of Tumor Response to Cetuximab (Erbitux),” Proceedings of the American Association for Cancer Research, 2004, Abstract 2032, pp. 45.
  • Fornier M., et al., “Trastuzumab in Combination with Chemotherapy for the Treatment of Metastatic Breast Cancer, Discussion 92-100,” Seminars in Oncology, 2000, vol. 27 (6 Suppl. 11), pp. 38-45.
  • Foulon C.F., et al., “Positively Charged Templates for Labeling Internalizing Antibodies: Comparison of N-Succinimidyl 5-lodo-3-Pyridinecarboxylate and the D-Amino Acid Peptide KRYRR,” Nuclear Medicine and Biology, 2001, vol. 28 (7), pp. 769-777.
  • Foulon C.F., et al., “Radioiodination via D-Amino Acid Peptide Enhances Cellular Retention and Tumor Xenograft Targeting of an Internalizing Anti-Epidermal growth Factor Receptor Variant III Monoclonal Antibody,” Cancer Research, 2000, vol. 60 (16), pp. 4453-4460.
  • Fowler K.J., et al., “A Mutation in the Epidermal Growth Factor Receptor in Waved-2 Mice has a Profound Effect on Receptor Biochemistry that Results in Impaired Lactation,” Proceedings of the National Academy of Sciences, 1995, vol. 92 (5), pp. 1465-1469.
  • Fox S.B., et al., “Tumour Angiogenesis,” The Journal of Pathology, 1996, vol. 179 (3), pp. 232-237.
  • Fraker P.J., et al., “Protein and Cell Membrane Iodinations with a Sparingly Soluble Chloroamide, 1,3,4,6-Tetrachloro-3a,6a-Diphrenylglycoluril,” Biochemical and Biophysical Research Communications, 1978, vol. 80 (4), pp. 849-857.
  • Frame M.C., “Newest Findings on the Oldest Oncogene How Activated Srcdoes it,” Journal of Cell Science, 2004, vol. 117 (Pt 7), pp. 989-998.
  • Francisco J.A., et al., “Cac10-Vcmmae, an Anti-Cd30-Monomethyl Auristatin E Conjugate with Potent and Selective Antitumor Activity,” Blood, 2003, vol. 102 (4), pp. 1458-1465.
  • Frank R., et al., “SPOT Synthesis. Epitope Analysis with Arrays of Synthetic Peptides Prepared on Cellulose Membranes,” Methods in Molecular Biology, 1996, vol. 66, pp. 149-169.
  • Franklin, et al., “Association Between Activation of Erbb Pathway Genes and Survival Following Gefitinib Treatment in Advanced Bac (Swog 0126),” Proceedings of the Annual Meeting of the American Society of Clinical Oncology, 2004, vol. 620s (Abstract 7015), pp. 22.
  • Frederick L., et al., “Analysis of Genomic Rearrangements Associated with Egrfviii Expression Suggests Involvement of Alu Repeat Elements,” Neuro-Oncology, 2000, vol. 2 (3), pp. 159-163.
  • Frederick L., et al., “Diversity and Frequency of Epidermal Growth Factor Receptor Mutations in Human Glioblastomas,” Cancer Research, 2000, vol. 60 (5), pp. 1383-1387.
  • Friedman H.S., et al., “Glioblastoma Multiforme and the Epidermal Growth Factor Receptor,” The New England Journal of Medicine, 2005, vol. 353 (19), pp. 1997-1999.
  • Friedman H.S., et al., “Temozolomide and Treatment of Malignant Glioma,” Clinical Cancer Research, 2000, vol. 6 (7), pp. 2585-2597.
  • Friedman L.M., et al., “Synergistic Down-Regulation of Receptor Tyrosine Kinases by Combinations of Mabs: Implications for Cancer Immunotherapy,” Proceedings of the National Academy of Sciences, 2005, vol. 102 (6), pp. 1915-1920.
  • Friess T., et al., “Combination Treatment with Erlotinib and Pertuzumab against Human Tumor Xenografts is Superior to Monotherapy,” Clinical Cancer Research, 2005, vol. 11 (14), pp. 5300-5309.
  • Fry D.W., et al., “A Specific Inhibitor of the Epidermal Growth Factor Receptor Tyrosine Kinase,” Science, 1994, vol. 265 (5175), pp. 1093-1095.
  • Fry D.W., et al., “Biochemical and Antiproliferative Properties of 4-[Ar(Alk)Ylamino]Pyridopyrimidines, a New Chemical Class of Potent and Specific Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor,” Biochemical Pharmacology, 1997, vol. 54 (8), pp. 877-887.
  • Fry D.W., et al., “Site-Directed Irreversible Inhibitors of the Erbb Family of Receptor Tyrosine Kinases as Novel Chemotherapeutic Agents for Cancer,” Anti-Cancer Drug Design, 2000, vol. 15 (1), pp. 3-16.
  • Fry D.W., “Inhibition of the Epidermal Growth Factor Receptor Family of Tyrosine Kinases as an Approach to Cancer Chemotherapy: Progression from Reversible to Irreversible Inhibitors,” Pharmacology & Therapeutics, 1999, vol. 82 (2-3), pp. 207-218.
  • Fry D.W., “Specific, Irreversible Inactivation of the Epidermal Growth Factor Receptor and Erbb2, by a New Class of Tyrosine Kinase Inhibitor,” Proceedings of the National Academy of Sciences, 1998, vol. 95 (20), pp. 12022-12027.
  • Fujino S., et al., “A Comparison of Epidermal Growth Factor Receptor Levels and Other Prognostic Parameters in Non-Small Cell Lung Cancer,” European Journal of Cancer, 1996, vol. 32A (12), pp. 2070-2074.
  • Fukai J., et al., “Antitumor Activity of Cetuximab Against Malignant Glioma Cells Overexpressing EGFR Deletion Mutant Variant III,” Cancer Science, 2008, vol. 99 (10), pp. 2062-2069.
  • Fukuoka M., et al., “Final Results from a Phase II Trial of Z01839 (‘Iressa’) for Patients with Advanced Non-small-cell lung Cancer (Ideal 1),” Proceedings of American Society of Clinical Oncology, 2002, vol. 21, pp. 298A.
  • Fukuoka M., et al., “Multi-Institutional Randomized Phase Ii Trial of Gefitinib for Previously Treated Patients with Advanced Non-Small-Cell Lung Cancer (The Ideal 1 Trial) [Corrected],” Journal of Clinical Oncology, 2003, vol. 21 (12), pp. 2237-2246.
  • Gadella Jr T.W., et al., “Oligomerization of Epidermal Growth Factor Receptors on A431 Cells Studied by Time-Resolved Fluorescence Imaging Microscopy. A Stereochemical Model for Tyrosine Kinase Receptor Activation,” Journal of Cell Biology, 1995, vol. 129 (6), pp. 1543-1558.
  • Gamou S., et al., “Glycosylation of the Epidermal Growth Factor Receptor and Its Relationship to Membrane Transport and Ligand Binding,” Journal of Biochemistry, 1988, vol. 104 (3), pp. 388-396.
  • Gan H.K., et al., “Targeting a Unique Egfr Epitope with Monoclonal Antibody 806 Activates Nf-Kappab and Initiates Tumour Vascular Normalization,” Journal of Cellular and Molecular Medicine, 2009, vol. 13 (9B), pp. 3993-4001.
  • Gan H.K., et al., “The EGFR VIII Variant in Glioblastoma Multiforme,” Journal of Clinical Neuroscience, 2009, vol. 16 (6), pp. 748-754.
  • Gan H.K., et al., “The Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor Ag1478 Increases the Formation of Inactive Untethered EGFR Dimers: Implications for Combination Therapy with Monoclonal Antibody 806,” Journal of Biological Chemistry, 2007, vol. 282 (5), pp. 2840-2850.
  • Garcia I.E., et al., “Expression of Mutated Epidermal growth Factor Receptor by Non-Small Cell lung Carcinomas,” Cancer Research, 1993, vol. 53 (14), pp. 3217-3220.
  • Garinchesa P., et al., “Organ-Specific Expression of the Colon Cancer Antigen A33, a Cell Surface Target for Antibody-Based Therapy,” International Journal of Oncology, 1996, vol. 9 (3), pp. 465-471.
  • Garrett T.P., et al., “Antibodies Specifically Targeting a Locally Misfolded Region of Tumor Associated EGFR,” Proceedings of the National Academy of Sciences, 2009, vol. 106 (13), pp. 5082-5087.
  • Garrett T.P., et al., “Crystal Structure of a Truncated Epidermal Growth Factor Receptor Extracellular Domain Bound to Transforming Growth Factor Alpha,” Cell, 2002, vol. 110 (6), pp. 763-773.
  • Garrett T.P., et al., “The Crystal Structure of a Truncated ErbB2 Ectodomain Reveals an Active Conformation, Poised to Interact with other ErbB Receptors,” Molecular Cell, 2003, vol. 11 (2), pp. 495-505.
  • Ge H., et al., “Evidence of High Incidence of Egfrviii Expression and Coexpression with Egfr in Human Invasive Breast Cancer by Laser Capture Microdissection and Immunohistochemical Analysis,” International Journal of Cancer, 2002, vol. 98 (3), pp. 357-361.
  • George J., et al., “Differential Effects of Anti-Beta2-Glycoprotein I Antibodies on Endothelial Cells and on the Manifestations of Experimental Antiphospholipid Syndrome,” Circulation, 1998, vol. 97, pp. 900-906.
  • Giaccone G., et al., “Combination Therapy With Zd1839 (Iressa),an Orally Active, Selective, Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor (Egfrtki), Gemcitabine and Cisplatin, In Patients with Advanced Solid Tumors: Promising Preliminary Results on Tolerability, Efficacy, and Pharmacokinetics,” Clinical Cancer Research, 2001, vol. 7, pp. 3765S.
  • Gibson T.B., et al., “Randomized Phase III Trial Results of PanitumumAb, a Fully Human Anti-Epidermal Growth Factor Receptor Monoclonal Antibody, in Metastatic Colorectal Cancer,” Clinical Colorectal Cancer, 2006, vol. 6 (1), pp. 29-31.
  • Gill G.N., et al., “Monoclonal Anti-Epidermal Growth Factor Receptor Antibodies Which are Inhibitors of Epidermal Growth Factor Binding and Antagonists of Epidermal Growth Factor Binding and Antagonists of Epidermal Growth Factor-Stimulated Tyrosine Protein Kinase Activity,” Journal of Biological Chemistry, 1984, vol. 259 (12), pp. 7755-7760.
  • Gill G.N., et al., “Relationship Between Production of Epidermal Growth Factor Receptors, Gene Amplification, and Chromosome 7 Translocation in Variant A431 Cells,” Somatic Cell and Molecular Genetics, 1985, vol. 11 (4), pp. 309-318.
  • Gill S., et al., “New Targeted Therapies in Gastrointestinal Cancers,” Current Treatment Options in Oncology, 2003, vol. 4 (5), pp. 393-403.
  • Giusti A.M., et al., “Somatic Diversification of S107 from an Antiphosphocholine to an Anti-DNA Autoantibody is due to a Single Base Change in its Heavy Chain Variable Region,” Proceedings of the National Academy of Sciences, 1987, vol. 84 (9), pp. 2926-2930.
  • Glennie M.J., et al., “Clinical Trials of Antibody Therapy,” Immunology Today, 2000, vol. 21 (8), pp. 403-410.
  • Glennie M.J., et al., “Renaissance of Cancer Therapeutic Antibodies,” Drug Discovery Today, 2003, vol. 8 (11), pp. 503-510.
  • Gold D.V., et al., “Localization of Pancreatic Cancer with Radiolabeled Monoclonal Antibody PAM4,” Critical Reviews in Oncology/Hematology, 2001, vol. 39 (1-2), pp. 147-154.
  • Goldberg R.M., “Cetuximab,” Nature Reviews Drug Discovery, 2005, Suppl. S10-S11.
  • Goldenberg A., et al., “Imaging of Human Tumor Xenografts with an Indium-111-Labeled Anti-Epidermal Growth Factor Receptor Monoclonal Antibody,” Journal of the National Cancer Institute , 1989, vol. 81 (21), pp. 1616-1625.
  • Goldenberg D.M., “Advancing Role of Radiolabeled Antibodies in the Therapy of Cancer,” Cancer Immunology, Immunotherapy, 2003, vol. 52 (5), pp. 281-296.
  • Goldenberg D.M., et al., “Targeted Therapy of Cancer with Radiolabeled Antibodies,” Journal of nuclear medicine, 2002, vol. 43 (5), pp. 693-713.
  • Goldenberg D.M., “The Role of Radiolabeled Antibodies in the Treatment of Non Hodgkins Lymphoma the coming of Age of Radioimmunotherapy,” Critical Reviews in Oncology Hematology, 2001, vol. 39 (1-2), pp. 195-201.
  • Goldman C.K.,et al., “Epidermal Growth Factor Stimulates Vascular Endothelial Growth Factor Production by Human Malignant Glioma Cells: A Model of Glioblastoma Multiforme Pathophysiology ,” Molecular Biology of the Cell, 1993, vol. 4 (1), pp. 121-133.
  • Goldman R., et al., “Heterodimerization of the Erbb-1 and ERRB-2 Receptors in Human Breast Carcinoma Cells: a Mechanism for Receptor Transregulation,” Biochemistry, 1990, vol. 29 (50), pp. 11024-11028.
  • Goldstein N.I., et al., “Biological Efficacy of a Chimeric Antibody to the Epidermal Growth Factor Receptor in a Human Tumor Xenograft Model,” Clinical Cancer Research, 1995, vol. 1 (11), pp. 1311-1318.
  • Gonzalez G., et al., “Epidermal Growth Factor-Based Cancer Vaccine for Non-Small-Cell Lung Cancer Therapy,” Annals of Oncology, 2003, vol. 14 (3), pp. 461-466.
  • Gorgoulis V.G., et al., “Molecular and Immunohistochemical Evaluation of Epidermal Growth Factor Receptor and C-Erb-B-2 Gene Product in Transitional Cell Carcinomas of the Urinary Bladder: a Study in Greek Patients,” Modern Pathology, 1995, vol. 8 (7), pp. 758-764.
  • Gorre M.E., et al., “Clinical Resistance to Sti-571 Cancer Therapy Caused by Bcr-Abl Gene Mutation or Amplification,” Science , 2001, vol. 293 (5531), pp. 876-880.
  • Goss G.D., et al., “Final Results of the Dose Escalation Phase of a Phase I Pharmacokinetics (Pk), Pharmacodynamic (PD) and Biological Activity Study of ZD1839 NCIC CTG in,” Proceedings of American Society of Clinical Oncology, 2001, vol. 20 (Abstract. 335), pp. 85A.
  • Graeven U., et al., “Phase I Study of the Humanised Anti-EGFR Monoclonal Antibody MatuzumAb (EMD 72000) Combined With Gemcitabine in Advanced Pancreatic Cancer,” British Journal of Cancer, 2006, vol. 94 (9), pp. 1293-1299.
  • Grandal M.V., et al., “EGFR VIIIEscapes Down-Regulation Due to Impaired Internalization and Sorting to Lysosomes,” Carcinogenesis, 2007, vol. 28 (7), pp. 1408-1417.
  • Grandis J.R., et al., “Elevated Levels of Transforming Growth Factor Alpha and Epidermal Growth Factor Receptor Messenger Rna Are Early Markers of Carcinogenesis in Head and Neck Cancer,” Cancer Research, 1993, vol. 53 (15), pp. 3579-3584.
  • Grandis J.R., et al., “Levels of TGP-alpha and EOFR Protein in Head and Neck Squamous Cell Carcinoma and Patient Survival,” Journal of the National Cancer Institute, 1998, vol. 90 (11), pp. 824-832.
  • Graness A., et al., “Protein-tyrosine-phosphatase-mediated Epidermal Growth Factor (Egf) Receptor Transinactivation and EGF Receptor-Independent Stimulation of Mitogen-Activated Protein Kinase by Bradykinin in A431 Cells,” Biochemical Journal, 2000, vol. 347 (Pt 2), pp. 441-447.
  • Graus-Porta D., et al., “Single-Chain Antibody-Mediated Intracellular Retention of Erbb-2 Impairs Neu Differentiation Factor and Epidermal Growth Factor Signaling,” Molecular and Cellular Biology, 1995, vol. 15 (3), pp. 1182-1191.
  • Green M.C. et al., “Monoclonal Antibody Therapy for Solid Tumors,” Cancer Treatment Reviews, 2000, vol. 26 (4), pp. 269-286.
  • Greenspan N.S., et al., “Defining Epitopes: Its not as Easy as it seems,” Nature Biotechnology, 1999, vol. 17 (10), pp. 936-937.
  • Groner B., et al., “Therapeutic Antibodies,” Current Molecular Medicine, 2004, vol. 4 (5), pp. 539-547.
  • Grunwald V., et al., “Development of the Epidermal Growth Factor Receptor Inhibitor Osi-774,” Seminars in Oncology , 2003, vol. 30 (3) (Suppl. 6), pp. 23-31.
  • Gschwind A., et al., “The Discovery of Receptor Tyrosine Kinases: Targets for Cancer Therapy,” Nature Reviews Cancer, 2004, vol. 4 (5), pp. 361-370.
  • Gullick W.J., et al., “A New Model for the Interaction of Egf-Like Ligands with their Receptors: the New One-Two,” European Journal of cancer , 1994, vol. 30A (14), pp. 2186.
  • Gullick W.J., et al., “Growth Factors, Growth Factor Receptors and Neoplasia,” Human & Experimental Toxicology, 1991, vol. 10 (6), pp. 398-400.
  • Gullick W.J., et al., “Prevalence of Aberrant Expression of the Epidermal Growth Factor Receptor in Human Cancers,” British Medical Bulletin, 1991, vol. 47 (1), pp. 87-98.
  • Gullick W.J., “Type I Growth Factor Receptors: Current Status and Future Work,” Biochemical Society Symposium, 1998, vol. 63, pp. 193-198.
  • Gulliford T., et al. , “Intensification of Growth Factor Receptor Signalling by Phorbol Treatment of Ligand-primed Cells Implies a Dimer-Stabilizing Effect of Protein Kinase C-dependent Juxtamembrane Domain Phosphorylation,” Cell Signalling, 1999, vol. 11 (4), pp. 245-252.
  • Gunnett, et al., “Phase II Study of Antiepidermal Growth Factor Receptor (EGFR) Antibody C225 Alone in Patients (Pts) with Metastatic Renal Carcinoma (RCC),” Annual Meeting of the American Society of Clinical Oncology, 1999, vol. 340a (Abstract 1309), pp. 18.
  • Gunther N. et al., “The Secreted Form of the Epidermal Growth Factor Receptor. Characterization and Crystallization of the Receptor Ligand Complex,” Journal of Biological Chemistry, 1990, vol. 265 (36), pp. 22082-5.
  • Gupta, et al., “Development of an EGFR VIIISpecific Recombinant Antibody,” BMC Biotechnology, 2010, vol. 72, pp. 10.
  • Gussow D., et al., “Humanization of Monoclonal Antibodies,” Methods in Enzymology, 1991, vol. 203, pp. 99-121.
  • Haas-Kogan D.A., et al., “Epidermal Growth Factor Receptor, Protein Kinase B/AKT, and Glioma Response to Erlotinib,” Journal of the National Cancer Institute, 2005, vol. 97 (12), pp. 880-887.
  • Haber D.A., et al., “Molecular Targeted Therapy of Lung Cancer: EGFR Mutations and Response to EGFR Inhibitors,” Cold Spring Harbor Symposia on Quantitative Biology, 2005, vol. 70, pp. 419-426.
  • Hackel P.O., et al., “Epidermal Growth Factor Receptors: Critical Mediators of Multiple Receptor Pathways,” Current Opinion in Cell Biology , 1999, vol. 11 (2), pp. 184-189.
  • Haigler H., et al., “Visualization by Fluorescence of the Binding and Internalization of Epidermal Growth Factor in Human Carcinoma Cells A-431,” Proceedings of the National Academy of Sciences united states of america, 1978, vol. 75 (7), pp. 3317-3321.
  • Halaisch M.E., et al., “Marked Inhibition of Glioblastoma Target Cell Tumorigenicity in Vitro by Retrovirus-Mediated Transfer of a Hairpin Ribozyme Against Deletion-Mutant Epidermal Growth Factor Receptor Messenger RNA,” Journal of Neurosurgery, 2000, vol. 92 (2), pp. 297-305.
  • Halatsch M.E., et al. , “Inverse Correlation of Epidermal Growth Factor Receptor Messenger RNA Induction and Suppression of AnchOrageindependentgrowth by OSI-774, an Epidermal Growth Factor Receptor Tyrosine Kmase Inhibitor in Glioblastoma Multiforme Cell Lines,” Journal of Neurosurgery, 2004, vol. 100 (3), pp. 523-533.
  • Halatsch M.E., et al., “Epidermal Growth Factor Receptor Inhibition for the Treatment of Glioblastoma Multiforme and other Malignant Brain Tumours,” Cancer Treatment Reviews, 2006, vol. 32 (2), pp. 74-89.
  • Haley J., et al., “The Human EGF Receptor Gene: Structure of the 110 Kb Locus and Identification of Sequences Regulating its Transcription,” Oncogene Research, 1987, vol. 1 (4), pp. 375-396.
  • Haley J.D., “Regulation of Epidermal Growth Factor Receptor Expression and Activation: A Brief Review,” Symposia of the Society for Experimental Biology, 1990, vol. 44, pp. 21-37.
  • Hambek M., et al., “Tumor Necrosis Factor a Sensitizes Low Epidermal Growth Factor Receptor (EGFR)-Expressing Carcinomas for Anti-EGFR Therapy,” Cancer Research, 2001, vol. 61 (3), pp. 1045-1049.
  • Hamblett K.J., et al., “Effects of Drug Loading on the Antitumor Activity a Monoclonal Antibody Drug Conjugate,” Clinical Cancer Research, 2004, vol. 10 (20), pp. 7063-7070.
  • Han S.W., et al., “Predictive and Prognostic Impact of Epidermal Growth Factor Receptor Mutation in Non-Small-Cell Lung Cancer Patients Treated with Gefitinib,” Journal of Clinical Oncology, 2005, vol. 23 (11), pp. 2493-2501.
  • Han Y., et al., “Tyrphostin Ag1478 Preferentially Inhibits Human Glioma Cells Expressing Truncated Rather than Wild-Type Epidermal Growth Factor Receptors,” Journal Cancer Research, 1996, vol. 56 (17), pp. 3859-3861.
  • Hanahan D., et al., “The Hallmarks of Cancer,” Cell, 2000, vol. 100 (1), pp. 57-70.
  • Hanks S.K., et al., “The Protein Kinase Family: Conserved Features and Deduced Phylogeny of the Catalytic Domains,” Science , 1988, vol. 241 (4861), pp. 42-52.
  • Hanna N., et al., “Phase II Trial of Cetuximab in Patients with Previously Treated Non-Small-Cell Lung Cancer,” Journal of Clinical Oncology, 2006, vol. 24 (33), pp. 5253-5258.
  • Harari D., et al., “Molecular Mechanisms Underlying ErbB2/Her2 Action in Breast Cancer,” Oncogene, 2000, vol. 19 (53), pp. 6102-6114.
  • Harari, et al., “Combining Radiation with Molecular Blockade of the EGF Receptor in Cancer Therapy,” Proceedings of the American Association for Cancer Research, 1999, 3747s, 5.
  • Harari P.M., “Epidermal Growth Factor Receptor Inhibition Strategies in Oncology,” Endocrine Related Cancer, 2004, vol. 11 (4), pp. 689-708.
  • Harari P.M., et al., “Head and Neck Cancer as a Clinical Model for Molecular Targeting of Therapy: Combining Egfr Blockade with Radiation,” International Journal of Radiation Oncology, Biology, Physics , 2001, vol. 49 (2), pp. 427-433.
  • Harries M., et al., “The Development and Clinical Use of Trastuzumab (Herceptin),” Endocrine-Related Cancer, 2002, vol. 9 (2), pp. 75-85.
  • Harris, et al., “Epidermal Growth Factor Receptor: A Marker of Early Relapse in Breast Cancer and Tumor Stage Progression in Bladder Cancer; Interactions with Meu” in: the Molecular Diagnostics of Human Cancer, Furth, et al., eds., Cold Spring Harbor Laboratory, New York, 1989, pp. 353-357.
  • Harris, et al., “The Role of ErbB2 Extracellular Domain in Predicting Response to Chemotherapy in Breast Cancer Patients,” Proceedings of the Annual Meeting of the American Society of Clinical Oncology, 1996, vol. 108 (Abstract 96), pp. 15.
  • Harris W.J., et al., “Therapeutic antibodies—the Coming of Age,” Trends Biotechnol, 1993, vol. 11 (2), pp. 42-44.
  • Hatanpaa K.J., et al., “Epidermal Growth Factor Receptor in Glioma: Signal Transduction, Neuropathology, Imaging, and Radioresistance,” Neoplasia, 2010, vol. 12 (9), pp. 675-684.
  • Hayman M.J., et al., “Cell Transformation by the Epidermal Growth Factor Receptor and V-Erbb,” Cancer Cells, 1991, vol. 3 (8), pp. 302-307.
  • He Y., et al., “Inhibition of Human Squamous Cell Carcinoma Growth In Vivo by Epidermal Growth Factor Receptor Antisense RNA Transcribed From the U6 Promoter,” Journal of the National Cancer Institute, 1998, vol. 90 (14), pp. 1080-1087.
  • Heath J.K., et al., “The Human A33 Antigen is a Transmembrane Glycoprotein and a Novel Member of the Immunoglobulin Superfamily,” Proceedings of the National Academy of Sciences united states of america, 1997, vol. 94 (2), pp. 469-474.
  • Hecht, et al., “ABX-EGF Monotherapy in Patients (Pts) with Metastatic Colorectal Cancer (mCRC) (An updated analysis),” Proceedings of the American Society of Clinical Oncology, 2004, vol. 247s (Abstract 3511), pp. 23.
  • Heimberger A.B., et al., “Epidermal Growth Factor Receptor VIII Peptide Vaccination is Efficacious Against Established Intracerebral Tumors,” Clinical Cancer Research, 2003, vol. 9 (11), pp. 4247-4254.
  • Heimberger A.B., et al., “Prognostic Effect of Epidermal Growth Factor Receptor and EGFRVIII in Glioblastoma Multiforme Patients,” Clinical Cancer Research, 2005, vol. 11 (4), pp. 1462-1466.
  • Heimberger A.B., et al., “The Natural History of EGFR and EGFRvIII in Glioblastoma Patients,” Journal of translational medicine, 2005, vol. 38, pp. 3.
  • Heimberger, “Brain Tumors in Mice Are Susceptible to Blockade of Epidermal Growth Factor Receptor (EGFR) with the Oral, Specific, EGFR-Tyrosine Kinase Inhibitor ZD1839 (Iressa)”, Clinical Cancer Research, 2002, 8, 3496-3502.
  • Helin K., et al., “Internalization and Down-Regulation of the Human' Epidermal Growth Factor Receptor are Regulated by the Carboxyl-Terminal Tyrosines,” Journal of Biological Chemistry 1991, vol. 266 (13), pp. 8363-8368.
  • Helin K., et al., “The Biological activity of the Human Epidermal Growth Factor Receptor is Positively Regulated by its C-Terminal Tyrosines,” Oncogene, 1991, vol. 6 (5), pp. 825-832.
  • Hendler F.J., et al., “Human Squamous Cell Lung Cancers Express Increased Epidermal Growth Factor Receptors,” Journal of clinical investigation, 1984, vol. 74 (2), pp. 647-651.
  • Henn W., et al., “Polysomy of Chromosome 7 is Correlated with Overexpression of the Erbb Oncogene in Human Glioblastoma Cell Lines,” Human Genetics , 1986, vol. 74 (1), pp. 104-106.
  • Henry M.D., et al., “A Prostate-Specific Membrane Antigen-Targeted Monoclonal Antibody-Chemotherapeutic Conjugate Designed for the Treatment of Prostate Cancer,” Cancer Research, 2004, vol. 64, pp. 7995-8001.
  • Hens M., et al., “Anti-EGFRVIII Monoclonal Antibody Armed with 1771.U: In Vivo Comparison of Macrocyclic and Acyclic Ligands,” Nuclear Medicine and Biology, 2010, vol. 37 (7), pp. 741-750.
  • Hens M., et al., “Labeling Internalizing Anti-Epidermal Growth Factor Receptor Variant III Monoclonal Antibody With (177)Lu: in Vitro Comparison of Acyclic and Macrocyclic Ligands,” Nuclear Medicine and Biology, 2009, vol. 36 (2), pp. 117-128.
  • Herbertson R.A., et al., “Phase I Biodistribution and Pharmacokinetic Study of Lewis Y-Targeting Immunoconjugate Cmd-193 in Patients with Advanced Epithelial Cancers,” Clinical Cancer Research, 2009, vol. 15 (21), pp. 6709-6715.
  • Herbst et al.,“Regulation of Postendocytic Trafficking of the Epidermal Growth Factor Receptor Through Endosomal Retention,” Journal of Biological Chemistry, 1994, 269 (17), pp. 12865-12873.
  • Herbst R.S., et al., “Dose-Comparative Monotherapy Trials of Zd1839 in Previously Treated Non-Small Cell Lung Cancer Patients,” Seminars in Oncology , 2003, vol. 30 (1) Suppl. 1), pp. 30-38.
  • Herbst R.S., et al., “Selective Oral Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Zd1839 is Generally Well-Tolerated and has Activity in Non-Small-Cell Lung Cancer and Other Solid Tumors: Results of a Phase I Trial,” Journal of Clinical Oncology, 2002, vol. 20 (18), pp. 3815-3825.
  • Herbst R.S., et al., “Epidermal Growth Factor Receptors as a Target for Cancer Treatment: The Emerging Role of IMC-C225 in the Treatment of Lung and Head and Neck Cancers,” Seminars in Oncology, 2002, vol. 29, pp. 27-36.
  • Herbst R.S., et al., “Erlotinib (Tarceva): an Update on the Clinical Trial Program,” Seminars in Oncology , 2003, vol. 30 (3) (Suppl. 7), pp. 34-46.
  • Herbst R.S., et al., “IMC-C225, An Anti-Epidermal Growth Factor Receptor Monoclonal Antibody, for Treatment of Head and Neck Cancer,” Expert Opinion on Biological, 2001, vol. 1 (4), pp. 719-732.
  • Herbst R.S., et al., “IMC-C225, an Anti-Epidermal Growth Factor Receptor Monoclonal Antibody for Treatment of Head and Neck Cancer,” Seminars in Oncology , 2002, vol. 29 (5) (Suppl. 14), pp. 18-30.
  • Herbst R.S., et al., “Monoclonal Antibodies to Target Epidermal Growth Factor Receptor-Positive Tumors: A New Paradigm for Cancer Therapy.,” Cancer, 2002, vol. 94 (5), pp. 1593-1611.
  • Herbst R.S., et al., “Phase II Multicenter Study of the Epidermal Growth Factor Receptor Antibody Cetuximab and Cisplatin for Recurrent and Refractory Squamous Cell Carcinoma of the Head and Neck.,” Journal of Clinical Oncology, 2005, vol. 23 (24), pp. 5578-5587.
  • Herbst R.S., et al., “Phase I/II Trial Evaluating the Anti-Vascular Endothelial Growth Factor Monoclonal Antibody Bevacizumab in Combination With the Her-1/Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Erlotinib for Patients with Recurrent Non-Small-Cell Lung Cancer,” Journal of Clinical Oncology, 2005, vol. 23 (11), pp. 2544-2555.
  • Herbst R.S., “Targeted Therapy in Non-Small-Cell Lung Cancer,” Oncology, 2002, vol. 16 (9), pp. 19-24.
  • Hertler A.A., et al., “Immunotoxins: A Clinical Review of their Use in the Treatment of Malignancies,” Journal of Clinical Oncology, 1989, vol. 7 (12), pp. 1932-1942.
  • Hidalgo, et al., “Phase 1 Trial of EKB-569, An Irreversible Inhibitor of the Epidermal Growth Factor Receptor (EGFR), in Patients with Advanced Solid Tumors,” Proceedings of the American Society of Clinical Oncology, 2002, vol. 17a (Abstract 65), pp. 21.
  • Hidalgo M., et al., “Phase I and Pharmacologic Study of OSI-774, an Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, in Patients With Advanced Solid Malignancies,” Journal of Clinical Oncology, 2001, vol. 9 (13), pp. 3267-3279.
  • Hills D., et al., “Specific Targeting of a Mutant, Activated FGF Receptor Found in Glioblastoma Using a Monoclonal Antibody,” International Journal of Cancer, 1995, vol. 63 (4), pp. 537-543.
  • Hirata A., et al., “Zd1839 (Iressa) Induces Antiangiogenic Effects Through Inhibition of Epidermal Growth Factor Receptor Tyrosine Kinase,” Cancer research , 2002, vol. 62 (9), pp. 2554-2560.
  • Hird, et al., “Immunotherapy with Monoclonal Antibodies” In: Genes and Cancer, Chapter 17, Carry, eds., John Wiley & Sons, Ltd., 1990, pp. 183-189.
  • Hirsch F.R., et al., “Epidermal Growth Factor Receptor in Non-Small-Cell Lung Carcinomas: Correlation Between Gene Copy Number and Protein Expression and Impact on Prognosis,” Journal of Clinical Oncology, 2003, vol. 21 (20), pp. 3798-3807.
  • Hirsch F.R., et al., “Increased Epidermal Growth Factor Receptor Gene Copy Number Detected by Fluorescence In Situ Hybridization Associates With Increased Sensitivity to Gefitinib in Patients With Bronchioloalveolar Carcinoma Subtypes: A Southwest Oncology Group Study,” Journal of Clinical Oncology, 2005, vol. 23 (28), pp. 6838-6845.
  • Hoffman, et al., “Phase I Trials of CDR-Grafted Humanized Monoclonal Antibody Hu3S193 in Patients with Lewis-Y Expressing Solid Tumors,” American Society of Clinical Oncology, 2001, Abstract 2634, pp. 20.
  • Hoffmann T., et al., “Antitumor Activity of Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies and Cisplatin in Ten Human Head and Neck Squamous Cell Carcinoma Lines,” Anticancer Research, 1997, vol. 17 (6D), pp. 4419-4425.
  • Hogg P.J., “Disulfide Bonds as Switches for Protein Function,” Trends in biochemical sciences, 2003, vol. 28 (4), pp. 210-214.
  • Holbro T., et al., “ErbB Receptors: Directing Key Signaling Networks Throughout Life,” Annual Review of Pharmacology and Toxicology, 2004, vol. 44, pp. 195-217.
  • Holbro T., et al., “The Erbb Receptors and their Role in Cancer Progression,” Experimental Cell Research , 2003, vol. 284 (1), pp. 99-110.
  • Holbrook M.R., et al., “Thermodynamic Mixing of Molecular States of the Epidermal Growth Factor Receptor Modulates Macroscopic Ligand Binding Affinity,” Biochemical Journal, 2000, vol. 352 (Pt 1), pp. 99-108.
  • Holland E.C., et al., “A Constitutively Active Epidermal Growth Factor Receptor Cooperates With Disruption of G1 Cell-Cycle Arrest Pathways to Induce Glioma-Like Lesions in Mice,” 1998, vol. 12 (23), pp. 3675-3685.
  • Holland E.C., et al., “Glioblastoma Multiforme: The Terminator,” Proceedings of the National Academy of Sciences united states of america, 2000, vol. 97 (12), pp. 6242-6244.
  • Holliger P., et al., ““Diabodies”: Small Bivalent and Bispecific Antibody Fragments,” Proceedings of the National Academy of Sciences of the USA, 1993, vol. 90 (14), pp. 6444-6448.
  • Hollstein M.C., et al., “Amplification of Epidermal Growth Factor Receptor Gene but no Evidence of Ras Mutations in Primary Human Esophageal Cancers,” Cancer research , 1988, vol. 48 (18), pp. 5119-5123.
  • Holm P., et al., “Functional Mapping and Single Chain Construction of the Anti-Cytokeratin & Monoclonal Antibody TS1,” Molecular Immunology, 2007, vol. 44, pp. 1075-1084.
  • Holmes M.A., et al., “Structural Consequences of Humanizing an Antibody1,” The Journal of Immunology, 1997, vol. 158 (5), pp. 2192-2201.
  • Holt L.J., et al., “Domain Antibodies: Proteins for Therapy,” Trends in Biotechnology, 2003, vol. 21 (11), pp. 484-490.
  • Honegger A., et al., “Biological Activities of EGF-Receptor Mutants with Individually Altered Autophosphorylation Site,” The EMBO Journal, 1988, vol. 7 (10), pp. 3045-3052.
  • Hong, et al., “Efficacy and Safety of the Anti-Epidermal Growth Factor Antibody (EGFR) Imc-225, In Combination with Cisplatin in Patients with Recurrent Squamous Cell Carcinoma of the Head and Neck (SCCHN) Refractory to Cisplatin Containing Chemotherapy,” Proceedings of the American Society of Clinical Oncology, 2001, vol. 224a (Abstract 895), pp. 20.
  • Hooft R.W., et al., “Errors in Protein Structures,” Nature, 1996, vol. 381 (6580), pp. 272.
  • Hortobagyi G.N., “Overview of Treatment Results with Trastuzumab (Herceptin) in Metastatic Breast Cancer ,” Seminars in Oncology , 2001, vol. 28 (6 Suppl. 18), pp. 43-47.
  • Hosoi K., et al., “Exogenous ATP and other Nucleoside Phosphates Modulate Epidermal Growth Factor Receptors of A-431 Epidermoid Carcinoma Cells,” Proceedings of the National Academy of Sciences, 1989, vol. 86 (12), pp. 4510-4514.
  • Huang H.S., et al., “The Enhanced Tumorigenic Activity of a Mutant Epidermal Growth Factor Receptor Common in Human Cancers is Mediated by Threshold Levels of Constitutive Tyrosine Phosphorylation and Unattenuated Signaling,” Journal of Biological Chemistry, 1997, vol. 272 (5), pp. 2927-2935.
  • Huang P.H., et al., “Phosphotyrosine Signaling Analysis of Site-Specific Mutations on EGFRvIII Identifies Determinants Governing Glioblastoma Cell Growth,” Molecular BioSystems, 2010, vol. 6 (7), pp. 1227-1237.
  • Huang P.H., et al., “Quantitative Analysis of EGFRvIII Cellular Signaling Networks Reveals a Combinatorial Therapeutic Strategy for Glioblastoma,” Proceedings of the National Academy of Sciences, 2007, vol. 104 (31), pp. 12867-12872.
  • Huang P.H., et al., “Uncovering Therapeutic Targets for Glioblastoma: A Systems Biology Approach,” Cell Cycle, 2007, vol. 6 (22), pp. 2750-2754.
  • Huang S., et al. “Dual-Agent Molecular Targeting of the Epidermal Growth Factor Receptor (Egfr) Combining Anti -Egfr Anti Body with Tyrosine Kinase Inhibitor,” Cancer Research, 2004, vol. 64, pp. 5355-5362.
  • Huang S.M., et al., “Epidermal Growth Factor Receptor Blockade with C225 Modulates Proliferation, Apoptosis, and Radiosensitivity in Squamous Cell Carcinomas of the Head and Neck,” Cancer research , 1999, vol. 59 (8), pp. 1935-1940.
  • Huang S.M., et al., “Epidermal Growth Factor Receptor Inhibition in Cancer Therapy: Biology, Rationale and Preliminary Clinical Results,” Investigational New Drugs, 1999, vol. 17 (3), pp. 259-269.
  • Huang S.M., et al., “Modulation of Radiation Response after Epidermal Growth Factor Receptor Blockade in Squamous Cell Carcinomas: Inhibition of Damage Repair, Cell Cycle Kinetics, and Tumor Angiogenesis,” Clinical Cancer Research, 2000, vol. 6 (6), pp. 2166-2174.
  • Huang S.M., et al., “Modulation of Radiation Response and Tumor-Induced Angiogenesis After Epidermal Growth Factor Receptor Inhibition by ZD1839 (Iressa),” Cancer Research, 2002, vol. 62 (15), pp. 4300-4306.
  • Hubbard S.R., “EGF Receptor Inhibition: Attacks on Multiple Fronts,” Cancer Cell, 2005, vol. 7 (4), pp. 287-288.
  • Hubbard S.R., et al., “Protein Tyrosine Kinase Structure and Function,” Annual Review of Biochemistry, 2000, vol. 69, pp. 373-398.
  • Huber P.E., et al., “Trimodal Cancer Treatment: Beneficial Effects of Combined Antiangiogenesis, Radiation, and Chemotherapy,” Cancer Research, 2005, vol. 65 (9), pp. 3643-3655.
  • Hudson P.J., et al., “Engineered Antibodies,” Nature medicine, 2003, vol. 9 (1), pp. 129-134.
  • Humphrey P.A., et al., “Amplification and Expression of the Epidermal Growth Factor Receptor Gene in Human Glioma Xenografts,” Cancer research , 1988, vol. 48 (8), pp. 2231-2238.
  • Humphrey P.A., et al., “Anti-Synthetic Peptide Antibody Reacting at the Fusion Junction of Deletion-Mutant Epidermal Growth Factor Receptors in Human Glioblastoma,” Proceedings of the National Academy of Sciences, 1990, vol. 87, pp. 4207-4211.
  • Humphrey P.A., et al., “Deletion-Mutant Epidermal Growth Factor Receptor in Human Gliomas: Effects of Type li Mutation on Receptor Function,” Biochemical and Biophysical Research Communications , 1991, vol. 178 (3), pp. 1413-1420.
  • Humphreys D.P., et al., “Therapeutic Antibody Production Technologies: Molecules, Applications, Expression and Purification ,” Therapeutic Antibody Production Technologies Humphreys & Glover, 2001, vol. 4 (2), pp. 172-185.
  • Hunts J., et al., “Hyperproduction and Gene Amplification of the Epidermal Growth Factor Receptor in Squamous Cell Carcinomas,” Japanese Journal of Cancer Research 1985, vol. 76 (8), pp. 663-666.
  • Hurtt M.R., et al., “Amplification of Epidermal Growth Factor Receptor Gene in Gliomas: Histopathology and Prognosis,” Journal of Neuropathology & Experimental Neurology, 1992, vol. 51 (1), pp. 84-90.
  • Hurwitz H., et al., “Bevacizumab Plus Irinotecan, Fluorouracil, and Leucovorin for Metastatic Colorectal Cancer,” The New England Journal of Medicine, 2004, vol. 350 (23), pp. 2335-2342.
  • Huston J.S., et al., “Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia Coli,” Proceedings of the National Academy of Sciences of the USA, 1988, vol. 85 (16), pp. 5879-5883.
  • Hynes N.E., et al., “ERBB Receptors and Cancer: The Complexity of Targeted Inhibitors,” Nature Reviews Cancer, 2005, vol. 5 (5), pp. 341-354.
  • Illidge T.M., et al., “Antibody Therapy of Lymphoma,” Expert Opinion on Pharmacotherapy , 2001, vol. 2 (6), pp. 953-961.
  • Inoue K., et al., “Paclitaxel Enhances the Effects of the Anti-Epidermal Growth Factor Receptor Monoclonal Antibody Imclone C225 in Mice with Metastatic Human Bladder Transitional Cell Carcinoma,” Clinical Cancer Research, 2000, vol. 6 (12), pp. 4874-4884.
  • International Preliminary Report on Patentability and Written Opinion for Application No. PCT/US07/19988, mailed on Mar. 17, 2009, 4 pages.
  • International Preliminary Report on Patentability and Written Opinion for Application No. PCT/US2010/024407, mailed on Aug. 23, 2011, 14 pages.
  • International Preliminary Report on Patentability for Application No. PCT/US02/15185, mailed on Nov. 3, 2003, 4 pages.
  • International Preliminary Report on Patentability for Application No. PCT/US08/03369, mailed on Sep. 15, 2009, 1 page.
  • International Preliminary Report on Patentability for Application No. PCT/US2005/005155, mailed on Aug. 22, 2006, 4 pages.
  • International Preliminary Report on Patentabilty for Application No. PCT/US2008/01024, mailed on Jul. 28, 2009, 1 page.
  • International Written Opinion for Application No. PCT/US2010/050559, mailed on May 10, 2011, 23 pages.
  • Invitation to Pay Additional Fees for Application No. PCT/US2010/024407 mailed on Jun. 4, 2010.
  • Ishida T., et al., “The Expression Technology of Chimeric and Humanized Antibodies,” Nippon Rinsho, 2002, vol. 60 (3), pp. 439-444.
  • Ishitoya J., et al., “Gene Amplification and Overexpression of Egf Receptor in Squamous Cell Carcinomas of the Head and Neck,” British Journal of Cancer 1989, vol. 59 (4), pp. 559-562.
  • Ishizawar R., et al., “C-src and Cooperating Partners in Human Cancer,” Cancer Cell, 2004, vol. 6 (3) pp. 209-214.
  • Italiano A., “Targeting the Epidermal Growth Factor Receptor in Colorectal Cancer: Advances and Controversies,” Oncology, 2006, vol. 70 (3), pp. 161-167.
  • Iznaga-Escobar N., et al. , “Technetium-99m-Antiepidermal Growth Factor-Receptor Antibody in Patients with Tumors of Epithelial Origin: Part ii Pharmacokinetics and Clearances,” Journal of Nuclear Medicine, 1998, vol. 39 (11), pp. 1918-1927.
  • Jamnongjit M., et al., “Epidermal Growth Factor Receptor Signaling is Required for Normal Ovarian Steroidogenesis and Oocyte Maturation,” Proceedings of the National Academy of Sciences united states of america , 2005, vol. 102 (45), pp. 16257-16262.
  • Janmaat M.L., et al., “Response to Epidermal Growth Factor Receptor Inhibitors in Non- Small Cell Lung Cancer Cells Limited Antiproliferative Effects and Absence of Apoptosis Associated with Persistent Activity of Extracellular Signal-regulated Kinase or Akt Kinase Pathways ,” Clinical Cancer Research, 2003, vol. 9 (6), pp. 2316-2326.
  • Janne P.A., et al., “Epidermal Growth Factor Receptor Mutations in Non-Small-Cell Lung Cancer: Implications for Treatment and Tumor Biology,” Journal of Clinical Oncology, 2005, vol. 23 (14), pp. 3227-3234.
  • Jaros E., et al., “Prognostic Implications of P53 Protein, Epidermal Growth Factor Receptor, and Ki-67 Labelling in Brain Tumours,” British Journal of Cancer, 1992, vol. 66 (2), pp. 373-385.
  • Jay E.T., et al., “Chemical Synthesis of a Biologically Active Gene for Human Immune Interferon-y, Prospect for Site-Specific Mutagenesis and Structure-Function Studies,” Journal of Biological Chemistry, 1984, 259 (10), pp. 6311-6317.
  • Ji H., et al., “Epidermal Growth Factor Receptor Variant III Mutations in Lung Tumorigenesis and Sensitivity to Tyrosine Kinase Inhibitors,” Proceedings of the National Academy of Sciences, 2006, vol. 103 (20), pp. 7817-7822.
  • Ji H., et al., “Perspective EGFR Targeted Therapy View From Biological Standpoint,” Cell Cycle, 2006, vol. 5 (18), pp. 2072-2076.
  • Ji H., et al., “The Impact of Human EGFR Kinase Domain Mutations on Lung Tumorigenesis and in Vivo Sensitivity to EGFR-Targeted Therapies,” Cancer Cell, 2006, vol. 9 (6), pp. 485-495.
  • Jiang B., et al., “A Novel Peptide Isolated from a Phage Display Peptide Library with TrastuzumAb can Mimic Antigen Epitope of HER-2,” The Journal of Biological Chemistry, 2005, vol. 280 (6), pp. 4656-4662.
  • Jiang H., et al., “Growth Suppression of Human Hepatocellular Carcinoma Xenografts by a Monoclonal Antibody CH12 Directed to Epidermal Growth Factor Receptor Variant III,” Journal of Biological Chemistry, 2011, vol. 286 (7), pp. 5913-5920.
  • Johns, et al., “A Novel Antibody Directed to the Epidermal Growth Factor Receptor (EGFR) Displays Additive and Synergistic Anti-Tumor Activity when used in Combination with Standard EGFR Therapeutics,” Proceedings of the International Symposium sponsored by the Cancer Research Institute, 2002, Abstract P-08.
  • Johns, et al., “A Novel Antibody Directed to the Epidermal Growth Factor Receptor (EGFR) Displays Additive and Synergistic Anti-Tumour Activity When Used in Combination with Standard EGFR Therapeutics,” The Proceedings of the 15th Annual Lorne Cancer Conference, 2003, Abstract P212.
  • Johns, et al., “Biological Properties of the Glioma Associated Delta 2-7 Epidermal Growth Factor Receptor,” the 11th International Conference on Second Messengers and Phosphoproteins, 2001, Abstract P183.
  • Johns et al., “Ludwig Au Annual Branch Report,” 2000, pp. 118-119.
  • Johns T.G., et al., “A Novel Antibody Directed to the Epidermal Growth Factor Receptor (EGFR) Displays Additive and Synergistic Anti-Tumor Activity When Used in Combination with Standard EGFR Therapeutics,” Proceedings of the American Association for Cancer Research, 2002, vol. 43, pp. 580-581.
  • Johns T.G., et al., “Antitumor Efficacy of Cytotoxic Drugs and the Monoclonal Antibody 806 is Enhanced by the EGF Receptor Inhibitor AG1478,” Proceedings of the National Academy of Sciences, 2003, vol. 100 (26), pp. 15871-15876.
  • Johns T.G., et al., “Drug Development Targeting the Transition State,” Science, 2004, vol. 2004 (242), pp. tw259.
  • Johns T.G., et al., “Identification of the Epitope for the Epidermal Growth Factor Receptor-Specific Monoclonal Antibody 806 Reveals That It Preferentially Recognizes an Untethered Form of the Receptor,” Journal of Biological Chemistry, 2004, vol. 279 (29), pp. 30375-30384.
  • Johns T.G., et al., “MAb 806 Enhances the Efficacy of Ionizing Radiation in Glioma Xenografts Expressing the de2-7 Epidermal Growth Factor Receptor,” International Journal of Radiation Oncology, Biology, Physics, 2010, vol. 78 (2), pp. 572-578.
  • Johns T.G., et al., “Novel Monoclonal Antibody Specific for the de2-7 Epidermal Growth Factor Receptor (EGFR) that also Recognizes the EGFR Expressed in Cells Containing Amplification of the EGFR Gene,” International Journal of Cancer, 2002, vol. 98 (3), pp. 398-408.
  • Johns T.G., et al., “The Antitumor Monoclonal Antibody 806 Recognizes a High-Mannose form of the EGF Receptor that Reaches the Cell Surface when Cells Over-Express the Receptor,” FASEB Journal, 2005, vol. 19 (7), pp. 780-782.
  • Johns T.G., et al., “The Efficacy of EGFR-Specific Antibodies Against Glioma Xenografts is Influenced by Receptor Levels, Activation Status and Heterodimerization,” Clinical Cancer Research, 2007, vol. 13 (6), pp. 1911-1925.
  • Jones R.B., et al., “A Quantitative Protein Interaction Network for the Erbb Receptors Using Protein Microarrays,” Nature, 2006, vol. 439 (7073), pp. 168-174.
  • Jorgensen, et al., “Immunoconjugates: A Therapy Whose Time Has Come”, Preclinica, 2004, vol. 2, pp. 1-4.
  • Jorissen R.N., et al., “Characterization of a Comparative Model of the Extracellular Domain of the Epidermal Growth Factor Receptor,” Protein Science, 2000, vol. 9 (2), pp. 310-324.
  • Jorissen R.N., et al., “Epidermal Growth Factor Receptor: Mechanisms of Activation and Signalling,” Experimental Cell Research, 2003, vol. 284 (1), pp. 31-53.
  • Jost M., et al., “The EGF Receptor—An Essential Regulator of Multiple Epidermal Functions,” European Journal of Dermatology, 2000, vol. 10 (7), pp. 505-510.
  • Jung G., et al., “Local Immunotherapy of Glioma Patients with a Combination of 2 Bispecific Antibody Fragments and Resting Autologous Lymphocytes: Evidence for in Situ T-Cell Activation and Therapeutic Efficacy,” International Journal of Cancer , 2001, vol. 91 (2), pp. 225-230.
  • Jungbluth A.A., et al., “A Monoclonal Antibody Recognizing Human Cancers with Amplification/Overexpression of the Human Epidermal Growth Factor Receptor,” Proceedings of the National Academy of Sciences, 2003, vol. 100 (2), pp. 639-644.
  • Jutten B., et al., “Binding of Cetuximab to the EGFRvIII Deletion Mutant'and Its Biological Consequences in Malignant Glioma Cells,” Radiotherapy and oncology, 2009, vol. 92 (3), pp. 393-398.
  • Kalofonos H.P., et al., “Antibody Guided Diagnosis and Therapy of Brain Gliomas Using Radiolabeled Monoclonal Antibodies against Epidermal Growth Factor Receptor and Placental Alkaline Phosphatase,” The Journal of Nuclear Medicine, 1989, vol. 30 (10), pp. 1636-1645.
  • Kamat V., et al., “Enhanced Egfr Inhibition and Distinct Epitope Recognition by EGFR Antagonistic.mAbs C225 and 425,” Cancer Biology & Therapy, 2008, vol. 7 (5), pp. 726-733.
  • Kamb A., et al., “A Cell Cycle Regulator Potentially Involved in Genesis of Many Tumor Types,” Science, 1994, vol. 264 (5157), pp. 436-440.
  • Kaminski M.S., et al., “Iodine-131-Anti-B1 Radioimmunotherapy for B-Cell Lymphoma,” Journal of Clinical Oncology, 1996, vol. 14 (7), pp. 1974-1981.
  • Karnes W.E.Jr., et al., “Autonomous Proliferation of Colon Cancer Cells that Coexpress Transforming Growth Factor Alpha and Its Receptor Variable Effects of Receptor-Blocking Antibody,” Gastroenterology, 1992, vol. 102 (2), pp. 474-485.
  • Karnes W.E.Jr., et al., “Inhibition of Epidermal Growth Factor Receptor Kinase Induces Protease-Dependent Apoptosis in Human Colon Cancer Cells,” Gastroenterology, 1998, vol. 114 (5), pp. 930-939.
  • Karpel-Massler G., et al., “Therapeutic Inhibition of the Epidermal Growth Factor Receptor in High-Grade Gliomas: Where do we Stand”, Molecular Cancer Research, 2009, vol. 7 (7), pp. 1000-1012.
  • Kashmiri S.V., et al., “Development of a Minimally Immunogenic Variant of Humanized Anti-Carcinoma Monoclonal Antibody Cc49,” Critical Reviews in Oncology / Hematology , 2001, vol. 38 (1), pp. 3-16.
  • Kasprzyk P.G., et al., “Therapy of an Animal Model of Human Gastric Cancer using a Combination of Anti-erbB-2 Monoclonal Antibodies,” Cancer Research, 1992, vol. 52 (10), pp. 2771-2776.
  • Katzel J.A., et al., “Recent Advances of Novel Targeted Therapy in Non-Small Cell Lung Cancer,” Journal of Hematology & Oncology, 2009, vol. 2, pp. 2.
  • Kawagoe K., et al., “Immunohistochemical Demonstration of Epidermal Growth Factor (Egf) Receptors in Normal Human Placental Villi,” Placenta, 1990, vol. 11 (1), pp. 7-15.
  • Kawamoto T., et al., “Growth Stimulation of A431 Cells by Epidermal Growth Factor Identification of High Affinity Receptors for Epidermal Growth Factor by an Anti-receptor Monoclonal Antibody,” Proceedings National Academy of Science, 1983, vol. 80 (5), pp. 1337-1341.
  • Kawamoto T., et al., “Relation of Epidermal Growth Factor Receptor Concentration to Growth of Human Epidermoid Carcinoma A431 Cells,” Journal of Biological Chemistry, 1984, vol. 259 (12), pp. 7761-7766.
  • Ke L.D., et al., “Differential Expression of Epidermal Growth Factor Receptor in Human Head and Neck Cancers,” Head & neck, 1998, vol. 20 (4), pp. 320-327.
  • Kelly, et al., “ZD1839 (Iressa), An Oral EGFR-TKI (Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor): Pharmacokinetic Results of a Phase I Study in Patients with Advanced Cancer,” Proceedings of the Annual Meeting of the American Association for Cancer Research, 2000, vol. 41 (Abstract 3896), pp. 612-613.
  • Kelly M.P., et al., “Therapeutic Efficacy of 177lu-Chx-A”-Dtpa-Hu3s193 Radioimmunotherapy in Prostate Cancer is Enhanced by Egfr Inhibition or Docetaxel Chemotherapya, Prostate, 2009, vol. 69 (1), pp. 92-104.
  • Khazaeli, et al., “Low Immunogenicity of a Chimeric Monoclonal Antibody (MoAb), IMC-C225, Used to Treat Epidermal Growth Factor Receptor-Positive Tumors,” Proceedings of the American Society of Clinical Oncology, 2000, vol. 207a (Abstract 808), pp. 19.
  • Khazaeli M.B., et al., “Human immune response to monoclonal antibodies,” Journal of immunotherapy with emphasis on tumor immunology, 1994, vol. 15 (1), pp. 42-52.
  • Khazaeli M.B., et al., “Pharmacokinetics and Immune Response of 1311-Chimeric Mouse/Human 872.3 (human y 4) Monoclonal Antibody in Humans,” Cancer Research, 1991, vol. 51 (20), pp. 5461-5466.
  • Khazaie K., et al., “EGF Receptor in Neoplasia and Metastasis,” Cancer and Metastasis Reviews, 1993, vol. 12 (3-4), pp. 255-274.
  • Kies, et al., “Final Report of the Efficacy and Safety of the Anti-Epidermal Growth Factor Antibody Erbitux (Imc-C225), in Combination with Cisplatin in Patients with Recurrent Squamous Cell Carcinoma of the Head and Neck (Scchn) Refractory to Cisplatin Containing Chemotherapy,” Proceedings of the American Society of Clinical Oncology, 2002, vol. 232a (Abstract 925), pp. 21.
  • Kikkawa K., et al., “Immunohistochemical and Histopathological Study of Expression of Epidermal Growth Factor Receptors in Gastric Cancer,” Nippon Geka Gakkai Zasshi, 1993, vol. 94 (12), pp. 1231-1238.
  • Kil S.J., et al., “A Leucine-Based Determinant in the Epidermal Growth Factor Receptor Juxtamembrane Domain is Required for the Efficient Transport of Ligand-Receptor Complexes to Lysosomes,” Journal of Biological Chemistry, 1999, vol. 274 (5), pp. 3141-3150.
  • Kim E.S., et al., A phase II Study of Erhitux (IMC-C225), an Epidermal Growth Factor Receptor(EGFR) Blocking Antibody, in Combination with Docetaxel in Chemotherapy Refractory/resistant Patients with Advanced Non-small Cell, Proceedings of the American Society for Clinical Oncology, 2002, vol. 21, pp. 293A.
  • Kim E.S., et al., “Epidermal Growth Factor Receptor Biology (Imc-C225),” Current Opinion in Oncology, 2001, vol. 13 (6), pp. 506-513.
  • Kim J., et al., “Regulation of Epidermal Growth Factor Receptor Internalization by G Protein-Coupled Receptors,” Biochemistry, 2003, vol. 42 (10), pp. 2887-2894.
  • Kim J.C., et al., “Enhancement of Colorectal Tumor Targeting Using a Novel Biparatopic Monoclonal Antibody Against Carcinoembryonic Antigen in Experimental Radioimmunoguided Surgery,” International Journal of Cancer, 2002, vol. 97 (4), pp. 542-547.
  • King D.J., et al., “Preparation and Preclinical Evaluation of Humanised A33 Immunoconjugates for Radioimmunotherapy,” British Journal of Cancer, 1995, vol. 72 (6), pp. 1364-1372.
  • Kiyota A., et al., “Anti-Epidermal Growth Factor Receptor Monoclonal Antibody 225 Upregulates P27(Kip1) and P15(Ink4b) and Induces G1 Arrest in Oral Squamous Carcinoma Cell Lines,” Oncology, 2002, vol. 63 (1), pp. 92-98.
  • Kiyota A., et al., “Expression of a Truncated Epidermal Growth Factor Receptor in Oral Squamous Cell Carcinomas,” Cancer Letters, 2000, vol. 161 (1), pp. 9-15.
  • Klapper L.N., et al., “Biochemical and Clinical Implications of the Erbb/Her Signaling Network of Growth Factor Receptors,” Advances in Cancer Research 2000, vol. 77, pp. 25-79.
  • Klapper L.N., et al., “Tumor-Inhibitory Antibodies to Her-2/Erbb-2 May Act by Recruiting C-Cbl and Enhancing Ubiquitination of HER-2,” Cancer Research, 2000, vol. 60 (13), pp. 3384-3388.
  • Klijn J.G., et al., “The Clinical Significance of Epidermal Growth Factor Receptor (Egf-R) in Human Breast Cancer: a Review on 5232 Patients,” Endocrine Reviews, 1992, vol. 13 (1), pp. 3-17.
  • Klijn J.G., et al., “The Prognostic Value of Epidermal Growth Factor Receptor (Egf-R) In Primary Breast Cancer: Results of a 10 Year Follow-up Study,” Breast Cancer Research and Treatment, 1994, vol. 29 (1), pp. 73-83.
  • Klingbeil C.K., et al., “Analysis of Substrate Recognition Determinants in a Synthetic Peptide Containing the Tyr 1173 Autophosphorylation Site of the Epidermal Growth Factor Receptor,” Archives of biochemistry and biophysics, 1995, vol. 316 (2), pp. 745.
  • Klingler-Hoffmann M., et al., “Inhibition of Phosphatidylinositol 3-Kinase Signaling Negates the Growth Advantage Imparted by a Mutant Epidermal Growth Factor Receptor on Human Gliobiastoma Cells,” International Journal of Cancer 2003, vol. 105 (3), pp. 331-339.
  • Klohs W.D., et al., “Inhibitors of Tyrosine Kinase,” Current Opinion in Oncology, 1997, vol. 9 (6), pp. 562-568.
  • Knecht R., et al., “Carcinomas Unresponsive to Either Cisplatinum or Anti-Egfr Therapy can be Growth Inhibited by Combination Therapy of Both Agents,” Anticancer Research, 2003, vol. 23 (3B), pp. 2577-2583.
  • Knutson V.P., et al., “Rapid, Reversible Internalization of Cell Surface Insulin Receptors. Correlation with Insulin-Induced Down-Regulation,” Journal of Biological Chemistry, 1983, vol. 258 (20), pp. 12139-12142.
  • Kobayashi S., et al., “An Alternative Inhibitor Overcomes Resistance Caused by a Mutation of the Epidermal Growth Factor Receptor,” Cancer Research, 2005, vol. 65 (16), pp. 7096-7101.
  • Kobayashi S., et al., “EGFR Mutation and Resistance of Non-Small-Cell Lung Cancer to Gefitinib,” The New England Journal of Medicine, 2005, vol. 352 (8), pp. 786-792.
  • Kondo I., et al., “Mapping of the Human Gene for Epidermal Growth Factor Receptor (Egfr) on the P13 Leads to Q22 Region of Chromosome 7,” Cytogenetics and Cell Genetics, 1983, vol. 35 (1), pp. 9-14.
  • Kopetz Synergistic effects of combination therapy with anti-EGFR and anti-Src therapy in vitro in colon cancer. In: Gastrointestinal Cancers Symposium Jan. 19, 2007 Abstract # 406. Retrieved from the Internet on Jun. 3, 2008 at <URL: http://www.asco.org/portal/site/AS CO/menuite m.34d60f5624 ba07fd506fe310ee37a01d/vgnextoid=7618201eb61a7010VgnVCM100000ed730ad1RCRD&vmview=abst—detail—view&confID=45 &abstractID=10679>; abstract.
  • Koprivica V., et al., “EGFR Activation Mediates Inhibition of Axon Regeneration by Myelin and Chondroitin Sulfate Proteoglycans,” Science , 2005, vol. 310 (5745), pp. 106-110.
  • Korshunov A., et al., “Prognostic Value of Tumor Associated Antigen Immunoreactivity and Apoptosis in Cerebral Glioblastomas: An Analysis of 163 Cases,” Journal of Clinical Pathology, 1999, vol. 52 (8), pp. 574-580.
  • Kosaka T., et al., “Mutations of the Epidermal Growth Factor Receptor Gene in Lung Cancer: Biological and Clinical Implications,” Cancer Research, 2004, vol. 64 (24), pp. 8919-8923.
  • Kramer A., et al., “Regulation of Daily Locomotor Activity and Sleep by Hypothalamic Egf Receptor Signaling,” Science , 2001, vol. 294 (5551), pp. 2511-2515.
  • Kris M.G., et al., “Objective regressions in non-small cell lung cancer patients treated in Phase I trials of oral ZD1839 (IressaTM), a selective tyrosine kinase inhibitor that blocks the epidermal growth factor receptor (EGFR), Abstract 233,” Chemotherapy, 2000, pp. 72.
  • Kris R.B., et al., “A phase II trial of Z01839 (‘Iressa’) in advanced non-small cell lung cancer (NSCLC) patients who had failed platinum- and docetaxel-based regimens (Ideal 2),” Proceedings of American Society of Clinical Oncology , 2002, vol. 21, pp. 21.
  • Krug L.M., et al., “Targeting Lewis Y (Le(Y)) in Small Cell Lung Cancer with a Humanized Monoclonal Antibody, Hu3s193: a Pilot Trial Testing Two Dose Levels,” Journal of Thoracic Oncology, 2007, vol. 2 (10), pp. 947-952.
  • Kuan C.T., et al., “1251-Labeled Anti-Epidermal Growth Factor Receptor-Yin Single-Chain Fv Exhibits Specific and High-Level Targeting of Glioma Xenografts,” Clinical Cancer Research, 1999, vol. 5 (6), pp. 1539-1549.
  • Kuan C.T., et al., “EGF Mutant Receptor vIII as a Molecular Target in Cancer Therapy,” Endocrine-Related Cancer, 2001, vol. 8 (2), pp. 83-96.
  • Kuan C.T., et al., “EGFRvIII as a Promising Target for Antibody-Based Brain Tumor Therapy,” Brain Tumor Pathology, 2000, vol. 17 (2), pp. 71-78.
  • Kuan C.T., et al., “Increased Binding Affinity Enhances Targeting of Glioma Xenografts by EGFRvIII-Specific scFv,” International Journal of Cancer, 2000, vol. 88 (6), pp. 962-969.
  • Kubo S., et al., “Three-Dimensional Magnetic Resonance Microscopy of Pulmonary Solitary Tumors in Transgenic Mice,” Magnetic Resonance in Medicine, 2006, vol. 56 (3), pp. 698-703.
  • Kumar R., et al., “Regulation of Phosphorylation of the C-Erbb-2/Her2 Gene Product by a Monoclonal Antibody and Serum Growth Factor(S) in Human Mammary Carcinoma Cells,” Molecular and Cellular Biology, 1991, vol. 11 (2), pp. 979-986.
  • Kunkel M.W., et al., “Inhibition of the Epidermal Growth Factor Receptor Tyrosine Kinase by Pd153035 in Human A431 Tumors in Athymic Nude Mice,” Investigational New Drugs, 1996, vol. 13 (4), pp. 295-302.
  • Kurpad S.N., et al., “Tumor Antigens in Astrocytic Gliomas,” Glia, 1995, vol. 15 (3), pp. 244-256.
  • Kwak E.L., et al., “Irreversible Inhibitors of the EGF Receptor May Circumvent Acquired Resistance to Gefitinib,” Proceedings of the National Academy of Sciences, 2005, vol. 102 (21), pp. 7665-7670.
  • Kwok T.T., et al., “Cell Cycle Dependence of Epidermal Growth Factor Induced Radiosensitization,” International Journal of Radiation Oncology Biology Physics, 1992, vol. 22 (3), pp. 525-527.
  • Kwok T.T., et al., “Differences in EGF Related Radiosensitisation of Human Squamous Carcinoma Cells with High and Low Numbers of EGF Receptors,” British Journal of Cancer, 1991, vol. 64 (2), pp. 251-254.
  • Lackmann M., et al., “Eph, a Protein Family Coming of Age: More Confusion, Insight, or Complexity,” Science Signaling, 2008, vol. 1 (15), pp. re2.
  • Lacouture M.E., “Mechanisms of Cutaneous Toxicities to EGFR Inhibitors,” Nature Reviews Cancer, 2006, vol. 6 (10), pp. 803-812.
  • Laderoute K.R., et al., “Epidermal Growth Factor Modifies Cell Cycle Control in A431 Human Squamous Carcinoma Cells Damaged by Ionizing Radiation,” Cancer Research, 1994, vol. 54 (6), pp. 1407-1411.
  • Lakowicz J.R., “Principles of Fluorescence Spectroscopy,” 2nd Edition, Kluwer Academic/Plenum Publishers, 1999, Table of Contents.
  • Lal A., et al., “Mutant Epidermal Growth Factor Receptor Up-Regulates Molecular Effectors of Tumor Invasion,” Cancer Research, 2002, vol. 62 (12), pp. 3335-3339.
  • Lammering G., et al., “EGFRVIII-Mediated Radioresistance Through a Strong Cytoprotective Response,” Oncogene, 2003, vol. 22 (36), pp. 5545-5553.
  • Lammering G., et al., “Inhibition of the Type III Epidermal Growth Factor Receptor Variant Mutant Receptor by Dominant-Negative EGFR-CD533 Enhances Malignant Glioma Cell Radiosensitivity,” Clinical Cancer Research, 2004, vol. 10 (19), pp. 6732-6743.
  • Lammering G., et al., “Radiosensitization of Malignant Glioma Cells through Overexpression of Dominant-Negative Epidermal Growth Factor Receptor,” Clinical Cancer Research, 2001, vol. 7 (3), pp. 682-690.
  • Lammerts Van Bueren J.J., et al., “Effect of Target Dynamics on Pharmacokinetics of a Novel Therapeutic Antibody Against the Epidermal Growth Factor Receptor: Implications for the Mechanisms of Action,” Cancer Research, 2006, vol. 66 (15), pp. 7630-7638.
  • Lammerts Van Bueren J.J., et al., “The Antibody Zalutumumab Inhibits Epidermal Growth Factor Receptor Signaling by Limiting Intra and Intermolecular Flexibility,” Proceedings of the National Academy of Sciences of the United States of America, 2008, vol. 105 (16), pp. 6109-6114.
  • Landry R.C., et al., “Antibody Recognition of a Conformational Epitope in a Peptide Antigen: Fv-Peptide Complex of an Antibody Fragment Specific for the Mutant EGF Receptor, EGFRvIII,” Journal of Molecular Biology, 2001, vol. 308 (5), pp. 883-893.
  • Langedijk J.P., et al., “Antigenic Structure of the Central Conserved Region of Protein G of Bovine Respiratory Syncytial Virus,” Journal of Virology, 1997, vol. 71 (5), pp. 4055-4061.
  • Lango M.N., et al., “Targeting Growth Factor Receptors: Integration of Novel Therapeutics in the Management of Head and Neck Cancer,” Current Opinion in Oncology, 2001, vol. 13 (3), pp. 168-175.
  • Lanzetti L., et al., “The Eps8 Protein Coordinates Egf Receptor Signalling Through Rac and Trafficking through Rab5,” Nature, 2000, vol. 408 (6810), pp. 374-377.
  • Lapthorn A.J., et al., “Cystine Nooses and Protein Specificity,” Nature Structural Biology, 1995, vol. 2 (4), pp. 266-268.
  • Larysz D., et al., “Epidermal Growth Factor Receptor Gene Expression in High Grade Gliomas”, Folia Neuropathol 2011, vol. 49 (1), pp. 28-38.
  • Laskowski R.A., “Procheck: A Program to Check the Sterochemical Quality of Protein Structures,” Journal of Applied Crystallography, 1993, vol. 26, pp. 283-291.
  • Lassman A.B., et al., “Response of Glioblastomas to EGFR Kinase Inhibitors,” The New England Journal of Medicine, 2006, vol. 354 (5), pp. 525-526.
  • Lautrette A., et al., “Angiotensin II and EGF Receptor Cross-Talk in Chronic Kidney Diseases: A New Therapeutic Approach,” Nature Medicine, 2005, vol. 11 (8), pp. 867-874.
  • Lawrentschuk N., et al., “Assessing Regional Hypoxia in Human Renal Tumours using 18f-Fluoromisonidazole Positron Emission Tomography,” British Journal of Urology International, 2005, vol. 96 (4), pp. 540-546.
  • Lax I., et al., “Epidermal Growth Factor (EGF) Induces Oligomerization of Soluble, Extracellular, Ligand-Binding Domain of EGF Receptor. A Low Resolution Projection Structure of the Ligand-Binding Domain,” Journal of Biological Chemistry, 1991, vol. 266 (21), pp. 13828-13833.
  • Lax I., et al., “Functional Analysis of the Ligand Binding Site of EGF-Receptor Utilizing Chimeric Chicken/Human Receptor Molecules,” EMBO Journal, 1989, vol. 8 (2), pp. 421-427.
  • Lax L., et al., “Noncontiguous Regions in the Extracellular Domain of EGF Receptor Define Ligand-Binding Specificity,” Cell Regulation, 1991, vol. 2 (5), pp. 337-345.
  • Leahy D.J., et al., “A Mammalian Expression Vector for Expression and Purification of Secreted Proteins for Structural Studies,” Protein Expression and Purification, 2000, vol. 20 (3), pp. 500-506.
  • Learn C.A., et al., “Resistance to Tyrosine Kinase Inhibition by Mutant Epidermal Growth Factor Receptor Variant III Contributes to the Neoplastic Phenotype of Glioblastoma Multiforme ,” Clinical Cancer Research, 2004, vol. 10 (9), pp. 3216-3224.
  • Lee F., et al., “Immunopet Detection of Xenografts Expressing De2-7 EGFR Using Iodine-124 Labelled Ch806 Via Residualising Ligand Impr4,” The Journal of Nuclear Medicine, 2006, vol. 47 (5), pp. 429.
  • Lee F.T., et al., “Enhanced Efficacy of Radioimmunotherapy with 90Y-CHX-A-DTPA-hu3S193 by Inhibition of Epidermal Growth Factor Receptor (EGFR) Signaling with EGFR Tyrosine Kinase Inhibitor AG1478,” Clinical Cancer Research, 2005, vol. 11 (19 pt 2), pp. 7080s-7086s.
  • Lee F.T., et al., “Immuno-Pet for Tumor Targeting,” Journal of Nuclear Medicine, 2003, vol. 44 (8), pp. 1282-1283.
  • Lee F.T., et al., “Immuno-Pet of Human Colon Xenograft- Bearing Balb/C Nude Mice using 124i-Cdr-Grafted Humanized A33 Monoclonal Antibody,” Journal of Nuclear Medicine, 2001, vol. 42 (5), pp. 764-769.
  • Lee F.T., et al., “Immuno-PET Quantitation of de2-7 Epidermal Growth Factor Receptor Expression in Glioma Using 1241-IMP-R4—Labeled Antibody ch806,” Journal of Nuclear Medicine, 2010, vol. 51 (6), pp. 967-972.
  • Lee Y.S., et al., “Therapeutic Efficacy of Antiglioma Mesenchymal Extracellular Matrix 131-Radiolabeled Murine Monoclonal Antibody in a Human Glioma Xenograft Model,” Cancer Research, 1988, vol. 48 (3), pp. 559-566.
  • Legge F.S., “Computational Design of Humanized Antibodies against the Epidermal Growth Factor Receptor (Project Report),” The Ludwig Institute for Cancer Research, University of Melbourne, 2003, 1-321.
  • Lei W., et al., “Enhancement of Chemosensitivity and Programmed Cell Death by Tyrosine Kinase Inhibitors Correlates with Egfr Expression in Non-Small Cell Lung Cancer Cells,” Anticancer Research, 1999, vol. 19 (1A), pp. 221-228.
  • Lenferink A.E., et al., “Blockade of the Epidermal Growth Factor Receptor Tyrosine Kinase Suppresses Tumorigenesis in Mmtv/Neu + Mmtv/Tgf-Alpha Bigenic Mice,” Proceedings of the National Academy of Sciences, 2000, vol. 97 (17), pp. 9609-9614.
  • Lenz, et al., “Consistent Response to Treatment with Cetuximab Monotherapy in Patients with Metastatic Colorectal Cancer,” Journal of Clinical Oncology, ASCO Annual Meeting Proceedings, 2005, Abstract 3536, 23 (16S).
  • Lenz H.J., et al., “Multicenter Phase II and Translational Study of Cetuximab in Metastatic Colorectal Carcinoma Refractory to Irinotecan, Oxaliplatin, and Fluoropyrimidines,” Journal of Clinical Oncology, 2006, vol. 24 (30), pp. 4914-4921.
  • Leon S.P., et al., “Genetic Aberrations in Human Brain Tumors,” Neurosurgery, 1994, vol. 34 (4), pp. 708-722.
  • Leu T.H., et al., “Functional Implication of the Interaction Between Egf Receptor and C-Src,” Frontiers in Bioscience, 2003, vol. 8, pp. S28-S38.
  • Levitzki A., et al., “Tyrosine Kinase Inhibition: An Approach to Drug Development,” Science 1995, vol. 267 (5205), pp. 1782-1788.
  • Lewis Phillips G.D., et al., “Targeting HER2-Positive Breast Cancer With Trastuzumab-Dm1, An Antibody-Cytotoxic Drug Conjugate,” Cancer Research 2008, vol. 68 (22), pp. 9280-9290.
  • Li B., et al., “Mutant Epidermal Growth Factor Receptor Displays Increased Signaling Through the Phosphatidylinositol-3 kinase/AKT Pathway and Promotes Radioresistance in Cells of Astrocytic Origin,” Oncogene, 2004, vol. 23 (26), pp. 4594-4602.
  • Li B., et al., “Resistance to Small Molecule Inhibitors of Epidermal Growth Factor Receptor in Malignant Gliomas,” Cancer Research, 2003, vol. 63 (21), pp. 7443-7450.
  • Li D., et al., “Therapeutic Anti-EGFR Antibody 806 Generates Responses in Murine de Novo EGFR Mutant-Dependent Lung Carcinomas,” Journal of Clinical Investigation, 2007, vol. 117 (2), pp. 346-352.
  • Li G., et al., “EGF Receptor Variant III as a Target Antigen for Tumor Immunotherapy,” Expert Review of Vaccines, 2008, vol. 7 (7), pp. 977-985.
  • Li S., et al., “Structural Basis for EGF Receptor Inhibition by the Therapeutic Antibody Imc- 11F8,” Structure, 2008, vol. 16 (2), pp. 216-227.
  • Li S., et al., “Structural Basis for Inhibition of the Epidermal Growth Factor Receptor by CetuximAb,” Cancer Cell, 2005, vol. 7 (4), pp. 301-311.
  • Libermann T.A., et al., “Amplification, Enhanced Expression and Possible Rearrangement of EGF Receptor Gene in Primary Human Brain Tumours of Glial Origin,” Nature, 1985, vol. 313 (5998), pp. 144-147.
  • Libermann T.A., et al., “Expression of Epidermal Growth Factor Receptors in Human Brain Tumors,” Cancer Research 1984, vol. 44 (2), pp. 753-760.
  • Lichtner R.B., et al., “Signaling inactive Epidermal Growth Factor Receptor Ligand Complexes in Intact Carcinoma Cells by Quinazoline Tyrosine Kinase Inhibitors,” Cancer Research, 2001, vol. 61 (15), pp. 5790-5795.
  • Lin C.R., et al., “Expression Cloning of Human Egf Receptor Complementary Dna: Gene Amplification and Three Related Messenger Rna Products in A431 Cells,” Science, 1984, vol. 224 (4651), pp. 843-848.
  • Lindmo T., et al., “Determination of the Immunoreactive Fraction of Radiolabeled Monoclonal Antibodies by Linear Extrapolation to Binding at Infinite Antigen Excess,” Journal of Immunological Methods, 1984, vol. 72 (1), pp. 77-89.
  • Lipton, et al., “Elevated Serum HER-2/neu Level Predicts Decreased Response to Hormone Therapy in Metastatic Breast Cancer,” Proceedings of the American Society of Clinical Oncology, 2000, vol. 71a (Abstract 274), pp. 19.
  • Little M., et al., “Of Mice and Men: Hybridoma and Recombinant Antibodies,” Immunology Today, 2000, vol. 21 (8), pp. 364-370.
  • Liu B., et al., “Epidermal Growth Factor Receptor Activation: An Upstream Signal for Transition of Quiescent Astrocytes into Reactive Astrocytes after Neural Injury,” Journal of Neuroscience, 2006, vol. 26 (28), pp. 7532-7540.
  • Liu L., et al., “Clinical Significance of EGFR Amplification and the Aberrant EgfrvIII Transcript in Conventionally Treated Astrocytic Gliomas,” Journal of Molecular Medicine 2005, vol. 83 (11), pp. 917-926.
  • Liu M., et al., “The Effect of Epidermal Growth Factor Receptor Variant III on Glioma Cell Migration by Stimulating ERK Phosphorylation Through the Focal Adhesion Kinase Signaling Pathway,” Archives of Biochemistry and Biophysics, 2010, vol. 502 (2), pp. 89-95.
  • Liu X.Y., et al., “Engineering Therapeutic Monoclonal Antibodies,” Immunological Reviews, 2008, vol. 222, pp. 9-27.
  • Liu Z., et al., “Generation of Anti-Idiotype Antibodies for Application in Clinical Immunotherapy Laboratory Analyses,” Hybridoma and Hybridomics, 2003, vol. 22 (4), pp. 219-228.
  • Livneh E., et al., “Reconstitution of Human Epidermal Growth Factor Receptors and Its Deletion Mutants in Cultured Hamster Cells,” Journal of Biological Chemistry, 1986, vol. 261 (27), pp. 12490-12497.
  • Lo H.W., “EGFR-Targeted Therapy in Malignant Glioma: Novel Aspects and Mechanisms of Drug Resistance,” Current Molecular Pharmacology, 2010, vol. 3 (1), pp. 37-52.
  • Lobuglio A.F., et al., “Mouse/Human Chimeric Monoclonal Antibody in Man: Kinetics and Immune Response,” Proceedings of the National Academy of Sciences, 1989, vol. 86 (11), pp. 4220-4224.
  • Loew S., et al., “The Epidermal Growth Factor Receptor as a Therapeutic Target in Glioblastoma Multiforme and Other Malignant Neoplasms,” Anti-Cancer Agents in Medicinal Chemistry, 2009, vol. 9 (6), pp. 703-715.
  • Lofts F.J., et al., “C-erbB2 Amplification and Overexpression in Human Tumors,” Cancer Treatment and Research, 1992, vol. 61, pp. 161-179.
  • Lorimer I.A., et al., “Activation of Extracellular-Regulated Kinases by Normal and Mutant Egf Receptors,” Biochimica et Biophysica Acta, 2001, vol. 1538 (1), pp. 1-9.
  • Lorimer I.A., et al., “Immunotoxins that Target an Oncogenic Mutant Epidermal Growth Factor Receptor Expressed in Human Tumors,” Clinical Cancer Research, 1995, vol. 1, pp. 859-864.
  • Lorimer I.A., et al., “Recombinant Immunotoxins Specific for a Mutant Epidermal Growth Factor Receptor: Targeting with a Single Chain Antibody Variable Domain Isolated by Phage Display,” Proceedings of the National Academy of Sciences, 1996, vol. 93, pp. 14815-14820.
  • Lorimer I.A., “Mutant Epidermal Growth Factor Receptors as Targets for Cancer Therapy,” Current Cancer Drug Targets, 2002, vol. 2 (2), pp. 91-102.
  • Lorusso P.M., et al., “Improvements in Quality of Life and Disease-Related Symptoms in Phase I Trials of the Selective Oral Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Zd1839 in Non-Small Cell Lung Cancer and Other Solid Tumors,” Clinical Cancer Research, 2003, vol. 9 (6), pp. 2040-2048.
  • Lu K.V., et al., “Fyn and SRC are Effectors of Oncogenic Epidermal Growth Factor Receptor Signaling in Glioblastoma Patients,” Cancer Research, 2009, vol. 69 (17), pp. 6889-6898.
  • Ludwig Institute for Cancer Research Press Release, “Clinical Trial Confirms Novel EGFR Antibody Targets Tumours but not Normal Tissues.” (Jun. 5, 2006).
  • Lui V.W., et al., “EGFR-Mediated Cell Cycle Regulation,” Anticancer Research, 2002, 22 (1A), pp. 1-11.
  • Lund K.A., et al., “Phosphorylation of the Epidermal Growth Factor Receptor at Threonine 654 Inhibits Ligand-Induced Internalization and Down-Regulation,” Journal of Biological Chemistry, 1990, vol. 265 (33), pp. 20517-20523.
  • Luwor, et al., “A Soluble Form of the Epidermal Growth Factor Receptor (EGFR) Specific Tyrosine Kinase Inhibitor AG1478 Enhances the Efficacy of Chemotherapy,” Proceedings of the American Association for Cancer Research, 2002, vol. 784 (Abstract 3885), pp. 43.
  • Luwor, et al., “Monoclonal Antibody 806 Inhibits the Growth of Tumor Xenografts Expressing either the de2-7 or Amplified Epidermal Growth Factor Receptor (EGFR) but not Wild-Type EGFR,” Austin and Repatriation Medical Centre Research Week, 2000, Abstract 88.
  • Luwor, et al., “The 806 Antibody Inhibits the Growth of Tumor Xenografts Expressing either the de2-7 or Amplified Epidermal Growth Factor Receptor (EGFR) but not Wild-Type EGFR,” Austin and Repatriation Medical Centre Research Week, 2001, Abstract 46.
  • Luwor, et al., “The 806 Antibody Inhibits the Growth of Tumor Xenografts Expressing either the de2-7 or Amplified Epidermal Growth Factor Receptor (EGFR) but not Wild-Type EGFR,” Proceedings of the 13th Annual Lorne Cancer Conference, 2001, Abstract 208.
  • Luwor R.B., et al., “Monoclonal Antibody 806 Inhibits the Growth of Tumor Xenografts Expressing Either the de2-7 or Amplified Epidermal Growth Factor Receptor (EGFR) but not Wild-Type EGFR,” Cancer Research, 2001, vol. 61 (14), pp. 5355-5361.
  • Luwor R.B., et al., “The Tumor-Specific De2-7 Epidermal Growth Factor Receptor (EGFR) Promotes Cells Survival and Heterodimerizes with the Wild-Type EGFR,” Oncogene, 2004, vol. 23 (36), pp. 6095-6104.
  • Luwor R.B., “The monoclonal antibody 806 and tyrosine kinase inhibitor ag1478: novel epidermal growth factor receptor therapeutics (Project Report),” in: Tumour Targeting Program Ludwig Institute for Cancer Research, 2003.
  • Lyall, et al., “EGF Induces Receptor Down-regulation with no Receptor Recycling in KB Cells,” Chemical Abstracts, 1985, vol. 102 (7), pp. 56832q.
  • Lydon N.B., et al., “A Potent Protein-Tyrosine Kinase Inhibitor which Selectively Blocks Proliferation of Epidermal Growth Factor Receptor-Expressing Tumor Cells in Vitro and in Vivo,” International Journal of Cancer, 1998, vol. 76 (1), pp. 154-163.
  • Lynch D.H., et al., “Therapeutic Potential of ABX-EGF: A Fully Human Anti-Epidermal Growth Factor Receptor Monoclonal Antibody for Cancer Treatment,” Seminars in Oncology, 2002, vol. 29 (1), pp. 47-50.
  • Lynch, et al., “A Phase II Trial of Cetuximab as Therapy for Recurrent Non-Small Cell Lung Cancer (NSCLC),” Proceedings of the American Society of Clinical Oncology, 2004, vol. 637s (Abstract 7084), pp. 23.
  • Lynch T.J., et al., “Activating Mutations in the Epidermal Growth Factor Receptor Underlying Responsiveness of Non-Small-Cell Lung Cancer to Gefitinib,” The New England Journal of Medicine , 2004, vol. 350, pp. 2129-2139.
  • MacCallum R.M., et al., “Antibody-Antigen Interactions: Contact Analysis and Binding Site Topography,” Journal of Molecular Biology, 1996, vol. 262 (5), pp. 732-745.
  • MacDiarmid J.A., et al., “Sequential Treatment of Drug-Resistant Tumors with Targeted Minicells Containing Sirna or a Cytotoxic Drug,” Nature Biotechnology, 2009, vol. 27 (7), pp. 643-651.
  • MacDonald A., et al., “Production and Response of a Human Prostatic Cancer Line to Transforming growth Factor-like Molecules,” British Journal of Cancer, 1990, vol. 62 (4), pp. 579-584.
  • Mach, “Monoclonal Antibodies” in: Oxford Textbook of Oncology, Chapter 1.8, Peckham, et al., eds., Oxford University Press, 1995, pp. 81-103.
  • Machiels J.P., et al., “Zalutumumab Plus Best Supportive Care Versus Best Supportive Care Alone in Patients with Recurrent or Metastatic Squamous-Cell Carcinoma of the Head and Neck After Failure of Platinum-Based Chemotherapy: An Open-Label, Randomised Phase 3 Trial,” Lancet Oncology, 2011, vol. 12 (4), pp. 333-343.
  • Maciag T., “The Human Epidermal Growth Factor Receptor-Kinase Complex,” Trends in Biochemical Sciences, 1982.
  • Maeda K., et al., “Ph-Dependent Receptor/Ligand Dissociation as a Determining Factor for Intracellular Sorting of Ligands for Epidermal Growth Factor Receptors in Rat Hepatocytes,” Journal of Controlled Release , 2002, vol. 82 (1), pp. 71-82.
  • Magne N., et al., “Influence of Epidermal Growth Factor Receptor (EGFR) p53 and Intrinsic Map Kinase Pathway Status of Tumour Cells on the Antiproliferative Effect of ZD1839 (Iressa),” British Journal of Cancer, 2002, vol. 86 (9), pp. 1518-1523.
  • Malden L.T., et al., “Selective Amplification of the Cytoplasmic Domain of the Epidermal Growth Factor Receptor Gene in Glioblastoma multiforme,” Cancer Research, 1988, vol. 48 (10), pp. 2711-2714.
  • Malik, et al., “Safety and Efficacy of Panitumumab Monotherapy in Patients with Metastatic Colorectal Cancer (mCRC),” Journal of Clinical Oncology, ASCO Annual Meeting Proceedings, 2005, Abstract 3520, 23 (16S).
  • Malik S.N., et al., “Pharmacodynamic Evaluation of the Epidermal Growth Factor Receptor Inhibitor OSI-774 in Human Epidermis of Cancer Patients,” Clinical Cancer Research, 2003, vol. 9 (7), pp. 2478-2486.
  • Maloney D.G., et al., “Idec-C2B8 (RituximAb) Anti-CD20 Monoclonal Antibody Therapy in Patients with Relapsed Low-Grade Non-Hodgkin”S Lymphoma, Blood, 1997, vol. 90 (6), pp. 2188-2195.
  • Mamot C., et al., “Epidermal Growth Factor Receptor (EGFR)-Targeted Immunoliposomes Mediate Specific and Efficient Drug Delivery to EGFR- and Egfrvill-Overexpressing Tumor Cells,” Cancer Research, 2003, vol. 63 (12), pp. 3154-3161.
  • Mamot C., et al., “Epidermal Growth Factor Receptor-Targeted Immunoliposomes Significantly Enhance the Efficacy of Multiple Anticancer Drugs in Vivo,” Cancer Research, 2005, vol. 65 (24), pp. 11631-11638.
  • Mano, et al., Phase I Trial of Zalutumumab and Irinotecan in Metastatic Colorectal Cancer Patients Who Have Failed Irinotecan-and Cetuximab-Based Therapy, ASCO Meeting, 2009.
  • Margolis B.L., et al., “All Autophbsphorylation Sites of Epidermal Growth Factor (EGF) Receptor and HER2/neu are Located in their Carboxyl-Terminal Tails. Identification of a Novel Site in EGF Receptor,” Journal of Biological Chemistry, 1989, vol. 264 (18), pp. 10667-10671.
  • Marie Y., et al., “Egfr Tyrosine Kinase Domain Mutations in Human Gliomas,” Neurology, 2005, vol. 64 (8), pp. 1444-1445.
  • Mariuzza R.A., et al., “The Structural Basis of Antigen-Antibody Recognition,” Annual Review of Biophysics and Biophysical Chemistry, 1987, vol. 16, pp. 139-159.
  • Markowitz S.D., et al., “Growth Stimulation by Coexpression of Transforming Growth Factor-Alpha and Epidermal Growth Factor-Receptor in Normal and Adenomatous Human Colon Epithelium,” Journal of Clinical Investigation, 1990, vol. 86 (1), pp. 356-362.
  • Marks J.D., et al., “By-Passing Immunization: Building High Affinity Human Antibodies by Chain Shuffling,” Biotechnology, 1992, vol. 10 (7), pp. 779-783.
  • Martinazzi M., et al., “Epidermal Growth Factor Receptor Immunohistochemistry in Different Histological Types of Infiltrating Breast Carcinoma,” Journal of Clinical Pathology, 1993, vol. 46 (11), pp. 1009-1010.
  • Maruo T., et al., “Immunohistochemical Demonstration of Elevated Expression of Epidermal Growth Factor Receptor in the Neoplastic Changes of Cervical Squamous Epithelium,” Cancer, 1992, vol. 69 (5), pp. 1182-1187.
  • Masui, et al., “Treatment with Anti-EGF Receptor Monoclonal Antibody Causes Regression of Difi Human Colorectal Carcinoma Xenografts,” Proceedings of the American Association for Cancer Research, 1991, vol. 394 (Abstract 2340), pp. 32.
  • Masui H., et al., “Cytotoxicity Against Human Tumor Cells Mediated by the Conjugate of Anti-Epidermal Growth Factor Receptor Monoclonal Antibody to Recombinant Ricin a Chain,” Cancer Research, 1989, vol. 49 (13), pp. 3482-3488.
  • Masui H., et al., “Enhanced Tumorigenesis of NR6 Cells which Express Non-Down-Regulating Epidermal Growth Factor Receptors,” Cancer Research, 1991, vol. 51 (22), pp. 6170-6175.
  • Masui H., et al., “Growth Inhibition of Human Tumor Cells in Athymic Mice by Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies,” Cancer Research, 1984, vol. 44, pp. 1002-1007.
  • Masui H., et al., “Mechanism of Antitumor Activity in Mice for Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies with Different Isotypes1,” Cancer Research, 1986, vol. 46 (11), pp. 5592-5598.
  • Matar P., et al. “Combined Epidermal Growth Factor Receptor Targeting with the Tyrosine Kinase Inhibitor Gefitinib (ZD1839) and the Monoclonal Anti Body Cetuximab (IMC-C225) : Superiority Over Single-Agent Receptor Targeting,” Clinical Cancer Research, 2004, vol. 10 (19), pp. 6487-6501.
  • Mateo C., et al., “Humanization of a Mouse Monoclonal Antibody that Blocks the Epidermal Growth Factor Receptor: Recovery of Antagonistic Activity,” Immunotechnology, 1997, vol. 3 (1), pp. 71-81.
  • Matsumoto, et al., “Blockade of EGF-R Signaling with Anti-EGFR Monoclonal Antibody (Mab) C225 Inhibits Matrix Metalloproteinase-9 (MMP-9) Expression and Invasion of Human Transitional Cell Carcinoma (TCC) in Vitro and in Vivo,” Proceedings of the American Association for Cancer Research, vol. 3 (Abstract 565), pp. 39.
  • Matsuo M., et al., “Zd1839, a Selective Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, Shows Antimetastatic Activity using a Hepatocellular Carcinoma Model,” Molecular Cancer Therapeutics, 2003, vol. 2 (6), pp. 557-561.
  • Mattoon D., et al., “The Tethered Configuration of the EGF Receptor Extracellular Domain Exerts only a Limited Control of Receptor Function,” Proceedings of the National Academy of Sciences, 2004, vol. 101 (4), pp. 923-928.
  • Maurizi M., et al., “Prognostic Significance of Epidermal Growth Factor Receptor in Laryngeal Squamous Cell Carcinoma,” British Journal of Cancer, 1996, vol. 74 (8), pp. 1253-1257.
  • Mayes E.L., et al., “Biosynthesis of the Epidermal Growth Factor Receptor in A431 Cells,” EMBO Journal, 1984, vol. 3 (3), pp. 531-537.
  • McCafferty J., et al., “Phage Antibodies: Filamentous Phage Displaying Antibody Variable Domains,” Nature, 1990, vol. 348, pp. 552-554.
  • McIntosh D.G., et al., “The Intraoperative Detection of Ovarian Adenocarcinoma using Radiolabeled CC49 Monoclonal Antibody and a Hand-Held Gamma-detecting Probe,” Cancer Biotherapy & Radiopharmaceuticals, 1997, vol. 12 (4), pp. 287-294.
  • McLeod H.L., et al., “In Vivo Pharmacology and Anti-Tumour Evaluation of the Tyrphostin Tyrosine Kinase Inhibitor RG13022,” British Journal of Cancer, 1996, vol. 74 (11), pp. 1714-1718.
  • Mehra V., et al., “Efficient Mapping of Protein Antigenic Determinants,” Proceedings of the National Academy of Sciences, 1986, vol. 83 (18), pp. 7013-7017.
  • Meikrantz W., et al., “Apoptosis and the Cell Cycle,” Journal of Cellular Biochemistry, 1995, vol. 58 (2), pp. 160-174.
  • Meilhoc E., et al., “High Efficiency Transformation of Intact Yeast Cells by Electric Field Pulses,” Biotechnology, 1990, vol. 8 (3), pp. 223-227.
  • Mellinghoff I.K., et al., “Molecular Determinants of the Response of Glioblastomas to EGFR Kinase Inhibitors,” The New England Journal of Medicine, 2005, vol. 353 (19), pp. 2012-2024.
  • Mellinghoff I.K., et al., “PTEN-Mediated Resistance to Epidermal Growth Factor Receptor Kinase Inhibitors,” Clinical Cancer Research, 2007, vol. 13 (2 pt 1), pp. 378-381.
  • Mellstedt H., “Monoclonal Antibodies in Human Cancer,” Drugs Today (Barc), 2003, vol. 39 (Suppl. C), pp. 1-16.
  • Mendelsohn, et al., “A Phase I Study of Chimerized Anti-Epidermal Growth Factor Receptor (EGFR) Monoclonal Antibody, C225, in Combination with Cisplatin (CDDP) in Patients (Pts) with Recurrent Head and Neck Squamous Cell Carcinoma (SCC),” Annual Meeting of the American Society of Clinical Oncology, 1999, vol. 389a (Abstract 1502), pp. 18.
  • Mendelsohn, et al., “Antibodies to Growth Factors and Receptors” in: Biologic Therapy of Cancer, Section 21.6, DeVita, et al., eds., JB Lippincott Co, 1995, pp. 607-623.
  • Mendelsohn, et al., “Principles of Molecular Cell Biology of Cancer: Growth Factors” in: Cancer: Principles and Practice of Oncology, Chapter 7, DeVita, et al., eds., J.B. Lippincott, Philadelphia, 1993, pp. 114-133.
  • Mendelsohn J., “Blockade of Receptors for Growth Factors: An Anticancer Therapy the Fourth Annual Joseph H Burchenal American Association of Cancer Research Clinical Research Award Lecture,” Clinical Cancer Research, 2000, vol. 6 (3), pp. 747-753.
  • Mendelsohn J., “Epidermal Growth Factor Receptor Inhibition by a Monoclonal Antibody as Anticancer Therapy,” Clincical Cancer Research, 1997, vol. 3 (12 pt 2), pp. 2703-2707.
  • Mendelsohn J., et al., “Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies May Inhibit A431 Tumor Cell Proliferation by Blocking an Autocrine Pathway,” Transactions of the Association of American Physicians, 1987, vol. 100, pp. 173-178.
  • Mendelsohn J., et al., “Epidermal Growth Factor Receptor Family and Chemosensitization,” Journal of the National Cancer Institute, 1997, vol. 89 (5), pp. 341-343.
  • Mendelsohn J., et al., “Epidermal Growth Factor Receptor Targeting in Cancer,” Seminars in Oncology, 2006, vol. 33 (4), pp. 369-385.
  • Mendelsohn J., et al., “Monoclonal antibodies against the receptor for epidermal growth factor as potential anticancer agents, New York: Alan R. Liss, Inc,” Cellular and Molecular Biology of Tumors and Preventative Clinical Applications, 1988, pp. 307-312.
  • Mendelsohn J., et al., “Status of Epidermal Growth Factor Receptor Antagonists in the Biology and Treatment of Cancer,” Journal of Clinical Oncology, 2003, vol. 21 (14), pp. 2787-2799.
  • Mendelsohn J., et al., “The EGF Receptor Family as Targets for Cancer Therapy,” Oncogene, 2000, vol. 19 (56), pp. 6550-6565.
  • Mendelsohn J., et al., “The Epidermal Growth Factor Receptor as a Target for Cancer Therapy,” Endocrine-Related Cancer, 2001, vol. 8 (1), pp. 3-9.
  • Mendelsohn J., et al. “The Willet F. Whitmore, Jr., Lectureship: Blockade of Epidermal Growth Factor Receptors as Anticancer Therapy,” Journal of Urology, 2001, vol. 165 (4), pp. 1152-1157.
  • Mendelsohn J., “Targeting the Epidermal Growth Factor Receptor for Cancer Therapy,” Journal of Clinical Oncology, 2002, vol. 20 (18), pp. 1S-13S.
  • Merlino G.T., et al., “Structure and Localization of Genes Encoding Aberrant and Normal Epidermal Growth Factor Receptor RNAS from A431 Human Carcinoma Cells,” Molecular and Cellular 1985, vol. 5 (7), pp. 1722-1734.
  • Messa C., et al., “EGF, TGF-Alpha, and EGF-R in Human Colorectal Adenocarcinoma,” Methods of Information in Medicine, 1998, vol. 37 (3), pp. 285-289.
  • Messing E.M., et al., “Epidermal Growth Factor—Interactions with Normal and Malignant Urothelium: In Vivo and in Situ Studies,” Journal of Urology, 1987, vol. 138 (5), pp. 1329-1335.
  • Mickey D.D., et al., “Heterotransplantation of a Human Prostatic Adenocarcinoma Cell Line in Nude Mice,” Cancer Research, 1977, vol. 37 (11), pp. 4049-4058.
  • Milano G., et al., “EGFR-Targeting Drugs in Combination with Cytotoxic Agents: from Bench to Bedside, a Contrasted Reality,” British Journal of Cancer, 2008, vol. 99 (1), pp. 1-5.
  • Milas L., et al., “In Vivo Enhancement of Tumor Radioresponse by C225 Antiepidermal Growth Factor Receptor Antibody,” Clinical Cancer Research, 2000, vol. 6, pp. 701-708.
  • Miller, et al., “A Pilot Trial Demonstrates the Safety of ZD1839 (Iressa), an Oral Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor (EGFR-TKI), in Combination with Carboplatin (C) and Paclitaxel (P) in Previously Untreated Advanced Non-Small Cell Lung Cancer (NSCLC),” 2001, Abstract 1301, pp. 20.
  • Mills L., et al., “Fully Human Antibodies to MCAM/MUC18 Inhibit Tumor Growth and Metastasis of Human Melanoma1 ,” Cancer Research, 2002, vol. 62 (17), pp. 5106-5114.
  • Mineo C., et al., “Regulated Migration of Epidermal Growth Factor Receptor from Caveolae,” Journal of Biological Chemistry, 1999, vol. 274 43), pp. 30636-30643.
  • Mischel P.S., et al., “Targeted Molecular Therapy of GBM,” Brain Pathology, 2003, vol. 13 (1), pp. 52-61.
  • Mishima K., et al., “A Peptide Derived from the Non-Receptor-Binding Region of Urokinase Plasminogen Activator Inhibits Glioblastoma Growth and Angiogenesis in Vivo in Combination with Cisplatin,” Proceedings of the National Academy of Sciences, 2000, vol. 97 (15), pp. 8484-8489.
  • Mishima K., et al., “Expression of a Tumor-Specific Mutant Epidermal Growth Factor Receptor Mediates Glioma Cell Invasion in Vivo,” Proceedings of the American Association for Cancer Research, 1999, vol. 40, pp. 519.
  • Mishima K., et al., “Growth Suppression of Intracranial Xenografted Glioblastomas Overexpressing Mutant Epidermal Growth Factor Receptors by Systemic Administration of Monoclonal Antibody (mAb) 806, A Novel Monoclonal Antibody Directed to the Receptor,” Cancer Research, 2001, vol. 61 (14), pp. 5349-5354.
  • Mitra S., et al., “Passive Antibody-Mediated Immunotherapy for the Treatment of Malignant Gliomas,” Neurosurgery Clinics of North America 2010, vol. 21 (1), pp. 67-76.
  • Moasser M.M., et al., “The Tyrosine Kinase Inhibitor ZD1839 (‘Iressa’) Inhibits Her2-Driven Signaling and Suppresses the Growth of Her2-Overexpressing Tumor Cells,” Cancer Research, 2001, vol. 61 (19), pp. 7184-7188.
  • Modjtahedi H., et al., “Antitumor Activity of Combinations of Antibodies Directed Against Different Epitopes on the Extracellular Domain of the Human EGF Receptor,” Cell Biophysics, 1993, vol. 22 (1-3), pp. 129-146.
  • Modjtahedi H., et al., “Differentiation or Immune Destruction: Two Pathways for Therapy of Squamous Cell Carcinomas With Antibodies to the Epidermal Growth Factor Receptor,” Cancer Research, 1994, vol. 54 (7), pp. 1695-1701.
  • Modjtahedi H., et al., “Egfr Blockade by Tyrosine Kinase Inhibitor or Monoclonal Antibody Inhibits Growth, Directs Terminal Differentiation and Induces Apoptosis in the Human Squamous Cell Carcinoma Hn5,” International Journal of Oncology, 1998, vol. 13 (2), pp. 335-342.
  • Modjtahedi H., et al., “Immunotherapy of Human Tumour Xenografts Overexpressing the Egf Receptor with Rat Antibodies that Block Growth Factor-Receptor Interaction,” British Journal of Cancer , 1993, vol. 67 (2), pp. 254-261.
  • Modjtahedi H., et al., “Phase I Trial and Tumour Localisation of the Anti-EGFR Monoclonal Antibody ICR62 in Head and Neck or Lung Cancer,” British Journal of Cancer, 1996, vol. 73 (2), pp. 228-235.
  • Modjtahedi H., et al., “Targeting of Cells Expressing Wild-type EGFR and Type-III Mutant EGFR (EGFRvIII) by Anti-EGFR MAb ICR62: A Two-pronged Attack for Tumour Therapy,” International Journal of Cancer, 2003, vol. 105 (2), pp. 273-280.
  • Modjtahedi H., et al., “The Human EGF Receptor as a Target for Cancer Therapy: Six New Rat mAbs Against the Receptor on the Breast Carcinoma MDA-MB 468,” British Journal of Cancer, 1993, vol. 67 (2), pp. 247-253.
  • Modjtahedi H., et al., “The Receptor for Egf and Its Ligands—Expression, Prognostic Value and Target for Therapy in Cancer (Review),” International Journal of Oncology, 1994, vol. 4 (2), pp. 277-296.
  • Moghal N., et al., “Multiple Positive and Negative Regulators of Signaling by the Egf-Receptor,” Current Opinion in Cell Biology, 1999, vol. 11 (2), pp. 190-196.
  • Montgomery R.B., et al., “Expression of Oncogenic Epidermal Growth Factor Receptor Family Kinases Induces Paclitaxel Resistance and Alters Beta-Tubulin Isotype Expression,” Journal of Biological chemistry, 2000, vol. 275 (23), pp. 17358-17363.
  • Morales A.A., et al., “Humanized Versus Murine Anti-Human Epidermal Growth Factor Receptor Monoclonal Antibodies for Immunoscintigraphic Studies,” Nuclear Medicine and Biology, 2000, vol. 27 (2), pp. 199-206.
  • Morea V., et al., “Antibody Structure, Prediction and Redesign,” Biophysical Chemistry, 1997, vol. 68 (1-3), pp. 9-16.
  • Moriki T., et al., “Activation of Preformed EGF Receptor Dimers by Ligand-induced Rotation of the Transmembrane Domain ,” Journal of Molecular Biology, 2001, vol. 311 (5), pp. 1011-1026.
  • Moroni M., et al., “Gene Copy Number For Epidermal Growth Factor Receptor (Egfr) and Clinical Response to Antiegfr Treatment in Colorectal Cancer: A Cohort Study,” The Lancet Oncology, 2005, vol. 6 (5), pp. 279-286.
  • Morrison S.L., et al., “Recombinant Chimeric Monoclonal Antibodies,” Important Advances in Oncology, 1990, pp. 3-18.
  • Moscatelio D.K., et al., “Frequent Expression of a Mutant Epidermal Growth Factor Receptor in Multiple Human Tumors,” Cancer Research, 1995, vol. 55, pp. 5536-5539.
  • Moscatello D.K., et al., “A Naturally Occurring Mutant Human Epidermal Growth Factor Receptor as a Target for Peptide Vaccine Immunotherapy of Tumors,” Cancer Research, 1997, vol. 57 (8), pp. 1419-1424.
  • Moscatello D.K., et al., “Constitutive Activation of Phosphatidylinositol 3-Kinase by a Naturally Occurring Mutant Epidermal Growth Factor Receptor,” Journal of Biological Chemistry, 1998, vol. 273 (1), pp. 200-206.
  • Moscatello D.K., et al., “Transformational and Altered Signal Transduction by a Naturally Occurring Mutant Egf Receptor,” Oncogene, 1996, vol. 13 (1), pp. 85-96.
  • Motoyama A.B., et al., “The Efficacy of ErbB Receptor-targeted Anticancer Therapeutics is Influenced by the Availability of Epidermal Growth Factor-related Peptides,” Cancer Research, 2002, vol. 62 (11), pp. 3151-3158.
  • Moulder S.L., et al., “Epidermal Growth Factor Receptor (HeR1) Tyrosine Kinase Inhibitor ZD1839 (Iressa) Inhibits HER2/neu (erbB2)-overexpressing Breast Cancer Cells in Vitro and in Vivo,” Cancer Research, 2001, vol. 61 (24), pp. 8887-8895.
  • Moyer J.D., et al., “Induction of Apoptosis and Cell Cycle Arrest by CP-358,774, An Inhibitor of Epidermal Growth Factor Receptor Tyrosine Kinase,” 1997, vol. 57 (21), pp. 4838-4848.
  • Murshudov G.N., et al., “Refinement of Macromolecular Structures by the Maximum-Likelihood Method,” Acta Crystallogr D Biol Crystallogr, 1997, vol. 53 (pt 3), pp. 240-255.
  • Murthy U., et al., “Binding of an Antagonistic Monoclonal Antibody to an Intact and Fragmented EGF-Receptor Polypeptide,” Archives of Biochemistry and Biophysics, 1987, vol. 252 (2), pp. 549-560.
  • Muthuswamy S.K., et al., “Controlled Dimerization of Erbb Receptors Provides Evidence for Differential Signaling by Homo- and Heterodimers,” 1999, vol. 19 (10), pp. 6845-6857.
  • Nagane M., et al., “A Common Mutant Epidermal Growth Factor Receptor Confers Enhanced Tumorigenicity on Human Glioblastoma Cells by Increasing Proliferation and Reducing Apoptosis,” Cancer Research, 1996, vol. 56 (21), pp. 5079-5086.
  • Nagane M., et al., “Aberrant Receptor Signaling in Human Malignant Gliomas: Mechanisms and Therapeutic Implications,” Cancer Letters, 2001, vol. 162, pp. S17-S21.
  • Nagane M., et al., “Drug Resistance of Human Glioblastoma Cells Conferred by a Tumor-Specific Mutant Epidermal Growth Factor Receptor Through Modulation of BCL-XL and Caspase-3-Like Proteases,” Proceedings of the National Academy of Sciences, 1998, vol. 95 (10), pp. 5724-5729.
  • Nagane M., et al., “Human Glioblastoma Xenografts Overexpressing a Tumor-Specific Mutant Epidermal Growth Factor Receptor Sensitized to Cisplatin by the Ag1478 Tyrosine Kinase Inhibitor,” Journal of Neurosurgery, 2001, vol. 95 (3), pp. 472-479.
  • Nair D.T., et al., “Crystal Structure of an Antibody Bound to an Immunodominant Peptide Epitope: Novel Features in Peptide-Antibody Recognition,” Journal of Immunology, 2000, vol. 165 (12), pp. 6949-6955.
  • Nakagawa, et al., “A phase I intermittent Dose-Escalation Trial of ZD1939 (iressa) in Japanese Patients with Solid Malignant Tumours,” Proceedings of the American Society of Clinical Oncology, 2000, vol. 183 (Abstract 711), pp. 19.
  • Nam S., et al., “Action of the Src Family Kinase Inhibitor, Dasatinib (Bms-354825), on Human Prostate Cancer Cells,” Cancer Research, 2005, vol. 65 (20), pp. 9185-9189.
  • Naramura M., et al., “Therapeutic Potential of Chimeric and Murine Anti-(Epidermal Growth Factor Receptor) Antibodies in a Metastasis Model for Human Melanoma,” Cancer Immunology, Immunotherapy, 1993, vol. 37 (5), pp. 343-349.
  • Narita Y., et al., “Mutant Epidermal Growth Factor Receptor Signaling Down-Regulates P27 Through Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway in Glioblastomas,” Cancer Research, 2002, vol. 62 (22), pp. 6764-6769.
  • Natale R.B., et al., “Zd1839 (Iressa): What's in it for the Patient,” Oncologist, 2002, vol. 7 (Suppl. 4), pp. 25-30.
  • Neal D.E., et al., “Epidermal-Growth-Factor Receptors in Human Bladder Cancer: Comparison of Invasive and Superficial Tumours,” Lancet, 1985, vol. 1 (8425), pp. 366-368.
  • Neal D.E., et al., “The Epidermal Growth Factor Receptor and the Prognosis of Bladder Cancer,” Cancer, 1990, vol. 65 (7), pp. 1619-1625.
  • Negri D.R., et al., “In Vitro and In Vivo Stability and Anti-Tumour Efficacy of an AntiEGFR/Anti-CD3 F(Ab')2 Bispecific Monoclonal Antibody,” British Journal of Cancer, 1995, vol. 72 (4), pp. 928-933.
  • Neidhardt F.C., et al., “Culture Medium for Enterobacteria,” Journal of bacteriology, 1974, vol. 119, pp. 736-747.
  • Nice E.C., et al., “Instrumental Biosensors: New Perspectives for the Analysis of Biomolecular Interactions,” BioEssays, 1999, vol. 21 (4), pp. 339-352.
  • Nicholson R.I., et al., “EGFR and Cancer Prognosis,” European Journal of Cancer, 2001, vol. 37 (Suppl. 4), pp. S9-S15.
  • Niikura H., et al., “Expression of Epidermal Growth Factor-Related Proteins and Epidermal Growth Factor Receptor in Common Epithelial Ovarian Tumors,” International Journal of Gynecological Pathology, 1997, vol. 16 (1), pp. 60-68.
  • Nishikawa R., et al., “A Mutant Epidermal Growth Factor Receptor Common in Human Glioma Confers Enhanced Tumorigenicity,” Proceedings of the National Academy of Sciences, 1994, vol. 91 (16), pp. 7727-7731.
  • Nishikawa R., et al., “Immunohistochemical Analysis of the Mutant Epidermal Growth Factor, AEGFR, in Glioblastoma,” Brain Tumor Pathology 2004, vol. 21 (2), pp. 53-56.
  • Noonberg S.B., et al., “Tyrosine Kinase Inhibitors Targeted to the Epidermal Growth Factor Receptor Subfamily: Role as Anticancer Agents,” Drugs, 2000, vol. 59 (4), pp. 753-767.
  • Normanno N., et al., “Cooperative Inhibitory Effect of ZD1839 (Iressa) in Combination with Trastuzumab (Herceptin) on Human Breast Cancer Cell Growth,” Annals of Oncology, 2002, vol. 13 (1), pp. 65-72.
  • Normanno N., et al., “Epidermal Growth Factor Receptor (EGFR) Signaling in Cancer,” Gene, 2006, vol. 366 (1), pp. 2-16.
  • Normanno N., et al., “Growth Inhibition of Human Colon Carcinoma Cells by Combinations of Anti-Epidermal Growth Factor-Related Growth Factor Antisense Oligonucleotides,” Clinical Cancer Research, 1996, vol. 2 (3), pp. 601-609.
  • Norton, et al., “Overall Survival (OS) Advantage to Simultaneous Chemotherapy (CRx) Plus the Humanized Anti-HER2 Monoclonal Antibody Herceptin (H) in HER2- Overexpressing (HER2+) Metastatic Breast Cancer (MBC),” Proceedings of the American Society of Clinical Oncology, 1999, vol. 127a (Abstract 483), pp. 18.
  • Notice of Oppositions to European Patent No. 1392359, filed by Genmab A/S on Jul. 21, 2010. With the Exception of Document D1b Referenced in the Noticed of Opposition, Documents Relied Upon in the Notice of Opposition are Submitted Herein or were Submitted in Information Disclosure filed in this Case on Dec. 23, 2008.
  • O-Charoenrat P., et al., “Overexpression of Epidermal Growth Factor Receptor in Human Head and Neck Squamous Carcinoma Cell Lines Correlates with Matrix Metalloproteinase-9 Expression and in Vitro Invasion,” International Journal of Cancer, 2000, vol. 86, pp. 307-317.
  • O-Charoenrat P., et al., “The Role of c-ErbB Receptors and Ligands in Head and Neck Squamous Cell Carcinoma,” Oral Oncology, 2002, vol. 38, pp. 627-640.
  • O-Charoenrat P., et al., “Vascular Endothelial Growth Factor Family Members are Differentially Regulated by c-ErbB Signaling in Head and Neck Squamous Carcinoma Cells,” Clinical and Experimental Metastasis, 2000, vol. 18, pp. 155-161.
  • Ochiai H., et al., “EGFRvIII-Targeted Immunotoxin Induces Antitumor Immunity that is Inhibited in the Absence of CD4+ and CD8+ T Cells,” Cancer Immunology, Immunotherapy, 2008, vol. 57 (1), pp. 115-121.
  • Oflazoglu E., et al., “Potent Anticarcinoma Activity of the Humanized Anti-Cd70 Antibody H1 F6 Conjugated to the Tubulin Inhibitor Auristatin Via an Uncleavable Linker,” Clinical Cancer Research, 2008, vol. 14 (19), pp. 6171-6180.
  • Ogiso H., et al., “Crystal Structure of the Complex of Human Epidermal Growth Factor and Receptor Extracellular Domains,” Cell, 2002, vol. 110, pp. 775-787.
  • Ohman L., et al., “A New Antibody Recognizing the VIII Mutation of Human Epidermal Growth Factor Receptor,” Tumor Biology, 2002, vol. 23, pp. 61-69.
  • Okamoto I., et al., “Expression of Constitutively Activated Egfrviii in Non-Small Cell Lung Cancer,” Cancer Science, 2003, vol. 94 (1), pp. 50-56.
  • Okamoto S., et al., “Monoclonal Antibody against the Fusion Junction of a Deletion-Mutant Epidermal Growth Factor Receptor,” British Journal of Cancer, 1996, vol. 73, pp. 1366-1372.
  • Olapade-Olaopa E.O., et al., “Evidence for the Differential Expression of a Variant EGF Receptor Protein in Human Prostate Cancer,” British Journal of Cancer, 2000, vol. 82 (1), pp. 186-194.
  • Olayioye M.A., et al., “ErbB-1 and ErbB-2 Acquire Distinct Signaling Properties Dependent Upon their Dimerization Partner,” Molecular and Cellular Biology, 1998, vol. 18 (9), pp. 5042-5051.
  • Olayioye M.A., et al., “The ErbB Signaling Network: Receptor Heterodimerization in Development and Cancer,” The EMBO Journal, 2000, vol. 19 (13), pp. 3159-3167.
  • Old L.J., “Immunotherapy for Cancer,” Scientific American, 1996, vol. 275 (3), pp. 136-143.
  • Olson J.E., et al., “Transmodulation of Epidermal Growth Factor Binding by Platelet-derived Growth Factor and 12-O-Tetradecanoylphorbol-13-acetate is not Sodium-dependent in Balb/c/3T3 Cells,” Journal of Biological Chemistry, 1990, vol. 265 (4), pp. 1847-1851.
  • Omidfar K., et al., “Production and Characterization of a New Antibody Specific for the Mutant EGF Receptor, EGFRvIII, in Camelus Bactrianus,” Tumor Biology, 2004, vol. 25 (4), pp. 179-187.
  • Omidfar K., et al., “Production of a Novel Camel Single-Domain Antibody Specific for the Type III Mutant EGFR,” Tumor Biology, 2004, vol. 25 (5-6), pp. 296-305.
  • Opresko L.K., et al., “Endocytosis and Lysosomal Targeting of Epidermal Growth Factor Receptors are Mediated by Distinct Sequences Independent of the Tyrosine Kinase Domain,” Journal of Biological Chemistry, 1995, vol. 270 (9), pp. 4325-4333.
  • Orntoft T.F., et al., “Clinical Aspects of Altered Glycosylation of Glycoproteins in Cancer,” Electrophoresis, 1999, vol. 20 (2), pp. 362-371.
  • Osband M.E., et al., “Problems in the Investigational Study and Clinical Use of Cancer Immunotherapy,” Immunology Today, 1990, vol. 11 (6), pp. 193-195.
  • Ostermann E., et al., “Effective Immunoconjugate Therapy in Cancer Models Targeting as Serine Protease of Tumor Fibroblasts,” Clinical Cancer Research, 2008, vol. 14 (14), pp. 4584-4592.
  • Otwinowski Z., et al., “Processing of X-Ray Diffraction Data Collected in Oscillation Mode,” Methods in Enzymology, 1997, vol. 276, pp. 307-326.
  • Overdijk, et al., “Role of ADCC in the in Vivo Antitumor Effects of Zalutumumab, a Human anti-EGF Receptor Antibody,” ASCO Meeting, 2010.
  • Overholser J.P., et al., “Epidermal Growth Factor Receptor Blockade by Antibody Imc-C225 Inhibits Growth of a Human Pancreatic Carcinoma Xenograft in Nude Mice,” Cancer, 2000, vol. 89 (1), pp. 74-82.
  • Owens R.J., et al., “The Genetic Engineering of Monoclonal Antibodies,” Journal of Immunological Methods , 1994, vol. 168 (2), pp. 149-165.
  • Ozanne B., et al., “Over-Expression of the EGF Receptor is a Hallmark of Squamous Cell Carcinomas,” The Journal of Pathology, 1986, vol. 149 (1), pp. 9-14.
  • Ozawa S., et al., “Prognostic Significance of Epidermal Growth Factor Receptor in Esophageal Cell Carcinomas,” Cancer, 1989, vol. 63 (11), pp. 2169-2173.
  • Padlan E.A., “A Possible Procedure for Recucing the Immunogenicity of Antibody Variable Domains While Preserving Their Ligand-Binding Properties,” Molecular Immunology, 1991, vol. 28 (4-5), pp. 489-498.
  • Padlan E.A., et al., “Anatomy of the Antibody Molecule,” Molecular Immunology, 1994, vol. 31 (3), pp. 169-217.
  • Padlan E.A., et al., “Identification of Specificity-determining Residues in Antibodies,” FASEB Journal, 1995, vol. 9 (1), pp. 133-139.
  • Padlan E.A., et al., “On the Nature of Antibody Combining Sites: Unusual Structural Features that may Confer on these Sites an Enhanced Capacity for Binding Ligands,” Proteins, 1990, vol. 7 (2), pp. 112-124.
  • Paez, et al., “EGFR Mutations in Lung Cancer: Correlation with Clinical Response to Gefitinib Therapy,” Science, 2004, vol. 304, pp. 1497-1500.
  • Paganelli G. et al., “Antibody-Guided Three-Step Therapy for High Grade Glioma with Yttrium-90 Biotin,” European Association of Nuclear Medicine, 1999, vol. 26 (4), pp. 348-357.
  • Pai L.H., et al., “The Use of Immunotoxins for Cancer Therapy,” European Journal of Cancer, 1993, vol. 29A (11), pp. 1606-1609.
  • Pai R., et al., “Prostaglandin E2 Transactivates EGF Receptor: a Novel Mechanism for Promoting Colon Cancer Growth and Gastrointestinal Hypertrophy,” Nature Medicine, 2002, vol. 8 (3), pp. 289-293.
  • Palacios R., et al., “Interleukin-3 Supports Growth of Mouse Pre-B-Cell Clones in Vitro,” Nature, 1984, vol. 309 (5964), pp. 126-131.
  • Panousis C., et al., “Engineering and Characterisation of Chimeric Monoclonal Antibody 806 (ch806) for Targeted Immunotherapy of Tumours Expressing de2-7 EGFR or Amplified EGFR,” British Journal of Cancer, 2005, vol. 92 (6), pp. 1069-1077.
  • Pao W., et al., “Acquired Resistance of Lung Adenocarcinomas to Gefitinib or Erlotinib is Associated with a Second Mutation in the EGFR Kinase Domain,” PLOS Medicine, 2005, vol. 2 (3), pp. e73.
  • Pao W., et al., “EGF Receptor Gene Mutations are Common in Lung Cancers From ‘Never Smokers’ and are Associated with Sensitivity of Tumors to Gefitinib and Erlotinib,” Proceedings of the National Academy of Sciences, 2004, vol. 101 (36), pp. 13306-13311.
  • Pao W., et al., “Epidermal Growth Factor Receptor Mutations, Small-Molecule Kinase Inhibitors, and Non-Small-Cell Lung Cancer: Current Knowledge and Future Directions,” Journal of Clinical Oncology, 2005, vol. 23 (11), pp. 2556-2568.
  • Pao W., et al., “KRAS Mutations and Primary Resistance of Lung Adenocarcinomas to Gefitinib or Erlotinib,” PLOS Medicine, 2005, vol. 2 (1), pp. e17.
  • Park K., et al., “A Review of the Benefit-Risk Profile of Gefitinib in Asian Patients with Advanced Non-Small-Cell Lung Cancer,” Current Medical Research and Opinion, 2006, vol. 22 (3), pp. 561-573.
  • Parker C., et al., “Preferential Activation of the Epidermal Growth Factor Receptor in Human Colon Carcinoma Liver Metastases in Nude Mice,” Journal of Histochemistry and Cytochemistry, 1998, vol. 46 (5), pp. 595-602.
  • Pastan I., “Targeted Therapy of Cancer with Recombinant Immunotoxins,” Biochimica et Biophysica Acta 1997, vol. 1333 (2), pp. C1-C6.
  • Patel D., et al., “Monoclonal Antibody Cetuximab Binds to and Down-Regulates Constitutively Activated Epidermal Growth Factor Receptor VIII on the Cell Surface,” Anticancer Research, 2007, vol. 27 (5A), pp. 3355-3366.
  • Pavelic K., et al., “Evidence for a Role of EGF Receptor in the Progression of Human Lung Carcinoma,” Anticancer Research, 1993, vol. 13 (4), pp. 1133-1137.
  • Pawson T., et al., “SH2 and SH3 Domains,” Current Biology, 1993, vol. 3 (7), pp. 434-442.
  • Pawson T., “Protein Modules and Signalling Networks,” Nature, 1995, vol. 373 (6515), pp. 573-580.
  • Pedersen M.W., et al., “Analysis of the Epidermal Growth Factor Receptor Specific Transcriptome: Effect of Receptor Expression Level and an Activating Mutation,” Journal of Cellular Biochemistry, 2005, vol. 96 (2), pp. 412-427.
  • Pedersen M.W., et al., “Mutations in the Epidermal Growth Factor Receptor: Structure and Biological Function in Human Tumors,” Ugeskrift for Laeger, 2006, vol. 168 (24), pp. 2354-2361.
  • Pedersen M.W., et al., “The Type III Epidermal Growth Factor Receptor Mutation. Biological Significance and Potential Target for Anti-Cancer Therapy,” Annals of Oncology, 2001, vol. 12 (6), pp. 745-760.
  • Pegram, et al., “Antibody Dependent Cell-Mediated Cytotoxicity in Breast Cancer Patients in Phase III Clinical Trials of a Humanized Anti-HER2 Antibody,” Proceedings of the Annual Meeting of the American Association for Cancer Research, 1997, vol. 602 (Abstract 4044), pp. 39.
  • Pegram M., et al., “Inhibitory Effects of Combinations of Her-2/Neu Antibody and Chemotherapeutic Agents Used for Treatment of Human Breast Cancers,” Oncogene, 1999, vol. 18 (13), pp. 2241-2251.
  • Pegram M.D, et al., “Phase II Study of Receptor-Enhanced Chemosensitivity Using Recombinant Humanized Anti-P185HER2/Neu Monoclonal Antibody Plus Cisplatin in Patients With HER2/Neu-Overexpressing Metastatic Breast Cancer Refractory to Chemotherapy Treatment,” Journal of Clinical Oncology, 1998, vol. 16 (8), pp. 2659-2671.
  • Pegram M.D, et al., “Rational Combinations of Trastuzumab with Chemotherapeutic Drugs Used in the Treatment of Breast Cancer,” Journal of the National Cancer Institute, 2004, vol. 96 (10), pp. 739-749.
  • Pegram M.D, et al., “The Effect of HER-2/neu Overexpression on Chemotherapeutic Drug Sensitivity in Human Breast and Ovarian Cancer Cells,” Oncogene, 1997, vol. 15 (5), pp. 537-547.
  • Pelloski C.E., et al., “Epidermal Growth Factor Receptor Variant III Status Defines Clinically Distinct Subtypes of Glioblastoma,” Journal of Clinical Oncology, 2007, vol. 25 (16), pp. 2288-2294.
  • Peng D., et al., “Anti-Epidermal Growth Factor Receptor Monoclonal Antibody 225 Up-Regulates p27KIP1 and Induces G1 Arrest in Prostatic Cancer Cell Line DU145,” Cancer Research, 1996, vol. 56, pp. 3666-3669.
  • Perera A.D., et al., “Requirement for the von Hippel-Lindau Tumor Suppressor Gene for Functional Epidermal Growth Factor Receptor Blockade by Monoclonal Antibody C225 in Renal Cell Carcinoma1,” Clinical Cancer Research, 2000, vol. 6 (4), pp. 1518-1523.
  • Perera, et al., “Internalisation and Trafficking of the Monoclonal Antibody 806 Reactive Epidermal Growth Factor Receptor,” Austin Health Research Week, 2003, Abstract 112.
  • Perera, et al., “The Influence of Epidermal Growth Factor Receptor (EGFR) Number and Activation on the Efficacy of Antibodies Directed to the Receptor,” Proceedings of the 14th Annual Lome Cancer Conference, 2002, Abstract 216.
  • Perera R.M., et al., “A Novel EGFR Antibody that Displays Synergistic Anti-Tumor Activity when Combined with Conventional EGFR Therapeutics,” Clinical Cancer Research, 2005, vol. 11 (17), pp. 6390-6399.
  • Perera R.M., et al., “Internalization, Intracellular Trafficking, and Biodistribution of Monoclonal Antibody 806: A Novel Anti-Epidermal Growth Factor Receptor Antibody,” Neoplasia, 2007, vol. 9 (12), pp. 1099-1110.
  • Perera R.M., “Therapeutic Efficacy and Intracellular Trafficking of Anti-Epidermal Growth Factor Receptor Antibodies (Project Report),” Tumour Targeting Program Ludwig Institute for Cancer Research, 2004.
  • Perez-Soler, et al., “A Phasell Trial of the Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor OSI-774, Following Platinum-Based Chemotherapy in Patients (Pts) with Advanced EGFR-Expressing, Non-Small Cell Lung Cancer (NSCLC),” Proceedings of the American Society of Clinical Oncology, 2001, vol. 310a (Abstract 1235), pp. 20.
  • Perez-Soler, et al., “Tumor Studies in Patients With Head & Neck Cancer Treated With Humanized Anti-Epidermal Growth Factor (Egfr) Monoclonal Antibody C225 in Combination With Cisplatin,” Proceedings of the American Society of Clinical Oncology, 1998, vol. 393a (Abstract 1514), pp. 17.
  • Perez-Soler R., et al., “HER1/EGFR Targeting: Refining the Strategy,” Oncologist, 2004, vol. 9 (1), pp. 58-67.
  • Perez-Soler R., et al., “Tumor Epidermal Growth Factor Receptor Studies in Patients With Non-Small-Cell Lung Cancer or Head and Neck Cancer Treated With Monoclonal Antibody RG 83852,” Journal of Clinical Oncology, 1994, vol. 12 (4), pp. 730-739.
  • Perl A.K., et al., “Conditional Gene Expression in the Respiratory Epithelium of the Mouse,” Transgenic Research, 2002, vol. 11 (1), pp. 21-29.
  • Perrotte P., et al., “Anti-Epidermal Growth Factor Receptor Antibody C225 Inhibits Angiogenesis in Human Transitional Cell Carcinoma Growing Orthotopically in Nude Mice,” Clinical Cancer Research, 1999, vol. 5 (2), pp. 257-265.
  • Petit A.M., et al., “Neutralizing Antibodies Against Epidermal Growth Factor and ErbB-2/Neu Receptor Tyrosine Kinases Down-Regulate Vascular Endothelial Growth Factor Production by Tumor Cells in Vitro and in Vivo: Angiogenic Implications for Signal Transduction Therapy of Solid Tumors,” The American Journal of Pathology, 1997, vol. 151 (6), pp. 1523-1530.
  • Petrides P.E., et al., “Modulation of Pro-Epidermal Growth Factor, Pro-Transforming Growth Factor Alpha and Epidermal Growth Factor Receptor Gene Expression in Human Renal Carcinomas,” Cancer Research, 1990, vol. 50 (13), pp. 3934-3939.
  • Pfister, et al., “A Phase I trial of the Epidermal Growth Factor Receptor (EGFR)-Directed Bispecific Antibody (BsAB) MDX-447 in Patients with Solid Tumors,” Proceedings of the American Society of Clinical Oncology, 1999, vol. 433a (Abstract 1667), pp. 18.
  • Pfosser A., et al., “Role of Target Antigen in Bispecific-Antibody-Mediated Killing of Human Glioblastoma Cells: A Pre-Clinical Study,” International Journal of Cancer, 1999, vol. 80 (4), pp. 612-616.
  • Pfreundschuh M., et al., “Serological Analysis of Cell Surface Antigens of Malignant Human Brain Tumors,” Proceedings of the National Academy of Sciences, 1978, vol. 75 (10), pp. 5122-5126.
  • Pietras R.J., et al., “Monoclonal Antibody to HER-2/Neureceptor Modulates Repair of Radiation-Induced DNA Damage and Enhances Radiosensitivity of Human Breast Cancer Cells Overexpressing this Oncogene,” Cancer Research, 1999, vol. 59 (6), pp. 1347-1355.
  • Pietras R.J., et al., “Remission of Human Breast Cancer Xenografts on Therapy with Humanized Monoclonal Antibody to her-2 Receptor and DNA-Reactive Drugs,” Oncogene, 1998, vol. 17 (17), pp. 2235-2249.
  • Pillay V., et al., “The Plasticity of Oncogene Addiction: Implications for Targeted Therapies Directed to Receptor Tyrosine Kinases1,” Neoplasia, 2009, vol. 11 (5), pp. 448-458.
  • Politi K., et al., “Lung Adenocarcinomas Induced in Mice by Mutant EGF Receptors Found in Human Lung Cancers Respond to a Tyrosine Kinase Inhibitor or to Down-Regulation of the Receptors,” Genes & Development, 2006, vol. 20 (11), pp. 1496-1510.
  • Ponten J., et al., “Long Term Culture of Normal and Neoplastic Human Glia,” Acta Pathologica et Microbiologica Scandinvica, 1968, vol. 74 (4), pp. 465-486.
  • Power B.E., et al., “Construction, Expression and Characterisation of a Single-Chain Diabody Derived from a Humanised Anti-Lewis Y Cancer Targeting Antibody using a Heat-Inducible Bacterial Secretion Vector,” Cancer Immunology, Immunotherapy, 2001, vol. 50 (5), pp. 241-250.
  • Power B.E., et al., “Noncovalent scFv Multimers of Tumor-Targeting Anti-Lewisy hu3S193 Humanized Antibody,” Protein Science, 2003, vol. 12 (4), pp. 734-747.
  • Power B.E., et al., “Synthesis of High Avidity Antibody Fragments (scFv multimers) for Cancer Imaging,” Journal of Immunological Methods, 2000, vol. 242 (1-2), pp. 193-204.
  • Prados M.D., et al., “Biology and Treatment of Malignant Glioma,” Seminars in Oncology, 2000, vol. 27 (3) (Suppl. 6), pp. 1-10.
  • Prenzel N., et al., “The Epidermal Growth Factor Receptor Family as a Central Element for Cellular Signal Transduction and Diversification,” Endocrine Related Cancer, 2001, vol. 8 (1), pp. 11-31.
  • Press O.W., et al., “Inhibition of Catabolism of Radiolabeled Antibodies by Tumor Cells Using Lysosomotropic Amines and Carboxylic Ionophores,,” Cancer Research, 1990, vol. 50 (4), pp. 1243-1250.
  • Press O.W., et al., “Ricin A-Chain Containing Immunotoxins Directed Against Different Epitopes on the CD2 Molecule Differ in Their Ability to Kill Normal and Malignant T Cells,” The Journal of Immunology, 1988, vol. 141, pp. 4410-4417.
  • Presta L.G., “Engineering of Therapeutic Antibodies to Minimize Immunogenicity and Optimize Function,” Advanced Drug Delivery Review, 2006, vol. 58 (5-6), pp. 640-656.
  • Presta L.G., et al., “Humanization of an Antibody Directed Against IgE,” Journal of Immunology, 1993, vol. 151 (5), pp. 2623-2632.
  • Presta L.G., et al., “Humanization of an Anti-Vascular Endothelial Growth Factor Monoclonal Antibody for the Therapy of Solid Tumors and Other Disorders,” Cancer Research, 1997, vol. 57 (20), pp. 4593-4599.
  • Presta L.G., “Molecular Engineering and Design of Therapeutic Antibodies,” Current Opinion in Immunology, 2008, vol. 20 (4), pp. 460-470.
  • Prewett M., et al., “Anti-Tumor and Cell Cycle Responses in KBCells Treated With a Chimeric Anti-Egfr Monoclonal Antibody in Combination with Cisplatin,” International Journal of Oncology, 1996, vol. 9 (2), pp. 217-224.
  • Prewett M., et al., “Mouse-Human Chimeric Anti-Epidermal Growth Factor Receptor Antibody C225 Inhibits the Growthof Human Renal Cell Carcinoma Xenografts in Nude Mice,” Clinical Cancer Research, 1998, vol. 4 (12), pp. 2957-2966.
  • Prewett M., et al., “The Biologic Effects of C225, a Chimeric Monoclonal Antibody to the EGFR, on Human Prostate Carcinoma,” Journal of Immunother Emphasis Tumor Immunol, 1996, vol. 19 (6), pp. 419-427.
  • Prewett M.C., et al., “Enhanced Antitumor Activity of Anti-Epidermal Growth Factor Receptor Monoclonal Antibody IMC-C225 in Combination With Irinotecan (CPT-11) Against Human Colorectal Tumor Xenografts,” Clinical Cancer Research, 2002, vol. 8 (5), pp. 994-1003.
  • Prigent S.A., et al., “Enhanced Tumorigenic Behavior of Glioblastonia Cells Expressing a Truncated Epidermal Growth Factor Receptor is Mediated Through the Ras-Shc-Grb2 Pathway,” Journal of Biological Chemistry 1996, vol. 271 (41), pp. 25639-25645.
  • Prigent S.A., et al., “The Type 1 (EGFR-Related) Family of Growth Factor Receptors and their Ligands,” Progress in Growth Factor Research 1992, vol. 4 (1), pp. 1-24.
  • Privalsky M.L., et al., “The Membrane Glycoprotein Encoded by the Retroviral Oncogene verb-B is Structurally Related to Tyrosine-Specific Protein Kinases,” Proceedings of the National Academy of Sciences, 1984, vol. 81 (3), pp. 704-707.
  • Pruss R.M., et al., “Variants of 3T3 Cells Lacking Mitogenic Response to Epidermal Growth Factor,” Proceedings National Academy of Science, 1977, vol. 74 (9), pp. 3918-3921.
  • Putz M.M., et al., “Functional fine-Mapping and Molecular Modeling of a Conserved Loop Epitope of the Measles Virus Hemagglutinin Protein,” European Journal of Biochemistry, 2003, vol. 270 (7), pp. 1515-1527.
  • Raben D., et al., “ZD1839, a Selective Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, Alone and in Combination With Radiation and Chemotherapy as a New Therapeutic Strategy in Non-Small Cell Lung Cancer,” Seminars in Oncology, 2002, vol. 29 (1) (Suppl. 4), pp. 37-46.
  • Raben, et al., “C225 Anti-EGFR Antibody Potentiates Radiation (RT) and Chemotherapy (Ct) Cytotoxicity in Human Non-Small Cell Lung Cancer (Nscic) Cells In Vitro and In Vivo,” Proceedings of the American Society of Clinical Oncology, 2001, vol. 257a (Abstract 1026), pp. 20.
  • Raben, et al., “Treatment of Human Intracranial Gliomas with Chimeric Monoclonal Antibody Against the Epidermal Growth Factor Receptor Increases Survival of Nude Mice when Treated Concurrently with Irradiation,” Proceedings of the American Association for Cancer Research, 1999, vol. 184 (Abstract 1224), pp. 40.
  • Radinsky R., et al., “Level and Function of Epidermal Growth Factor Receptor Predict the Metastatic Potential of Human Colon Carcinoma Cells1,” Clinical Cancer Research, 1995, vol. 1, pp. 19-31.
  • Raizer J.J., “HERVEGFR Tyrosine Kinase Inhibitors for the Treatment of Glioblastoma Multiforme,” Journal of Neuro-Oncology, 2005, vol. 74 (1), pp. 77-86.
  • Rakowicz-Szulcznska E.M., et al., “Epidermal Growth Factor (EGF) and Monoclonal Antibody to Cell Surface EGF Receptor Bind to the Same Chromatin Receptor,” Archives of Biochemistry and Biophysics, 1989, vol. 268 (2), pp. 456-464.
  • Ramnarain D.B., et al., “Differential Gene Expression Analysis Reveals Generation of an Autocrine Loop by a Mutant Epidermal Growth Factor Receptor in Glioma Cells,” Cancer Research, 2006, vol. 66 (2), pp. 867-874.
  • Ramos T.C., et al., “Treatment of High-Grade Glioma Patients with the Humanized Anti-Epidermal Growth Factor Receptor (EGFR) Antibody H-R3,” Cancer Biology and Therapy, 2006, vol. 5 (4), pp. 375-379.
  • Ramos-Suzarte M., et al., “99mTc-Labeled Antihuman Epidermal Growth Factor Receptor Antibody in Patients with Tumors of Epithelial Origin: Part III. Clinical Trials Safety and Diagnostic Efficacy,” The Journal of Nuclear Medicine, 1999, vol. 40 (5), pp. 768-775.
  • Ramsland P.A., et al., “Structural Convergence of Antibody Binding of Carbohydrate Determinants in Lewis Y Tumor Antigens,” Jouranal of Molecular Biology, 2004, vol. 340 (4), pp. 809-818.
  • Ranson M., “ZD1839 (IressaT): For More Than Just Non-Small Cell Lung Cancer,” Oncologist, 2002, vol. 7 (Suppl. 4), pp. 16-24.
  • Rao G.S., et al., “Radiosensitization of Human Breast Cancer Cells by a Novel ErbB Family Receptor Tyrosine Kinase Inhibitor,” International Journal of Radiation Oncology Biology Physics, 2000, vol. 48 (5), pp. 1519-1528.
  • Raymond C.K., et al., “General Method for Plasmid Construction Using Homologous Recombination,” BioTechniques, 1999, vol. 26 (1), pp. 134-138, 140-141.
  • Rayzman, et al., “Monoclonal Antibodies for Cancer Therapy,” Cancer Forum, 2002, vol. 26 (2), pp. 104-108.
  • Reardon D.A., et al., “Recent Advances in the Treatment of Malignant Astrocytoma,” Journal of Clinical Oncology, 2006, vol. 248 (8), pp. 1253-1265.
  • Reed J.C., “Dysregulation of Apoptosis in Cancer,” Journal of Clinical Oncology, 1999, vol. 17 (9), pp. 2941-2953.
  • Reese, et al., “Effects of the 4D5 Antibody on HER2/Neu Heterodimerization with other Class I-Receptors in Human Breast Cancer Cells,” Proceedings of the Annual Meeting of the American Association for Cancer Research, 1996, vol. 51 (Abstract 353), pp. 37.
  • Reilly R.M., et al., “A Comparison of EGFand MAb 528 Labeled with 111 in for Imaging Human Breast Cancer,” Journal of Nuclear Medicine, 2000, vol. 41 (5), pp. 903-911.
  • Reiss M., et al., “Activation of the Autocrine Transforming Growth Factor Alpha Pathway in Human Squamous Carcinoma Cells,” Cancer Research, 1991, vol. 51 (23 Pt 1), pp. 6254-6262.
  • Reist C.J., et al., “Astatine-211 Labeling of Internalizing Anti-EGFRvIII Monoclonal Antibody Using N-Succinimidyl 5-[211At]Astato-3-Pyridinecarboxylate,” Nuclear Medicine and Biology, 1999, vol. 26 (4), pp. 405-411.
  • Reist C.J., et al., “Improved Targeting of an Anti-Epidermal Growth Factor Receptor Variant III Monoclonal Antibody in Tumor Xenografts after Labeling Using N-Succinimidyl 5-lodo-3-Pyridinecarboxylate,” Cancer Research, 1997, vol. 57, pp. 1510-1515.
  • Reist C.J., et al., “In Vitro and in Vivo Behavior of Radiolabeled Chimeric Anti-EGFRvIII Monoclonal Antibody: Comparison with its Murine Parent,” Nuclear Medicine and Biology, 1997, vol. 24 (7), pp. 639-647.
  • Reist C.J., et al., “Radioiodination of Internalizing Monoclonal Antibodies Using N-Succinimidyl 5-lodo-3-Pyridinecarboxylate,,” Cancer Research, 1996, vol. 56 (21), pp. 4970-4977.
  • Reist C.J., et al., “Tumor-Specific Anti-Epidermal Growth Factor Receptor Variant III Monoclonal Antibodies: Use of the Tyramine-Cellobiose Radioiodination Method Enhances Cellular Retention and Uptake in Tumor Xenografts,” Cancer Research, 1995, vol. 55 (19), pp. 4375-4382.
  • Reiter J.L, et al., “Comparative Genomic Sequence Analysis and Isolation of Human and Mouse Alternative EGFR Transcripts Encoding Truncated Receptor Isoforms,” Genomics, 2001, vol. 71, pp. 1-20.
  • Rettig W.J., et al., “Immunogenetics of Human Cell Surface Differentiation,” Annual Review of Immunology, 1989, vol. 7, pp. 481-511.
  • Reynolds F.H., et al., “Human Transforming Growth Factors Induce Tyrosine Phosphorylation of EGF Receptors,” Nature, 1981, vol. 292 (5820), pp. 259-262.
  • Ribas, et al., “Systemic Delivery of Sirna via Targeted Nanoparticles in Patients with Cancer: Results:from a First-in-Class phase I Clinical Trial,” Journal of Clinical Oncology, 2010, vol. 28 (15s) (Abstract 3022).
  • Riemer A.B., et al., “Matching of TrastuzumAb (Herceptin) Epitope Mimics Onto the Surface of Her-2/neu—A New Method of Epitope Definition,” Molecular Immunology, 2005, vol. 42, pp. 1121-1124.
  • Riemer A.B., et al., “Mimotope Vaccines: Epitope Mimics Induce Anti-Cancer Antibodies,” Immunology Letters, 2007, vol. 113 (1), pp. 1-5.
  • Riemer A.B., et al., “Vaccination with Cetuximab Mimotopes and Biological Properties of Induced Anti-Epidermal Growth Gactor Receptor Antibodies,” Journal of National Cancer Institute, 2005, vol. 97 (22), pp. 1663-1670.
  • Riese D.J., et al., “Specificity within the EGF Family/ErbB Receptor Family Signaling Network,” Bioessays, 1998, vol. 20 (1), pp. 41-48.
  • Rieske P., et al., “A Comparative Study of Epidermal Growth Factor Receptor (EGFR) and MDM2 Gene Amplification and Protein Immunoreactivity in Human Glioblastomas,” Polish Society of Pathologists, 1998, vol. 49 (3), pp. 145-149.
  • Rinehart, et al.,“A Phase 1 Clinical and Pharmacokinetic Study of Oral CI-1033, A Pan-Erbb Tyrosine Kinase Inhibitor in Patients with Advanced Solid Tumors,” Proceedings of the American Society of Clinical Oncology, 2002, vol. 11a (Abstract 41), pp. 21.
  • Ringerike T., et al., “High-Affinity Binding of Epidermal Growth Factor (EGF) to EGF Receptor is Disrupted by Overexpression of Mutant Dynamin (K44A),” Journal of Biological Chemistry, 1998, vol. 273 (27), pp. 16639-16642.
  • Ritter G., et al., “Serological Analysis of Human Anti-Human Antibody Responses in Colon Cancer Patients Treated with Repeated Doses of Humanized Monoclonal Antibody A33,” Cancer Research, 2001, vol. 61 (18), pp. 6851-6859.
  • Riva P., et al., “Role of Nuclear Medicine in the Treatment of Malignant Gliomas: The Locoregional Radioimmunotherapy Approach,” European Journal of Nuclear Medicine, 2000, vol. 27 (5), pp. 601-609.
  • Rivera F., et al., “Current Situation of Panitumumab, Matuzumab, Nimotuzumab and Zalutumumab,” Acta Oncoloqica 2008, vol. 47 (1), pp. 9-19.
  • Ro J., et al., “Amplified and Overexpressed Epidermal Growth Factor Receptor Gene in Uncultured Primary Human Breast Carcinoma,” Cancer Research, 1988, vol. 48 (1), pp. 161-164.
  • Robert F., et al., “Phase I Study of Anti—Epidermal Growth Factor Receptor Antibody CetuximAb in Combination with Radiation Therapy in Patients with Advanced Head and Neck Cancer,” Journal of Clinical Oncology, 2001, vol. 19 (13), pp. 3234-3243.
  • Rocha-Lima C.M., et al., “EGFR Targeting of Solid Tumors,” Cancer Control, 2007, vol. 14 (3), pp. 295-304.
  • Rodeck U., et al., “Interactions between Growth Factor Receptors and Corresponding Monoclonal Antibodies in Human Tumors,” Journal of Cellular Biochemisty, 1987, vol. 35, pp. 315-320.
  • Rodeck U., et al., “Monoclonal Antibody 425 Inhibits Growth Stimulation of Carcinoma Cells by Exogenous EGF and Tumor-Derived EGF/TGF-c,” Journal of Cellular Biochemistry, 1990, vol. 44 (2), pp. 69-79.
  • Rodeck U., et al., “Tumor Growth Modulation by a Monoclonal Antibody to the Epidermal Growth Factor Receptor: Immunologically Mediated and Effector Cell-Independent Effects,” Cancer Research, 1987, vol. 47 (14), pp. 3692-3696.
  • Roepstorff K., et al., “Sequestration of Epidermal Growth Factor Receptors in Non-caveolar Lipid Rafts Inhibits Ligand Binding,” Journal of Biological Chemistry, 2002, vol. 277 (21), pp. 18954-18960.
  • Roguska M.A., et al., “A Comparison of Two Murine Monoclonal Antibodies Humanized by Cdr-Grafting and Variable Domain Resurfacing,” Protein Engineering, 1996, vol. 9 (10), pp. 895-904.
  • Roguska M.A., et al., “Humanization of Murine Monoclonal Antibodies through Variable Domain Resurfacing,” Proceedings of the National Academy of Sciences, 1994, vol. 91 (3), pp. 969-973.
  • Rosell, et al., “Randomized phase II study of cetuximab in combination with cisplatin (C) and vinorelbine (V) vs. CV alone in the first-line treatment of patients (pts) with epidermal growth factor receptor (EGFR)-expressing advanced non- small-cell lung cancer (NSCLC),” 2004, Abstract 7012, pp. 22.
  • Rosell, et al., “Randomized Phase II Study of Cetuximab in Combination with Cisplatin (C) and vinorelbine (V) vs. CV Alone in the First-Line Treatment of Patients (Pts) with Epidermal Growth Factor Receptor (EGFR)-Expressing Advanced Non-Small-Cell Lung Cancer (NSCLC),” Proceedings of the American Society of Clinical Oncology, 2004, vol. 620s (Abstract 7012), pp. 23.
  • Rosenberg, et al., “Erbitux (IMC-225) Plus Weekly Irinotecan (CPT-11), Fluorouricil (5FU) and Leucovorin (LV) in Colorectal Cancer (CRC) that Expresses the Epidermal Growth Factor Receptor (EGFR),” Proceedings of the American Society of Clinical Oncology, vol. 135a (Abstract 536), pp. 21.
  • Ross J.S., et al., “Anticancer Antibodies,” American Journal of Clinical Pathology, 2003, vol. 119 (4), pp. 472-485.
  • Rougier, et al., “Cetuximab + FOLFIRI as First-Line Treatment for Metastatic Colorectal CA,” Proceedings of the Annual Meeting of the American Society of Clinical Oncology, 2004, vol. 248s (Abstract 3513), pp. 22.
  • Rowinsky E.K., et al., “Safety, Pharmacokinetics, and Activity of ABX-EGF, a Fully Human Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Patients with Metastatic Renalcell Cancer,” Journal of Clinical Oncology, 2004, vol. 22 (15), pp. 3003-3015.
  • Rubin, et al., “Monoclonal Antibody (MoAb) IMC-C225, an Anti-Epidermal Growth Factor Receptor (EGFR), for Patients with EGFR-positive Tumors Refractory to or in Relapse from Previous Therapeutic Regimens,” Proceedings of the American Society of Clinical Oncology, 2000, vol. 474a (Abstract 1860), pp. 193.
  • Rubin, et al., “Monoclonal Antibody (Moab) IMC-C225, An Anti-Epidermal Growth Factor Receptor (EGFR,.For Patients (Pts) with EGFr-Positive Tumors Refractory to or in Relapse from Previous Therapeutic Regimens,” American Society of Clinical Oncology, 2000, Abstract 1860, pp. 19.
  • Rubin G.J., et al., “Inhibition of Epidermal Growth Factor Receptor Gene Expression and Function Decreases Proliferation of Head and Neck Squamous Carcinoma but not Normal Mucosal Epithelial Cells,” Oncogene , 1997, vol. 15 (4), pp. 409-416.
  • Rubin Grandis J., et al., “Quantitative Immunohistochemical Analysis of Transforming Growth Factor-Alpha and Epidermal Growth Factor Receptor in Patients with Squamous Cell Carcinoma of the Head and Neck,” Cancer, 1996, vol. 78 (6), pp. 1284-1292.
  • Rubio, et al., “Cetuximab in Combination with oxaliplatin/5-Fluorouracil (5-FU)/folinic Acid (FA) (FOLFOX-4) in the First-Line Treatment of Patients with Epidermal Growth Factor Receptor (EGFR)-Expressing Metastatic Colorectal Cancer: An International Phase II Study,” Journal of Clinical Oncology, 2005 ASCO Annual Meeting Proceedings, 2005, Abstract 3535, pp. 23.
  • Rusch V., et al., “Overexpression of the Epidermal Growth Factor Receptor and Its Ligand Transforming Growth Factor Alpha is Frequent in Resectable Non-Small Cell Lung Cancer but Does not Predict Tumor Progression,” Clinical Cancer Research, 1997, vol. 3 (4), pp. 515-522.
  • Rusnak D.K., et al., “The Effects of the Novel, Reversible Epidermal Growth Factor Receptor/ErbB-2 Tyrosine Kinase Inhibitor, GW2016, on the Growth of Human Normal and Tumor-Derived Cell Lines in Vitro and in Vivo,” Molecular Cancer Therapeutics, 2001, vol. 1 (2), pp. 85-94.
  • Rusnak, et al., “The Effects of the Novel EGFR/ErbB-2 Tyrosine Kinase Inhibitor, GW2016, on the Growth of Human Normal and Transformed Cell Lines,” Proceedings of the American Association for Cancer Research, 2001, vol. 803 (Abstract 4309), pp. 42.
  • Safa M.M., et al., “Adjuvant Immunotherapy for Melanoma and Colorectal Cancers,” Seminars in Oncology, 2001, vol. 28 (1), pp. 68-92.
  • Saikali S., et al., “Expression of Nine Tumour Antigens in a Series of Human Glioblastoma Multiforme: Interest of EGFRVIII, II-13ralpha2, GP100 and TRP-2 for Immunotherapy,” Journal of Neuro-Oncology, 2007, vol. 81 (2), pp. 139-148.
  • Sainsbury J.R., et al., “Epidermal-Growth-Factor Receptor Status as Predictor of Early Recurrence of and Death from Breast Cancer,” Lancet, 1987, vol. 1 (8547), pp. 1398-1402.
  • Sainsbury J.R., et al., “Presence of Epidermal Growth Factor Receptor as an Indicator of Poor Prognosis in Patients with Breast Cancer,” Journal of Clinical Pathology, 1985, vol. 38 (11), pp. 1225-1228.
  • Sako Y., et al., “Single-Molecule Imaging of EGFR Signalling on the Surface of Living Cells,” Nature Cell Biology, 2000, vol. 2 (3), pp. 168-172.
  • Sakurada A., et al., “Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Lung Cancer: Impact of Primary or Secondary Mutations,” Clinical Lung Cancer, 2006, vol. 7 (Supply 4), pp. S138-S144.
  • Salazar, et al., “Dose-Dependent Inhibition of the EGFR and Signalling Pathways with the Anti-EGFR Monoclonal Antibody (MAb) EMD 72000 Administered every Three Weeks (q3w). A Phase I Pharmacokinetic/Pharmacodynamic (PK/PD) Study to Define the Optimal Biological Dose (OBD),” 2004, vol. 127s (Abstract 2002), pp. 22.
  • Saleh M.N., et al., “Combined Modality Therapy of A431 Human Epidermoid Cancer Using Anti-EGFr Antibody C225 and Radiation,” Cancer Biother Radiopharm, 1999, vol. 14 (6), pp. 451-463.
  • Salomon D.S., et al., “Epidermal Growth Factor-Related Peptides and their Receptors in Human Malignancies,” Critical Reviews in Oncology/Hematology, 1995, vol. 19 (3), pp. 183-232.
  • Saltz, et al., “Cetuximab (IMC-225) Plus Irinotecan (CPT-11) is Active in CPT-11- Refractory Colorectal Cancer (CRC) that Expresses Epidermal Growth Factor Receptor (EGFR),” Proceedings of the American Society of Clinical Oncology, 2001, vol. 3a (Abstract 7), pp. 20.
  • Saltz, et al., “Single Agent IMC-C225 (Erbitux[TM]) has Activity in CPT-11 Refractory Colorectal Cancer that Expresses the Epidermal Growth Factor Receptor (EGFR),” Proceedings of the American Society of Clinical Oncology, 2002, vol. 127a (Abstract 504), pp. 21.
  • Saltz, et al., “Interim Report of Randomized Phase II Trial of Cetuximab/Bevacizumab/Irinotecan (CBI) versus Cetuximab/Bevacizumab (CB) in Irinotecan-Refractory Colorectal Cancer,” Proceedings of the American Society of Clinical Oncology, 2006, Abstract 169b.
  • Saltz, et al., “The Presence and Intensity of the Cetuximab-Induced Acne-Like Rash Predicts Increased Survival in Studies Across Multiple Malignancies,” Proceedings of American Society of Clinical Oncology, 2003, Abstract 817, pp. 22.
  • Saltz L.B., et al., “Phase II Trial of Cetuximab in Patients With Refractory Colorectal Cancer that Expresses the Epidermal Growth Factor Receptor,” Journal of Clinical Oncology, 2004, vol. 22 (7), pp. 1201-1208.
  • Sampson, et al., “An EGFRvIII Specific Peptide Vaccine Generates Antitumor Immunity Through a Humoral Pathway,” Neuro-Oncology, 1999, vol. S103 (Abstract 135).
  • Sampson J.H., et al., “Tumor-Specific Immunotherapy Targeting the EGFRVIII Mutation in Patients with Malignant Glioma,” Seminars in immunology, 2008, vol. 20 (5), pp. 267-275.
  • Sampson J.H., et al., “Unarmed, Tumor-Specific Monoclonal Antibody Effectively Treats Brain Tumors,” Proceedings of the National Academy of Sciences, 2000, vol. 97 (13), pp. 7503-7508.
  • Sanderson R.J., et al., “In Vivo Drug-Linker Stability of an Anti-CD30 Dipeptide-Linked Auristatin Immunoconjugate,” Clinical Cancer Research, 2005, vol. 11 (2 Pt 1), pp. 843-852.
  • Sandler A.B., “Nondermatologic Adverse Events Associated with Anti-EGFR Therapy,” Oncology, 2006, vol. 20 (5 Suppl. 2), pp. 35-40.
  • Santon J.B., et al., “Effects of Epidermal Growth Factor Receptor Concentration on Tumorigenicity of A431 Cells in Nude Mice,,” Cancer Research, 1986, vol. 46 (9), pp. 4701-4705.
  • Sartor, C.L., “Biological Modifiers as Potential Radiosensitizers: Targeting the Epidermal Growth Factor Receptor Family,” Seminars in Oncology, 2000, 27 (6 Suppl 11), pp. 15-20.
  • Sartor C.L., “Mechanisms of Disease: Radiosensitization.by Epidermal Growth Factor Receptor Inhibitors,” Nature Clinical Practice Oncology, 2004, vol. 1 (2), pp. 80-87.
  • Sarup J.C., et al., “Characterization of an Anti-P185HER2 Monoclonal Antibody that Stimulates Receptor Function and Inhibits Tumor Cell Growth,” Growth Regulation, 1991, vol. 1 (2), pp. 72-82.
  • Sato J.D., et al., “Biological Effects in Vitro of Monoclonal Antibodies to Human Epidermal Growth Factor Receptors,” Molecular Biology and Medicine, 1983, vol. 1 (5), pp. 511-529.
  • Sato J.D., et al., “Derivation and Assay of Biological Effects of Monoclonal Antibodies to Epidermal Growth Factor Receptors,” Methods Enzymology, 1987, vol. 46, pp. 63-81.
  • Sauter G., et al., “Patterns of Epidermal Growth Factor Receptor Amplification in Malignant Gliomas,” 1996, vol. 148 (4), pp. 1047-1053.
  • Scher H.I., et al., “Changing Pattern of Expression of the Epidermal Growth Factor Receptor and Transforming Growth Factor Alpha in the Progression of Prostatic Neoplasm,” Clinical Cancer Research, 1995, vol. 1 (5), pp. 545-550.
  • Schlegel J., et al., “Amplification of the Epidermal-Growth-Factor-Receptor Gene Correlates with Different Growth Behaviour in Human Glioblastoma,” International Journal of Cancer, 1994, vol. 56 (1), pp. 72-77.
  • Schlessinger J., “Cell Signaling by Receptor Tyrosine Kinases,” Cell, 2000, vol. 103 (2), pp. 211-225.
  • Schlessinger J., “Common and Distinct Elements in Cellular Signaling via EGF and FGF Receptors,” 2004, vol. 306 (5701), pp. 1506-1507.
  • Schlessinger J., “Ligand-Induced, Receptor-Mediated Dimerization and Activation of EGF Receptor,” Cell, 2002, vol. 110 (6), pp. 669-672.
  • Schmidt M., et al., “Expression of an Oncogenic Mutant Egf Receptor Markedly Increases the Sensitivity of Cells to an EGF-Receptor-Specific Antibody-Toxin,” International Journal of Cancer, 1998, vol. 75 (6), pp. 878-884.
  • Schmidt M., et al., “Suppression of Metastasis Formation by a Recombinant Single Chain Antibody-Toxin Targeted to Full-Length and Oncogenic Variant EGF Receptors,” Oncogene, 1999, vol. 18 (9), pp. 1711-1721.
  • Schmidt M.H., et al., “Epidermal Growth Factor Receptor Signaling Intensity Determines Intracellular Protein Interactions, Ubiquitination, and Internalization,” Proceedings of the National Academy of Sciences, 2003, vol. 100 (11), pp. 6505-6510.
  • Schmidt-Ullrich R.K., et al., “Radiation-Induced Proliferation of the Human A431 Squamous Carcinoma Cells is Dependent on EGFR Tyrosine Phosphorylation,” Oncogene, 1997, vol. 15 (10), pp. 1191-1197.
  • Schmiedel J. et al., “MatuzumAb Binding to EGFR Prevents the Conformational Rearrangement Required for Dimerization,” Cancer Cell, 2008, vol. 13 (4), pp. 365-373.
  • Schmitz K.R., et al., “Interaction of Antibodies with ErbB Receptor Extracellular Regions,” Experimental Cell Research 2009, vol. 315 (4), pp. 659-670.
  • Schneebaum S., et al., “Immunoguided Lymph Node Dissection in Colorectal Cancer: A New Challenge”, World Journal Surgery, 2001, vol. 25 (12), pp. 1495-1499.
  • Schnurch H.G., et al., “Growth Inhibition of Xenotransplanted Human Carcinomas by a Monoclonal Antibody Directed Against the Epidermal Growth Factor Receptor,” European Journal of Cancer, 1994, vol. 30A (4), pp. 491-496.
  • Schwechheimer K., et al., “EGFR Gene Amplification-Rearrangement in Human Glioblastomas,” International Journal of Cancer, 1995, vol. 62 (2), pp. 145-148.
  • Scott A.M., et al., “A Phase I Biodistribution and Pharmacokinetic Trial of Humanized Monoclonal Antibody Hu3s193 in Patients with Advanced Epithelial Cancers that Express the Lewis-Y Antigen,” Clinical Cancer Research, 2007, vol. 13 (11), pp. 3286-3292.
  • Scott A.M., et al., “A Phase I Clinical Trial with Monoclonal Antibody Ch806 Targeting Transitional State and Mutant Epidermal Growth Factor Receptors,” Proceedings of the National Academy of Sciences, 2007, vol. 104 (10), pp. 4071-4076.
  • Scott A.M., et al., “A Phase I Dose-Escalation Study of SibrotuzumAb in Patients with Advanced or Metastatic Fibroblast Activation Protein-Positive Cancer,” Clinical Cancer Research, 2003, vol. 9 (5), pp. 1639-1647.
  • Scott A.M., et al., “A Phase I Single Dose Escalation Trial of ch806 in Patients with Advanced Tumors Expressing the 806 Antigen,” Ludwig Institute for Cancer Research, ASCO Meeting Abstracts 24: 13028.
  • Scott A.M., et al., “A Phase I Trial of Humanized Monoclonal Antibody A33 in Patients with Colorectal Carcinoma: Biodistribution, Pharmacokinetics, and Quantitative Tumor Uptake,” Clinical Cancer Research, 2005, vol. 11 (13), pp. 4810-4817.
  • Scott A.M., et al., “Antibody-Based Immunological Therapies,” Current Opinion in Immunology, 1997, vol. 9 (5), pp. 717-722.
  • Scott A.M., et al., “Clinical Promise of Tumour Immunology,” Lancet, 1997, vol. 349 (Suppl. 2), pp. SII19-SII22.
  • Scott A.M., et al., “Comparison of Phase I Trials of Anti-epidermal Growth Factor Receptor (egfr) Monoclonal Antibodies (mats) 528 and 225 Labelled with 1-131 and in-111, Abstract 1005,” Journal of Nuclear Medicine, 1993, vol. 34 (5), pp. 213P.
  • Scott A.M., et al., “Construction, Production, and Characterization of Humanized Anti-Lewis Y Monoclonal Antibody 3S193 for Targeted Immunotherapy of Solid Tumors,” Cancer Research, 2000, vol. 60 (12), pp. 3254-3261.
  • Scott A.M., et al., “Specific Targeting, Biodistribution, and Lack of Immunogenicity of Chimeric Anti-GD3 Monoclonal Antibody KM871 in Patients with Metastatic Melanoma: Results of a Phase I Trial,” Journal of Clinical Oncology, 2001, vol. 19 (19), pp. 3976-3987.
  • Scott A.M., et al., “Tumor Imaging and Therapy,” Radiologic Clinics of North America, 1993, vol. 31 (4), pp. 859-879.
  • Scott, “Antibody Therapeutics,” Ludwig Institute Colon Cancer Initiative Symposium, Baltimore, MD, United States, 2010.
  • Scott, “Cell Surface and Intracellular Targets for Antibody Directed Cancer Therapeutics,” City of Hope Cancer Center, Los Angeles, CA, United States, 2007.
  • Scott, “Cell Surface Targets for Therapy,” LICR Brain Cancer Initiative Meeting, Rockville, MD, United States, 2009.
  • Scott, “Development of a Humanised Antibody Against a Novel Epitope of EGFR,” Australasian Vaccines & Immunotherapy Development Meeting, Melbourne, Australia, 2010.
  • Scott, “Development of a Novel Anti-EGFR Humanised Antibody—the Complex Path from Academia to Industry,” Lowy Symposium, Sydney, Australia, 2010.
  • Scott, “EGFR Targeted Therapeutics,” ComBio Brisbane, Australia, 2006.
  • Scott, “Epidermal Growth Factor Receptor Targeting for Cancer Therapy,” 3rd Barossa Meeting—Signalling Systems, Barossa Valley, South Australia, 2007.
  • Scott et al., “Immunological Effects of Chimeric Anti-Gd3 Monoclonal Antibody Km871 in Patients with Metastatic Melanoma,” Cancer immunity: a journal of the Academy of Cancer Immunology, 2005, vol. 3, pp. 5.
  • Scott, “Growth Factors and their implications in head and neck cancer,” Garnett Passe Scientific Meeting: Frontiers in Otorhinolarvnoologv, Noosa, Queensland, Australia, 2004.
  • Scott, “Implications of Antibody:Receptor Binding Structure and Signalling on Tumour Growth,” Discovery Science & Biotechnology Conference, Melbourne, Australia, 2006.
  • Scott, “Implications of Antibody:Receptor Binding Structure and Signalling on Tumour Growth,” Monash University—Department of Biochemistry & Molecular Biology, Melbourne, Australia, 2005.
  • Scott, “Molecular Targets for Cancer Therapeutics,” Baker Institute Seminar, Melbourne, Australia, 2002.
  • Scott, “Molecular Targets for Cancer Therapeutics,” Cambridge University Seminar, United Kingdom, 2002.
  • Scott, “Molecular Targets for Cancer Therapeutics,” Monash University Seminar, Melbourne, Australia, 2002.
  • Scott, “Novel Antibodies that Bind to a Conformational Epitope of EGFR,” IBC 20th Annual Antibody Engineering Conference, San Diego, CA, United States, 2009.
  • Scott, “Novel Antibody that Inhibits EGFR Activation,” Fifth International Congress on Monoclonal Antibodies in Cancer, Quebec City, Canada, 2005.
  • Scott, “Of Mice and Man—The Role of Growth Factor Receptors in Cancer,” Austin Hospital Division of Medicine Grand Round, Melbourne, Australia, 2007.
  • Scott, “Pathway Specific Therapeutics: from Cancer Biology to Targeted Therapy,” Garvan Signalling Symposium, Melbourne, Australia, 2010.
  • Scott, “Receptor Based Targets for Antibody Therapy of Solid Tumours,” The Second China International Symposium on Antibody Engineering: Current Status and Future Perspective of Antibody Therapeutics, Belling, China, 2005.
  • Scott, “Recombinant Antibodies for Immune and Cell Signalling Based Therapeutics,” AntibOZ 2 Conference, Heron Island, Queensland, Australia, 2004.
  • Scott, “Recombinant Antibody Therapy of Cancer—the LICR Antibody Program,” A Star Agency for Science, Technology and Research, ICMB, Singapore, 2008, D776.
  • Scott, “Targeted Cancer Therapeutics—the Role of Signalling and Immune Effector Mechanisms,” Royal North Shore Hospital Scientific Forum Sydney, NSW, Australia, 2003.
  • Scott, “Targeted Therapeutics—the Role of Signalling and Immune Effector Mechanism,” Centre for Immunology and Cancer Research, University of Queensland, Brisbane, Queensland, Australia, 2003.
  • Scott, “Targeted Therapeutics—the Role of Signalling and Immune Effector Mechanisms,” Peter MacCallum Cancer Immunology Program Seminar, Melbourne, Australia, 2003.
  • Scott, “Targeting a Novel EGFR Epitope on Cancer Cells,” Keystone Symposia: Antibodies as Drugs: Targeted Cancer Therapies, Whistler British Columbia, Canada, 2009.
  • Scott., “Targeting a Novel EGFR Epitope on Cancer Cells” Ludwig Institute for Cancer Research, LICR Translational Oncology Conference, Melbourne (Australia) (Nov. 6-8, 2009) Abstract No. abs#014.
  • Scott, “Targeting a Tumour Specific Epitope of the Epidermal Growth Factor Receptor,” Keystone Symposium: Antibodies as Drugs: From Basic Biology to the Clinic, Alberta, Canada, 2007.
  • Scott, “Targeting the Epiderm'al Growth Factor Receptor for Antibody Therapy of Solid Tumours,” 17th Annual IBC Antibody Engineering Conference, San Diego, CA, United States, 2006.
  • Scott, “Targeting the Epidermal Growth Factor Receptor for Antibody Therapy of Solid Tumours,” EGFR Cascade Meeting, San Diego, CA, United States, 2006.
  • Scott, Targeting the Epidermal Growth Factor Receptor for Antibody Therapy of Solid TuMours.7 Third International AntibOZ Conference, Heron, Island, Queensland, Australia, 2007.
  • Scott, “The Biology of EGFR in Normal and Diseased Tissues,” Australian Lung Cancer Conference, Surfers Paradise, Q1c1, Australia, 2008.
  • Scott, “Therapy of EGFR Expressing Cancers with a Novel Tumour Specific Antibody,” AHMRC Congress, Brisbane, Old Australia, 2008.
  • Scott, “Structural Biology and Molecular Imaging in Cancer Therapeutics,” Bosch Institute Annual Scientific Meeting, Sydney, Australia, 2010.
  • Scott, “Understanding the Biology of Targeted Therapies in Cancer,” University of Melbburne/Roval Melbourne HospitaVWestern Hospital Consortium Seminar, Melbourne, Australia 2008.
  • Sellers W.R., et al., “Apoptosis and Cancer Drug Targeting,” Journal of Clinical Investigation, 1999, vol. 104 (12), pp. 1655-1661.
  • Senter P.D., “Potent Antibody Drug Conjugates for Cancer Therapy,” Current Opinion in Chemical Biology, 2009, vol. 13 (3), pp. 235-244.
  • Senzer, et al., “Phase 2 Evaluation of OSI-774, A Potent Oral Antagonist of the EGFR-Tk in Patients with Advanced Squamous Cell Carcinoma of the Head and Neck,” Proceedings of the American Society of Clinical Oncology, 2001, 2a (Abstract 6), 20.
  • Sepp-Lorenzino, et al., “Farnesyl:Protein Transferase Inhibitors (Ftis) Block Tyrosine Kinase Signal Transduction and Act in Concert With an Anti-EGR Receptor Antibody to Inhibit Cancer Cell Growth,” Proceedings of the Annual of the American Association for Cancer Research, 1996, vol. 37 (Abstract 2877), pp. 421-422.
  • Seymour L., et al., “Novel Anti-Cancer Agents in Development: Exciting Prospects and New Challenges,” Cancer Treatment Reviews, 1999, vol. 25, pp. 301-312.
  • Sharafinski M.E., et al., “Epidermal Growth Factor Receptor Targeted Therapy of Squamous Cell Carcinoma of the Head and Neck,” Head Neck, 2010, vol. 32 (10), pp. 1412-1421.
  • She Q.B., et al., “The Bad Protein Integrates Survival Signaling by EGFR/MAPK and PI3K/Akt Kinase Pathways in PTEN-Deficient Tumor Cells,” Cancer Cell, 2005, vol. 8 (4), pp. 287-297.
  • Shepherd F.A., et al., “Unraveling the Mystery of Prognostic and Predictive Factors in Epidermal Growth Factor Receptor Therapy,” Journal of Clinical Oncology, 2006, vol. 24 (7), pp. 1219-1220.
  • Sherrill J.M., et al., “Activation of Epidermal Growth Factor Receptor by Epidermal Growth Factor,” Biochemistry, 1996, vol. 35 (18), pp. 5705-5718.
  • Shibata T., et al., “Enhancing Effects of Epidermal Growth Factor on Human Squamous Cell Carcinoma Motility and Matrix Degradation but Not Growth,” Tumor Biology, 1996, vol. 17 (3), pp. 168-175.
  • Shigematsu H., et al., “Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers,” Journal of the National Cancer Institute, 2005, vol. 97 (5), pp. 339-346.
  • Shigematsu H., et al., “Somatic Mutations of Epidermal Growth Factor Receptor Signaling Pathway in Lung Cancers,” International Journal of Cancer, 2006, vol. 118 (2), pp. 257-262.
  • Shimizu N., et al., “Detection of Epidermal Growth Factor Receptor Gene Amplification in Human Squamous Cell Carcinomas Using Fluorescence in Situ Hybridization,” Japanese Journal of Cancer Research, 1994, vol. 85 (6), pp. 567-571.
  • Shimizu N., et al., “Genetics of Cell Surface Receptors for Bioactive Polypeptides: Binding of Epidermal Growth Factor is Associated With the Presence of Human Chromosome 7 in Human-Mouse Cell Hybrids,” Proceedings of the National Academy of Sciences, 1980, vol. 77 (6), pp. 3600-3604.
  • Shin D.M., et al., “Dysregulation of Epidermal Growth Factor Receptor Expression in Premalignant Lesions During Head and Neck Tumorigenesis,” Cancer Research, 1994, vol. 54 (12), pp. 3153-3159.
  • Shin D.M., et al., “Epidermal Growth Factor Receptor-Targeted Therapy with C225 and Cisplatin in Patients with Head and Neck Cancer,” Clinical Cancer Research, 2001, vol. 7, pp. 1204-1213.
  • Shinkawa T., et al., “The Absence of Fucose but Not the Presence of Galactose or Bisecting N-Acetylglucosamine of Human IgG1 Complex-type Oligosaccharides Shows the Critical Role of Enhancing Antibody-dependent Cellular Cytotoxicity,” The Journal of Biological Chemistry, 2003, vol. 278 (5), pp. 3466-3473.
  • Shinojima N., et al., “Prognostic Value of Epidermal Growth Factor Receptor in Patients with Glioblastoma Multiforme,” Cancer Research, 2003, vol. 63 (20), pp. 6962-6970.
  • Shintani S., et al., “Gefitinib (‘Iressa’, ZD1839), an Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, Up-Regulates P27kip1 and Induces G1 Arrest in Oral Squamous Cell Carcinoma Cell Lines,” Oral Oncology, 2004, vol. 40 (1), pp. 43-51.
  • Shintani S., et al., “Intragenic Mutation Analysis of the Human Epidermal Growth Factor Receptor (Egfr) Gene in Malignant Human Oral Keratinocytes,” Cancer Research, 1999, vol. 59 (16), pp. 4142-4147.
  • Shusta E.V., et al., “Directed Evolution of a Stable Scaffold for T-Cell Receptor Engineering,” Nature Biotechnology, 2000, vol. 18 (7), pp. 754-759.
  • Shusta E.V., et al., “Yeast Polypeptide Fusion Surface Display Levels Predict Thermal Stability and Soluble Secretion Efficiency,” Journal of Molecular Biology, 1999, vol. 292 (5), pp. 949-956.
  • Sibilia M., et al., “The EGF Receptor Provides an Essential Survival Signal for SOS-Dependent Skin Tumor Development,” Cell, 2000, vol. 102 (2), pp. 211-220.
  • Siegel-Lakhai W.S., et al., “Current Knowledge and Future Directions of the Selective Epidermal Growth Factor Receptor Inhibitors Erlotinib (arceva) and Gefitinib (Iressa),” Oncologist, 2005, vol. 10 (8), pp. 579-589.
  • Silver J., et al., “Erbb is Linked to the Alpha-Globin Locus on Mouse Chromosome 11,” Molecular Cell Biology, 1985, vol. 5 (7), pp. 1784-1786.
  • Sirotnak, et al., “Potentiation of Cytotoxic Agents Against Human Tumors in Mice by Zd1839 (Iressa), An Inhibitor of Egfr Tyrosine Kinase, Does Not Require High Levels of Expression of EGFR,” Proceedings of the Annual Meeting of the American Association for Cancer Research, 2000, vol. 482 (Abstract 3076), pp. 41.
  • Sirotnak F.M., et al., “Efficacy of Cytotoxic Agents Against Human Tumor Xenografts is Markedly Enhanced by Coadministration of Zd1839 (Iressa), An Inhibitor of Egfr Tyrosine Kinase,” Clinical Cancer Research, 2000, vol. 6 (12), pp. 4885-4892.
  • Sivasubramanian A., et al., “Structural Model of the mAb 806-EGFR Complex Using Computational Docking Followed by Computational and Experimental Mutagenesis,” Structure, 2006, vol. 14, pp. 401-414.
  • Sizeland A.M., et al., “Anti-Sense Transforming Growth Factor Alpha Oligonucleotides Inhibit Autocrine Stimulated Proliferation of a Colon Carcinoma Cell Line,” Molecular Biology of the Cell, 1992, vol. 3 (11), pp. 1235-1243.
  • Sizeland A.M., et al., “The Proliferative and Morphologic Responses of a Colon Carcinoma Cell Line (LIM 1215) Require the Production of Two Autocrine Factors,” Molecular and Cellular Biology, 1991, vol. 11 (8), pp. 4005-4014.
  • Skov K., et al., “Interaction of Platinum Drugs with Clinically Relevant x-ray doses in Mammalian Cells: A Comparison of Cisplatin, Carboplatin, Iproplatin, and Tetraplatin.,” International Journal of Radiation Oncology Biology Physics, 1991, vol. 20 (2), pp. 221-225.
  • Slamon D.J., et al., “Studies of the Her-2/Neu Proto-Oncogene in Human Breast and Ovarian Cancer,” Science, 1989, vol. 244, pp. 707-712.
  • Slamon D.J., et al., “Use of Chemotherapy Plus a Monoclonal Antibody Against HER2 for Metastatic Breast Cancer that Overexpresses HER2,” The New England Journal of Medicine, 2001, vol. 344 (11), pp. 783-792.
  • Slamon et al., “Addition of Herceptin (Humanized Anti-Her2 Antibody) to First Line Chemotherapy for Her2 Overexpressing Metastatic Breast Cancer (Her2+/Mbc) Markedly Increases Anticancer Activity: A Randomized, Multinational Controlled Phase III Trial,” Proceedings of the Annual Meeting of the American Society of Clinical Oncology, 1998, vol. 98a (Abstract 377), pp. 17.
  • Slichenmyer W.J., et al., “Anticancer Therapy Targeting the ErbB Family of Receptor Tyrosine Kinases,” Seminars in Oncology, 2001, vol. 28 (Suppl. 16), pp. 67-79.
  • Slieker L.J., et al., “Post-Translational Processing of the Epidermal Growth Factor Receptor. Glycosylation-Dependent Acquisition of Ligand-Binding Capacity,” Journal of Biological Chemistry, 1985, vol. 260 (2), pp. 687-690.
  • Slieker L.J., et al., “Synthesis of Epidermal Growth Factor Receptor in Human a431 Cells Glycosylation-Dependent Acquisition of Ligand Binding Activity Occurs Post-Translationally in the Endoplasmic Reticulum,” Journal of Biological Chemistry, 1986, vol. 261 (32), pp. 15233-15241.
  • Sliwkowski M.X., et al., “Nonclinical Studies Addressing the Mechanism of Action of TrastuzumAb (Herceptin),”Seminars in Oncology, 1999, vol. 26 (Suppl. 12), pp. 60-70.
  • Smith J.S., et al., “PTEN Mutation, EGFR Amplification, and outcome in Patients with Anaplastic Astrocytoma and Glioblastoma Multiforme,” Journal of the National Cancer Institute , 2001, vol. 93 (16), pp. 1246-1256.
  • Snyder L.C., et al., “Overview of Monoclonal Antibodies and Small Molecules Targeting the Epidermal Growth Factor Receptor Pathway in Colorectal Cancer,” Clinical Colorectal Cancer, 2005, vol. 5 (Suppl. 2), pp. S71-S80.
  • Sobol R.E., et al., “Epidermal Growth Factor Receptor Expression in Human Lung Carcinomas Defined by a Monoclonal Antibody,” Journal of the National Cancer Institute, 1987, vol. 79 (3), pp. 403-407.
  • Soderquist A.M., et al., “Glycosylation of the Epidermal Growth Factor Receptor in a-431 Cells. The Contribution of Carbohydrate to Receptor Function,” Journal of Biological Chemistry, 1984, vol. 259 (20), pp. 12586-12594.
  • Sok J.C., et al., “Mutant Epidermal Growth Factor Receptor (EGFRvIII) Contributes to Head and Neck Cancer Growth and Resistance to EGFR Targeting,” Clinical Cancer Research, 2006, vol. 12 (17), pp. 5064-5073.
  • Solbach C., et al., “Antitumor Effect of MAb EMD 55900 Depends on EGF-R Expression and Histopathology1,” Neoplasia, 2002, vol. 4 (3), pp. 237-242.
  • Solomon B., et al., “EGFR Blockade with ZD1839 (Iressa) Potentiates the Antitumor Effects of Single and Multiple Fractions of Ionizing Radiation in Human A431 Squamous Cell Carcinoma,” International Journal of Radiation Oncology Biology Physics, 2003, vol. 55 (3), pp. 713-723.
  • Solomon B.M., et al., “Rash from EGFR Inhibitors: Opportunities and Challenges for Palliation,” Current Oncology Reports, 2008, vol. 10 (4), pp. 304-308.
  • Sonabend A.M., et al., “Targeting Epidermal Growth Factor Receptor Variant Iii: A Novel Strategy for the Therapy of Malignant Glioma,” Expert Review of Anticancer Therapy, 2007, vol. 7 (12 Suppl), pp. S45-S50.
  • Sorensen O.E., et al., “Injury-Induced Innate Immune Response in Human Skin Mediated by Transactivation of the Epidermal Growth Factor Receptor,” Journal of Clinical Investigation, 2006, vol. 116 (7), pp. 1878-1885.
  • Sorscher S.M., et al., “EGFR Mutations and Sensitivity to Gefitinib,” The New England Journal of Medicine, 2004, vol. 351 (12), pp. 1260-1261.
  • Soulieres D., et al., “Multicenter Phase II Study of Erlotinib, an Oral Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, in Patients With Recurrent or Metastatic Squamous Cell Cancer of the Head and Neck,” Journal of Clinical Oncology, 2004, vol. 22 (1), pp. 77-85.
  • Spurr N.K., et al., “Chromosomal localisation of the Human Homologues to the Oncogenes erb A and B,” The EMBO Journal, 1984, vol. 3 (1), pp. 159-169.
  • Spurr N. K., et al., “Mapping of cellular oncogenes; erb B on chromosome 7,” Cytogenet, 1984, vol. 37, pp. 590.
  • Sridhar S.S., et al., “Inhibitors of Epidermal-Growth-Factor Receptors: A Review of Clinical Research with a Focus on Non-Small-Cell Lung Cancer,” Lancet Oncology, 2003, vol. 4 (7), pp. 397-406.
  • Stabin M.G., et al., “Olinda/Exm: The Second-Generation Personal Computer Software for Internal Dose Assessment in Nuclear Medicine,” Journal of Nuclear Medicine, 2005, vol. 46 (6), pp. 1023-1027.
  • Stamos J., et al., “Structure of the Epidermal Growth Factor Receptor Kinase Domain Alone and in Complex with a 4-Anilinoquinazoline Inhibitor,” Journal of Biological Chemistry, 2002, vol. 277 (48), pp. 46265-46272.
  • Stancoviski I., et al., “Mechanistic Aspects of the Opposing Effects of Monoclonal Antibodies to the ErbB2 Receptor on Tumor Growth,” Proceedings of the National Academy of Sciences, 1991, vol. 88, pp. 8691-8695.
  • Steffens M.G., et al., “Targeting of Renal Cell Carcinoma with Iodine-131-Labeled Chimeric Monoclonal Antibody G250,” Journal of Clinical Oncology, 1997, vol. 15 (4), pp. 1529-1537.
  • Stein R., et al., “Combining Radioimmunotherapy and Chemotherapy for Treatment of Medullary Thyroid Carcinoma: Effectiveness of Dacarbazine,” Cancer, 2002, vol. 94 (1), pp. 51-61.
  • Stockert, Ludwig Institute for Cancer Research, 1995, pp. 226-227.
  • Stockert, Ludwig Institute for Cancer Research, 1997, pp. 212-213.
  • Stockhausen M.T., et al., “Maintenance of Egfr and EgfrVIII Expressions in an In Vivo and In Vitro Model of Human Glioblastoma Multiforme,” Experimental Cell Research, 2011, vol. 317 (11), pp. 1513-1526.
  • Stragliatto G., et al., “Multiple Infusions of Anti-Epidermal Growth Factor Receptor (EGFR) Monoclonal Antibody (EMD 55,900) in Patients with Recurrent Malignant Gliomas,” European Journal of Cancer, 1996, vol. 32A (4), pp. 636-640.
  • Studnicka G.M., et al., “Human-Engineered Monoclonal Antibodies Retain Full Specific Binding Activity by Preserving Non-CDR Complementarity-Modulating Residues,” Protein Engineering, 1994, vol. 7 (6), pp. 805-814.
  • Sturgis E.M., et al., “Effects of Antiepidermal Growth Factor Receptor Antibody 528 on the Proliferation and Differentiation of Head and Neck Cancer,” Otolaryngology Head and Neck Surgery, 1994, vol. 111 (5), pp. 633-643.
  • Sugawa N., et al., “Function of Aberrant EGFR in Malignant Gliomas,” Brain Tumor Pathology, 1998, vol. 15 (1), pp. 53-57.
  • Sugawa N., et al., “Identical Splicing of Aberrant Epidermal Growth Factor Receptor Transcripts from Amplified Rearranged Genes in Human Glioblastomas,” Proceedings of the National Academy of Sciences USA, 1990, vol. 87, pp. 8602-8606.
  • Sugimura M., et al., “Immunohistochemical study on the expression of epidermal growth factor receptor (EGF-R) in invasive cervical cancer of the uterus,” Nippon Sanka Fujinka Gakkai Zasshi, 1992, vol. 44 (6), pp. 689-694.
  • Sunada H., et al., “Monoclonal Antibody against Epidermal Growth Factor Receptor is Internalized without Stimulating Receptor Phosphorylation,” Proceedings of the National Academy of Sciences, 1986, vol. 83 (11), pp. 3825-3829.
  • Surmacz E., “Function of the IGF-I Receptor in Breast Cancer,” Journal of Mammary Gland Biology and Neoplasia, 2000, vol. 5 (1), pp. 95-105.
  • Sutherland M.S., et al., “Lysosomal Trafficking and Cysteine Protease Metabolism Confer Target-Specific Cytotoxicity by Peptide-Linked Anti-Cd30-Auristatin Conjugates,” Journal of Biological Chemistry, 2006, vol. 281 (15), pp. 10540-10547.
  • Suwa T., et al., “Epidermal Growth Factor Receptor-Dependent Cytotoxic Effect of Anti-Egfr Antibody-Ribonuclease Conjugate on Human Cancer Cells,” Anticancer Research, 1999, vol. 19 (5B), pp. 4161-4165.
  • Swaisland H., et al., “Pharmacokinetics and Tolerability of the Orally Active Selective Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor ZD1839 in Healthy Volunteers,” Clinical Pharmacokinetics, 2001, vol. 40 (4), pp. 297-306.
  • Tabernero, et al., “A phase I PK and serial tumor and skin pharmacodynamic (PD) study of weekly (q1w), every 2-week (q2w) or every 3-week (q3w) 1-hour (h) infusion EMD72000, a humanized monoclonal anti-epidermal growth factor receptor (EGFR) antibody, in patients (pt) with advanced tumors,” Proceedings of the American Society of Clinical Oncology, vol. 192 (Abstract 770), pp. 22.
  • Tabernero, et al., “An international phase II study of cetuxirriab in combination with .oxaliplatin/5-fluorouracil (5-FU)/folinic acid (FA) (FOLFOX-4) in the first-line treatment of patients with metastatic colorectal cancer (CRC) expressing epidermal growth factor receptor (EGFR),” Proceedings of the American Society of Clinical Oncology, 2004, vol. 248s (Abstract 3512), pp. 23.
  • Taetle, et al., “Effects of Anti-Epidermal Growth Factor (EGF) Receptor Antibodies and an Anti-Egf Receptor Recombinant-Ricina Chain Immunoconjugate on Growth of Human Cell,” Chemical Abstracts, 1988, vol. 109 (21), pp. 184218a.
  • Taetle R., et al., “Effects of Anti-Epidermal Growth Factor (Egf) Receptor Antibodies and an Anti-Egf Receptor Recombinant-Ricin a Chain Immunoconjugate on Growth of Human Cells,” National Cancer Institute, 1988, vol. 80 (13), pp. 1053-1059.
  • Tahtis K., et al., “Biodistribution Properties of 111 Indium-labeled C-Functionalized trans-Cyclohexyl Diethylenetriaminepentaacetic Acid Humanized 3S193 Diabody and F(ab )2 Constructs in a Breast Carcinoma Xenograft Model,” Clinical Cancer Research, 2001, vol. 7 (4), pp. 1061-1072.
  • Tai A.L.S., et al., “Co-Overexpression of Fibroblast Growth Factor 3 and Epidermal Growth Factor Receptor is Correlated with the Development of Nonsmall Cell Lung Carcinoma,” Cancer, 2006, vol. 106, pp. 146-155.
  • Takahashi H., et al., “Radioimmunodetection of Human Glioma Xenografts by Monoclonal Antibody to Epidermal Growth Factor Receptor,” Cancer Research, 1987, vol. 47 (14), pp. 3847-3850.
  • Takasu S., et al., “Antibody-based therapy for brain tumor,” Nihon Rinsho, 2005, vol. 63 (Suppl. 9), pp. 563-568.
  • Takasu S., et al., “Radioimmunoscintigraphy of Intracranial Glioma Xenograft with a Technetium-99m-Labeled Mouse Monoclonal Antibody Specifically Recognizing Type III Mutant Epidermal Growth Factor Receptor,” Journal of Neuro-Oncology, 2003, vol. 63 (3), pp. 247-256.
  • Tan A.R., et al., “Pharmacokinetics of CetuximAb after Administration of Escalating Single Dosing and Weekly Fixed Dosing in Patients with Solid Tumors,” Clinical Cancer Research, 2006, vol. 12 (21), pp. 6517-6522.
  • Tang C.K., et al., “Epidermal Growth Factor Receptor VIII Enhances Tumorigenicity in Human Breast Cancer,” Cancer Research, 2000, vol. 60 (11), pp. 3081-3087.
  • Tang P., et al., “The Autocrine Loop of Tgf-Alpha/Egfr and Brain Tumors,” Journal of Neuro Oncology, 1997, vol. 35 (3), pp. 303-314.
  • Tang P.A., et al., “Phase IIstudy of Ispinesib in Recurrent or Metastatic Squamous Cell Carcinoma of the Head and Neck,” Investigational New Drugs, 2008, vol. 26 (3), pp. 257-264.
  • Tannock I.F., “Treatment of Cancer with Radiation and Drugs,” Journal of Clinical Oncology, 1996, vol. 14 (12), pp. 3156-3174.
  • Tanswell P., et al., “Population Pharmacokinetics of Antifibroblast activation protein monoclonal antibody F19 in cancer patients,” British Journal of Clinical Pharmacology, 2001, vol. 51 (2), pp. 177-180.
  • Tateishi M., et al., “Prognostic Influence of the Co-Expression of Epidermal Growth Factor Receptor and c-erbB-2 Protein in Human Lung Adenocarcinoma,” Surgical Oncology, 1994, vol. 3 (2), pp. 109-113.
  • Temam, et al., “Epidermal Growth Factor Receptor Copy Number Alterations Correlate with Poor Clinical Outcome in Patients with Head and Neck Squamous Cancer,” Journal of Clinical Oncology, 2007, vol. 25 (16), pp. 2164-2170.
  • Temming K., et al., “Evaluation of RGD-Targeted Albumin Carriers for Specific Delivery of Auristatin E to Tumor Blood Vessels,” Bioconjugate Chemistry, 2006, vol. 17 (6), pp. 1385-1394.
  • Teramoto T., et al., “Inhibitory Effect of Antiepidermal Growth Factor Receptor Antibody on a Human Gastric Cancer,” Cancer, 1996, vol. 77 (8), pp. 1639-1645.
  • Tewes, et al., “Results of a phase I trial of the humanized anti epidermal growth factor receptor (EGFR) monoclonal antibody EMD 72000 in patients with EGFR expressing solid tumors,” Proceedings of the American Society of Clinical Oncology, 2002, vol. 95a (Abstract 378), pp. 21.
  • Thaung C., et al., “Novel ENU-Induced Eye Mutations in the Mouse: Models for Human Eye Disease,” Human Molecular Genetics, 2002, vol. 11 (7), pp. 755-767.
  • Thomas S.M., et al., “Pharmacokinetic and Pharmacodynamic Properties of EGFR Inhibitors Under Clinical Investigation,” Cancer Treatment Reviews, 2004, vol. 30 (3), pp. 255-268.
  • Thompson D.M., et al., “The EGF receptor: Structure, Regulation and Potential Role in Malignancy,” 1985, vol. 4 (4), pp. 767-788.
  • Tice D.A., et al., “Mechanism of Biological Synergy Between Cellular Src and Epidermal Growth Factor Receptor,” Proceedings of the National Academy of Sciences, 1999, vol. 96 (4), pp. 1415-1420.
  • Tietze L.F., et al., “Novel Analogues of cc-1065 and the Duocarmycins for the use in Targeted Tumour Therapies,” Anti-Cancer Agents in Medicinal Chemistry, 2009, vol. 9 (3), pp. 304-325.
  • Tochon-Danguy H.J., et al., “Imaging and Quantitation of the Hypoxic Cell Fraction of Viable Tumor in an Animal Model of Intracerebral High Grade Glioma Using [18F]Fluoromisonidazole (FMISO),” Nuclear Medicine and Biology, 2002, vol. 29 (2), pp. 191-197.
  • Todaro G.J., et al., “Transformation by Murine and Feline Sarcoma Viruses Specifically Blocks Binding of Epidermal Growth Factor to Cells,” Nature, 1976, vol. 264 (5581), pp. 26-31.
  • Todd R., et al., “Epidermal Growth Factor Receptor (EGFR) Biology and Human Oral Cancer,” Histol Histopathol, 1999, vol. 14 (2), pp. 491-500.
  • Toi M., et al., “Epidermal Growth Factor Receptor Expression as a Prognostic Indicator in Breast Cancer,” European Journal of Cancer, 1991, vol. 27 (8), pp. 977-980.
  • Tokuda Y., et al., “In Vitro and in Vivo Anti-Tumour Effects of a Humanised Monoclonal Antibody Against C-Erbb-2 Product,” British Journal of Cancer, 1996, vol. 73 (11), pp. 1362-1365.
  • Tokumo M., et al., “The Relationship Between Epidermal Growth Factor Receptor Mutations and Clinicopathologic Features in Non-Small Cell Lung Cancers,” Clinical Cancer Research, 2005, vol. 11 (3), pp. 1167-1173.
  • Torres L.A., et al., “Phase I/II Clinical Trial of the Humanized Anti-Egf-R Monoclonal Antibody H-R3 Labelled With 99mtc in Patients with Tumour of Epithelial Origin,” Nuclear medicine communications, 2005, vol. 26 (12), pp. 1049-1057.
  • Toth J., et al., “Analysis of EGFR Gene Amplification, Protein Over-Expression and Tyrosine Kinase Domain Mutation in Recurrent Glioblastoma,” Pathology & Oncology Research, 2009, vol. 15 (2), pp. 225-229.
  • Toyooka, et al., “EGFR Mutation and Response of Lung Cancer to Gefitinib,” The New England Journal of Medicine, 2005, vol. 352 (20), pp. 2136.
  • Trail P.A., et al., “Monoclonal Antibody Drug Conjugates in the Treatment of Cancer,” Current Opinion in Immunology, 1999, vol. 11, pp. 584-588.
  • Tran D.D., et al., “CAML is Required for Efficient EGF Receptor Recycling,” Developmental Cell, 2003, vol. 5 (2), pp. 245-256.
  • Traxler P., et al., “AEE788: A Dual Family Epidermal Growth Factor Receptor/Erbb2 and Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitor With Antitumor and Antiangiogenic Activity,” Cancer Research, 2004, vol. 64 (14), pp. 4931-4941.
  • Trigo, et al., “Cetuximab Monotherapy is Active in Patients (Pts) with Platinum-Refractory Recurrent/Metastatic Squamous Cell Carcinoma of the Head and Neck (Scchn) (Results of a Phase II Study),” Proceedings of the American Society of Clinical Oncology, 2004, vol. 488s (Abstract 5502), pp. 22.
  • Trummell, et al., “The Biological Effects of Anti-Epidermal Growth Factor Receptor and Ionizing Radiation in Human Head and Neck Tumor Cell Lines,” Proceedings of the American Association for Cancer Research, 1999, vol. 144 (Abstract 958), pp. 40.
  • Tsao M.S., et al., “Erlotinib in Lung Cancer-Molecular and Clinical Predictors of Outcome,” The New England Journal of Medicine, 2005, vol. 352 (2), pp. 133-144.
  • Tsuchihashi Z., et al., “Responsiveness to Cetuximab without Mutations in EGFR,” The New England Journal of Medicine, 2005, vol. 353 (2), pp. 208-209.
  • Tsugu, et al., “Localization of Aberrant Messenger RNA of Epidermal Growth Factor Receptor (EGFR) in Malignant Glioma,” Anticancer research , 1997, vol. 17 (3C), pp. 2225-2232.
  • Turkeri L.N., et al., “Impact of the Expression of Epidermal Growth Factor, Transforming Growth Factor Alpha, and Epidermal Growth Factor Receptor on the Prognosis of Superficial Bladder Cancer,” Urology, 1998, vol. 51 (4), pp. 645-649.
  • Turner T., et al., “EGF Receptor Signaling Enhances in Vivo Invasiveness of Du-145 Human Prostate Carcinoma Cells,” Clinical and Experimental Metastasis, 1996, vol. 14 (4), pp. 409-418.
  • Tzahar E., et al., “Bivalence of EGF-like Ligands Drives the ERBP Signaling Network,” The EMBO Journal, 1997, vol. 16 (16), pp. 4938-4950.
  • Uegaki K., et al., “Clinicopathological Significance of Epidermal Growth Factor and its Receptor in Human Pancreatic Cancer,” Anticancer research, 1997, vol. 17 (5B), pp. 3841-3847.
  • Uemura H.E., et al., “Internal Image Anti-Idiotype Antibodies Related to Renal- Cell Carcinoma-Associated Antigen G250.Int. J. Cancer,” International Journal of Cancer, 1994, vol. 56 (4), pp. 609-614.
  • Ullrich A., et al., “Human Epidermal Growth Factor Receptor CDNA Sequence and Aberrant Expression of the Amplified Gene in A431 Epidermoid Carcinoma Cells,” Nature, 1984, vol. 309 (5967), pp. 418-425.
  • Ullrich A., et al., “Signal Transduction by Receptors with Tyrosine Kinase Activity,” Cell, 1990, vol. 61 (2), pp. 203-212.
  • U.S. Appl. No. 60/290,410, filed May 11, 2001.
  • U.S. Appl. No. 60/326,019, filed Sep. 28, 2001.
  • U.S. Appl. No. 60/342,258, filed Dec. 21, 2001.
  • Ushiro H., et al., “Identification of Phosphotyrosine as a Product of Epidermal Growth GactorActivated Protein Kinase in A-431 Cell Membranes,” Journal of Biological Chemistry, 1980, vol. 255 (18), pp. 8363-8365.
  • Vagin A., et al., “MOLREP: An Automated Program for Molecular Replacement,” Journal of Applied Crystallography, 1997, vol. 30, pp. 1022-1025.
  • Vagin A.A., et al., “Spherically Averaged Phased Translation Function and its Application to the Search for Molecules and Fragments in Electron-Density Maps,” Acta Crystallographica, 2001, vol. 57 (10), pp. 1451-1456.
  • Vaidyanathan G., et al., “Improved Xenograft Targeting of Tumor-Specific Anti-Epidermal Growth Factor Receptor Variant III Antibody Labeled Using N-Succinimidyl 4-Guanidinomethyl-3-Iodobenzoate,” Nuclear Medicine and Biology, 2002, vol. 29, pp. 1-11.
  • Vajdos F.F., et al., “Comprehensive Functional Maps of the Antigen-binding Site of an AntiErbB2 Antibody Obtained with Shotgun Scanning Mutagenesis,” Journal of Molecular Biology, 2002, vol. 320 (2), pp. 415-428.
  • Van De Loosdrecht A.A., et al., “A Tetrazolium-Based Colorimetric MTT Assay to Quantitate Human Monocyte Mediated Cytotoxicity against Leukemic Cells from Cell Lines and Patients with Acute Myeloid Leukemia,” Journal of Immunological Methods, 1994, vol. 174 (1-2), pp. 311-320.
  • Van De Vijver M.J., et al., “Ligand-Induced Activation of A431 Cell Epidermal Growth Factor Receptors Occurs Primarily by an Autocrine Pathway that Acts Upon Receptors on the Surface Rather than Intracellularly,” Journal of Biological Chemistry, 1991, vol. 266 (12), pp. 7503-7508.
  • Van Den Eynde B.J., et al., “Tumor Antigens” in: Encyclopedia of Immunology, 2nd Edition, Academic Press Limited, 1998, pp. 2424-2431.
  • Van Der Heyden M.A., et al., “Identification of an Intracellular Domain of the EGF Receptor Required for High-Affinity Binding of EGF,” FEBS Letters, 1997, vol. 410 (2-3), pp. 265-268.
  • Van Dijk M.A., et al., “Human Antibodies as Next Generation Therapeutics,” Current Opinion in Chemical Biology, 2001, vol. 5 (4), pp. 368-374.
  • Van Doorn R., et al., “Follicular and Epidermal Alterations in Patients Treated with Zd1839 (Iressa), an Inhibitor of the Epidermal Growth Factor Receptor,” British Journal of Dermatology, 2002, vol. 147 (3), pp. 598-601.
  • Van Regenmortel M.H., et al., “Comparative Immunological Methods,” Methods in Enzymology, 1993, vol. 224, pp. 130-140.
  • Vanhoefer U., et al., “Phase I Study of the Humanized Antiepidermal Growth Factor Receptor Monoclonal Antibody EMD72000 in Patients with Advanced Solid Tumors that Express the Epidermal Growth Factor Receptor,” Journal of Clinical Oncology, 2004, vol. 22 (1), pp. 175-184.
  • Veale D., et al., “Epidermal Growth Factor Receptors in Non-Small Cell Lung Cancer,” British Journal of Cancer, 1987, vol. 55, pp. 513-516.
  • Veale D., et al., “The Relationship of Quantitative Epidermal Growth Factor Receptor Expression in Non-Small Cell Lung Cancer to Long Term Survival,” British Journal of Cancer, 1993, vol. 68 (1), pp. 162-165.
  • Velu T.J., et al., “Epidermal Growth-Factor-Dependent Transformation by a Human Egf Receptor Proto-Oncogene,” Science, 1987, vol. 238 (4832), pp. 1408-1410.
  • Venter D.J., et al., “Overexpression of the C-Erbb-2 Oncoprotein in Human Breast Carcinomas: Immunohistological Assessment Correlates With Gene Amplification,” Lancet, 1987, vol. 2 (8550), pp. 69-72.
  • Verbeek B.S., et al., “Overexpression of EGFR and c-erbB2 Causes Enhanced Cell Migration in Human Breast Cancer Cells and NIH3T3 Fibroblasts,” FEBS Letters, 1998, vol. 425 (1), pp. 145-150.
  • Vermorken, et al., “Cett.lximab (Erbitux®) in Recurrent/Metastatic (R&M) Squamous Cell Carcinoma of the Head and Neck (SCCHN) Refractory to First-Line Platinum- Based Therapies,” Journal of Clinical Oncology, 2005, Abstract 5505, pp. 23.
  • Vermorken J.B., et al., “Platinum-Based Chemotherapy Plus Cetuximab in Head and Neck Cancer,” The New England Journal of Medicine, 2008, vol. 359 (11), pp. 1116-1127.
  • Verveer P.J., et al., “Quantitative Imaging of Lateral ErbB1 Receptor Signal Propagation in the Plasma Membrane,” Science, 2000, vol. 290 (5496), pp. 1567-1570.
  • Viana-Pereira M., et al., “Analysis of Egfr Overexpression, EGFR Gene Amplification and the EEGFRvIII Mutation in Portuguese High-Grade Gliomas,” Anticancer Research, 2008, vol. 28 (2A), pp. 913-920.
  • Viloria-Petit A., et al., “Acquired Resistance to the Antitumor Effect of Epidermal Growth Factor Receptor-blocking Antibodies in vivo: A Role for Altered Tumor Angiogenesis,” Cancer Research, 2001, vol. 61 (13), pp. 5090-5101.
  • Vincent P.W., et al., “Anticancer Efficacy of the Irreversible EGFR Tyrosine Kinase Inhibitor PD 0169414 Against Human Tumor Xenografts,” Cancer Chemother Pharmacol, 2000, vol. 45 (3), pp. 231-238.
  • Vitali, et al., “Monoclonal Antibody 806 Inhibits the Growth of Subcutaneous and Intracranial Tumor Xenografts Expressing Either the De2.7 or Amplified Epidermal Growth Factor Receptor (Egfr) But Not Wild-Type EGFR,” Proceedings of the 14th Annual Lorne Cancer Conference, Abstract 221.
  • Voelzke W.R., et al., “Targeting the Epidermal Growth Factor Receptor in High-Grade Astrocytomas,” Current Treatment Options in Oncology, 2008, vol. 9 (1), pp. 23-31.
  • Vogel C.L., et al., “Efficacy and Safety of Trastuzumab as a Single Agent in First-Line Treatment of HER2-Overexpressing Metastatic Breast Cancer,” Journal of Clinical Oncology, 2002, vol. 20 (3), pp. 719-726.
  • Vogel C.L., et al., “First-Line Herceptin Monotherapy in Metastatic Breast Cancer,” Oncology, 2001, vol. 61 (Suppl. 2), pp. 37-42.
  • Vogt N., et al., “Relationships Linking Amplification Level to Gene Over-Expression in Gliomas,” PLoS One, 2010, vol. 5 (12), pp. e14249.
  • Voldborg B.R., et al., “Epidermal Growth Factor Receptor (EGFR) and EGFR Mutations, Function and Possible Role in Clinical Trials,” Annals of Oncology, 1997, vol. 8 (12), pp. 1197-1206.
  • Volm M., et al., “Prognostic Value of ERBB-1, VEGF, cyclin A, FOS, JUN and MYC in Patients with Squamous Cell Lung Carcinomas,” British Journal of Cancer, 1998, vol. 77 (4), pp. 663-669.
  • Wade J.D., et al., “An Automated Peptide and Protein Thiazolidine Coupling Chemistry for Biosensor Immobilization giving a Unique N-Terminal Orientation,” Analytical Biochemistry, 2006, vol. 348 (2), pp. 315-317.
  • Wakeling A.E., et al., “Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors,” Current Opinion in Pharmacology, 2002, vol. 2 (4), pp. 382-387.
  • Wakeling A.E., et al., “Specific Inhibition of Epidermal Growth Factor Receptor Tyrosine Kinase by 4-Anilinoquinazolines,” Breast Cancer Research and Treatment, 1996, vol. 38 (1), pp. 67-73.
  • Wakeling E.L., et al., “Human EGFR, a Candidate Gene for the Silver-Russell Syndrome, Is Biallelically Expressed in a Wide Range of Fetal Tissues,” European Journal of Human Genetics, 1998, vol. 6 (2), pp. 158-164.
  • Waksal H.W., “Role of an Anti-Epidermal Growth Factor Receptor in Treating Cancer,” Cancer Metastasis Reviews, 1999, vol. 18 (4), pp. 427-436.
  • Waldmann, et al.,“Monoclonal antibodies in diagnosis and therapy,” Science, 1991, 252 (5013), 1657-1662.
  • Walewski J., et al., “Rituximab (Mabthera, Rituxan) in Patients with Recurrent Indolent Lymphoma: Evaluation of Safety and Efficacy in a Multicenter Study,” Medical Oncology, 2001, vol. 18 (2), pp. 141-148.
  • Walker F., et al., “Activation of the Ras/Mitogen-Activated Protein Kinase Pathway by Kinase-Defective Epidermal Growth Factor Receptors Results in Cell Survival but Not Proliferation,” Molecular and Cellular Biology, 1998, vol. 18 (12), pp. 7192-7204.
  • Walker F., et al., “Biochemical Characterization of Mutant EGF Receptors Expressed in the Hemopoietic Cell Line BaF/3,” Growth Factors, 1998, vol. 16 (1), pp. 53-67.
  • Walker F., et al., “CR1/CR2 Interactions Modulate the Functions of the Cell Surface Epidermal Growth Factor Receptor,” Journal of Biological Chemistry, 2004, vol. 279 (21), pp. 22387-22398.
  • Walker F., et al., “Reconstitution of the High Affinity Epidermal Growth Factor Receptor on Cell-free Membranes after Transmodulation by Platelet-derived Growth Factor,” Journal of Biological Chemistry, 1991, vol. 266 (5), pp. 2746-2752.
  • Walker R.A., et al., “Expression of Epidermal Growth Factor Receptor MRNA and Protein in Primary Breast Carcinomas,” Breast Cancer Research and Treatment, 1999, vol. 53 (2), pp. 167-176.
  • Walton G.M., et al., “Analysis of Deletions of the Carboxyl Terminus of the Epidermal Growth Factor Receptor Reveals Self-Phosphorylation at Tyrosine 992 and Enhanced in Vivo Tyrosine Phosphorylation of Cell Substrates,” Journal of Biological Chemistry, 1990, vol. 265 (3), pp. 1750-1754.
  • Wang D.P., et al., “Immunohistochemical Localization of C-Erbb-2 Protein and Epidermal Growth Factor Receptor in Normal Surface Epithelium, Surface Inclusion Cysts, and Common Epithelial Tumours of the Ovary,” Virchows Arch a Pathol Anat Histopathol, 1992, vol. 421 (5), pp. 393-400.
  • Wang H., et al., “Epidermal Growth Factor Receptor VIII Enhances Tumorigenicity and Resistance to 5-Fluorouracil in Human Hepatocellular Carcinoma,” Cancer Letters, 2009, vol. 279 (1), pp. 30-38.
  • Wang X., et al., “Epidermal Growth Factor Receptor is a Cellular Receptor for Human Cytomegalovirus,” Nature, 2003, vol. 424 (6947), pp. 456-461.
  • Wang Z., et al., “Endocytosis Deficiency of Epidermal Growth Factor (EGF) Receptor-ERBB2 Heterodimers in Response to EGF Stimulation,” Molecular Biology of the Cell, 1999, vol. 10 (5), pp. 1621-1636.
  • Ward E.S., et al., “Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted from Escherichia Coli,” Nature, 1989, vol. 341 (6242), pp. 544-546.
  • Wargalla, et al., “Rate of Internalization of an Immunotoxin Correlates With Cytotoxic Activity Against Human Tumor Cells,” Proceedings of the National Academy of Sciences, 1989, vol. 86 (13), pp. 5146-5150.
  • Waterfield M.D., et al., “A Monoclonal Antibody to the Human Epidermal Growth Factor Receptor,” Journal of Cellular Biochemisty, 1982, vol. 20, pp. 149-161.
  • Waterman H., et al., “Alternative Intracellular Routing of ERBB Receptors May Determine Signaling Potency,” Journal of Biological Chemistry, 1998, vol. 273 (22), pp. 13819-13827.
  • Waterman H., et al., “Molecular Mechanisms Underlying Endocytosis and Sorting of ErbB Receptor Tyrosine Kinases,” FEBS Letters, 2001, vol. 490 (3), pp. 142-152.
  • Waugh M.G., et al., “Epidermal Growth Factor Receptor Activation is Localized Within Low-Buoyant Density, Non-Caveolar Membrane Domains,” Biochemical Journal, 1999, vol. 337 (pt 3), pp. 591-597.
  • Webster W.M., et al., “Engineering Antibody Affinity and Specificity,” International Journal of Cancer—Supplement, 1988, vol. 3, pp. 13-16.
  • Wedegaertner P.B., et al., “Activation of the Purified Protein Tyrosine Kinase Domain of the Epidermal Growth Factor Receptor,” Journal of Biological Chemistry, 1989, vol. 264 (19), pp. 11346-11353.
  • Wedegaertner P.B., et al., “Effect of Carboxyl Terminal Truncation on the Tyrosine Kinase Activity of the Epidermal Growth Factor Receptor,” Archives of Biochemistry and Biophysics, 1992, vol. 292 (1), pp. 273-280.
  • Weiner L.M., “An Overview of Monoclonal Antibody Therapy of Cancer,” Seminars in Oncology, 1999, vol. 26 (4 Suppl. 12), pp. 41-50.
  • Weiss G.A., et al., “Rapid Mapping of Protein Functional Epitopes by Combinatorial Alanine Scanning,” Proceedings of the National Academy of Sciences, 2000, vol. 97 (16), pp. 8950-8954.
  • Wells A., “EGF Receptor,” The International Journal of Biochemistry & Cell Biology, 1999, vol. 31 (6), pp. 637-643.
  • Wells A., et al., “Ligand-Induced Transformation by a Noninternalizing Epidermal Growth Factor Receptor,” Science, 1990, vol. 247 (4945), pp. 962-964.
  • Welt, et al., “Phase I Study of Humanized A33 (huA33) Antibody in Patients with Advanced Colorectal Cancer,” Proceedings of the Annual Meeting of the American Society of Clinical Oncology, 1977, vol. 436a (Abstract 1563), pp. 16.
  • Welt S., et al., “Antibody Targeting in Metastatic Colon Cancer: A Phase I Study of Monoclonal Antibody F19 Against a Cell-Surface Protein of Reactive Tumor Stromal Fibroblasts,” Journal of Clinical Oncology, 1994, vol. 12 (6), pp. 1193-1203.
  • Welt S., et al., “Phase I Study of Anticolon Cancer Humanized Antibody A33,” Clinical Cancer Research, 2003, vol. 9, pp. 1338-1346.
  • Welt S., et al., “Phase I/II Study of Iodine 125-Labeled Monoclonal Antibody A33 in Patients with Advanced Colon Cancer,” Journal of Clinical Oncology, 1996, vol. 14 (6), pp. 1787-1797.
  • Welt S., et al., “Phase I/II Study of Iodine 131-Labeled Monoclonal Antibody A33 in Patients with Advanced Colon Cancer,” Journal of Clinical Oncology, 1994, vol. 12 (8), pp. 1561-1571.
  • Welt S., et al., “Preliminary Report of a Phase I Study of Combination Chemotherapy and Humanized A33 Antibody Immunotherapy in Patients with Advanced Colorectal Cancer,” Clinical Cancer Research, 2003, vol. 9 (4), pp. 1347-1353.
  • Welt S., et al., “Quantitative Analysis of Antibody Localization in Human Metastatic Colon Cancer: A Phase I Study of Monoclonal Antibody A33,” Journal of Clinical Oncology, 1990, vol. 8 (11), pp. 1894-1906.
  • Wen, et al., “Potentiation of Antitumor Activity of PG-TXL with Anti-EGFR Monoclonal Antibody C225 in MDA-MB-468 Human Breast Cancer Xenograft,” Proceedings of the American Association for Cancer Research, 2000, vol. 323 (Abstract 2052), pp. 51.
  • Weppler S.A., et al., “Expression of EGFR Variant vIII promotes Both Radiation Resistance and Hypoxia Tolerance,” Radiotherapy and Oncology, 2007, vol. 83 (3), pp. 333-339.
  • Wersall P., et al., “Intratumoral Infusion of the Monoclonal Antibody, mAb 425, against the Epidermal-Growth-Factor Receptor in Patients with Advanced Malignant Gliomacancer Immunol,” Cancer Immunology Immunotherapy, 1997, vol. 44 (3), pp. 157-164.
  • Westwood J.A., et al., “Adoptive transfer of T cells modified with a humanized chimeric receptor gene inhibits growth of Lewis-Y-expressing tumors in mice,” Proceedings of the National Academy of Sciences, 2005, vol. 102 (52), pp. 19051-19056.
  • Wheeler D.L., et al., “Mechanisms of Acquired Resistance to Cetuximab: Role of HER (ErbB) Family Members,” Oncogene, 2008, vol. 27 (28), pp. 3944-3956.
  • Wheeler S.E., et al., “Epidermal Growth Factor Receptor Variant III Mediates Head and Neck Cancer Cell Invasion, via STAT3 Activation,” Oncogene, 2010, vol. 29 (37), pp. 5135-5145.
  • Whitson K.B., et al., “Functional Effects of Selective Glycosylation at Asn-579 of the Epidermal Growth Factor Receptor,” Biochemistry, 2005, vol. 44 (45), pp. 14920-14931.
  • Wikstrand C.J., et al., “Cell Surface Localization and Density of the Tumor-Associated Variant of the Epidermal Growth Factor Receptor, EGFRvIII,” Cancer Research, 1997, vol. 57, pp. 4130-4140.
  • Wikstrand C.J., et al., “Comparative Localization of Glioma-Reactive Monoclonal Antibodies in Vivo In an Athymic Mouse Human Glioma Xenograft Model,” Journal of Neuroimmunology, 1987, vol. 15 (1), pp. 37-56.
  • Wikstrand C.J., et al., “Investigation of a Synthetic Peptide as Immunogen for a Variant Epidermal Growth Factor Receptor Associated with Gliomas,” Journal of Neuroimmunology, 1993, vol. 46 (1-2), pp. 165-173.
  • Wikstrand C.J., et al., “Monoclonal Antibodies against EGFRvIII are Tumor Specific and React with Breast and Lung Carcinomas and Malignant Gliomas,” Cancer Research, 1995, vol. 55 (14), pp. 3140-3148.
  • Wikstrand C.J., et al., “Monoclonal Antibody Therapy of Human Gliomas: Current Status and Future Approaches,” Cancer Metastasis Reviews, 1999, vol. 18 (4), pp. 451-464.
  • Wikstrand C.J., et al., “Production and Characterization of Two Human Glioma Xenograft-Localizing Monoclonal Antibodies,” Cancer Research, 1986, vol. 46 (11), pp. 5933-5940.
  • Wikstrand C.J., et al., “The Class III Variant of the Epidermal Growth Factor Receptor (EGFRvIII): Characterization and Utilization as an Immunotherapeutic Target,” Journal of Neurovirology, 1998, vol. 4 (2), pp. 148-158.
  • Wikstrand, et al., “Antibodies and molecular immunology: immunohistochemistry and antigens of diagnostic significance” in: Russell and Rubinsteins Pathology of Tumors of the Nervous System, Chapter 8, Bigner, et al., eds., Arnold and Oxford University Press, Inc, 1998, pp. 251-304.
  • Wiley H.S., et al., “The Role of Tyrosine Kinase Activity in Endocytosis, Compartmentation, and Down-Regulation of the Epidermal Growth Factor Receptor,” Journal of Biological Chemistry, 1991, vol. 266 (17), pp. 11083-11094.
  • Wiley H.S., et al., “Trafficking of the ErbB Receptors and its Influence on Signaling,” Experimental Cell Research, 2003, vol. 284 (1), pp. 78-88.
  • Willett C.J., et al., “Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer,” Nature Medicine, 2004, vol. 10, pp. 145-147.
  • Williams, et al., “Combination of ZD1839 (IRESSA), An EGFR Tyrosine Kinase Inhibitor, and Radiotherapy Increases Antitumour Efficacy in a Human Colon Cancer Xenograft Model,” Proceedings of the American Association for Cancer Research, 2001, vol. 715 (Abstract 3840), pp. 42.
  • Williams K.J., et al., “ZD1839 (‘Iressa’), A Specific Oral Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor, Potentiates Radiotherapy in a Human Colorectal Cancer Xenograft Model,” British Journal of Cancer, 2002, vol. 86 (7), pp. 1157-1161.
  • Winer E.P., et al., “New Combinations with Herceptin in Metastatic Breast Cancer,” Oncology, 2001, vol. 61 (Suppl. 2), pp. 50-57.
  • Winkler M.E., et al. , “Epidermal Growth Factor and Transforming Growth Factor Alpha Bind Differently to the Epidermal Growth Factor Receptor ,” Biochemistry, 1989, vol. 28 (15), pp. 6373-6378.
  • Winter G., et al., “Man-Made Antibodies,” Nature, 1991, vol. 349 (6307), pp. 293-299.
  • Wollman R., et al., “Effect of Epidermal Growth Factor on the Growth and Radiation Sensitivity of Human Breast Cancer Cells In Vitro,” International Journal of Radiation Oncology, 1994, vol. 30 (1), pp. 91-98.
  • Woltjer R.L., et al., “Direct Identification of Residues of the Epidermal Growth Factor Receptor in Close Proximity to the Amino Terminus of Bound Epidermal Growth Factor,” Proceedings of the National Academy of Sciences, 1992, vol. 89 (16), pp. 7801-7805.
  • Wong A.J., et al., “Increased Expression of the Epidermal Growth Factor Receptor Gene in Malignant Gliomas is Invariably Associated with Gene Amplification,” Proceedings of the National Academy of Sciences, 1987, vol. 84 (19), pp. 6899-6903.
  • Wong A.J., et al., “Structural Alterations of the Epidermal Growth Factor Receptor Gene in Human Gliomas,” Proceedings of the National Academy of Sciences USA, 1992, vol. 89, pp. 2965-2969.
  • Wood E.R., et al., “A Unique Structure for Epidermal Growth Factor Receptor Bound to GW572016 (Lapatinib): Relationships among Protein Conformation, Inhibitor off-rate, and Receptor Activity in Tumor Cells,” Cancer Research, 2004, vol. 64 (18), pp. 6652-6659.
  • Woodburn, et al., “ZD1839, An Epidermal Growth Factor Tyrosine Kinase Inhibitor Selected for Clinical Development,” Proceedings of the Annual Meeting of the American Association for Cancer Research, 1997, vol. 633 (Abstract 4251), pp. 38.
  • Woodburn, et al., “ZD1839 (IRESSA) A Selective Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor (Egfr-Tki): Inhibition of CFOS MRNA, An Intermediate Marker of EGFR Activation,Corre- Lates with Tumor Growth Inhibition,” Proceedings of the Annual Meeting of the American Association for Cancer Research, 2000, vol. 402 (Abstract 2552), pp. 41.
  • Woodburn J.R., et al., “The Epidermal Growth Factor Receptor and Its Inhibition in Cancer Therapy,” Pharmacology and Therapeutics, 1999, vol. 82 (2-3), pp. 241-250.
  • Written Opinion for the Application No. PCT/US2008/009771, mailed on Jan. 5, 2009, 7 pages.
  • Wu D., et al. , “Human Epidermal Growth Factor (EGF) Receptor Sequence Recognized by EGF Competitive Monoclonal Antibodies. Evidence for the localization of the EGF-binding site ,” Journal of Biological Chemistry, 1989, vol. 264 (29), pp. 17469-17475.
  • Wu G., et al., “Targeted Delivery of Methotrexate to Epidermal Growth Factor Receptor-Positive Brain Tumors by means of Cetuximab (IMC-C225) Dendrimer Bioconjugates,” Molecular cancer therapeutic, 2006, vol. 5 (1), pp. 52-59.
  • Wu H., et al., “Humanization of a Murine Monoclonal Antibody by Simultaneous Optimization of Framework and CDR Residues,” Journal of Molecular Biology, 1999, vol. 294 (1), pp. 151-162.
  • Wu X., et al., “Apoptosis Induced by an Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in a Human Colorectal Carcinoma Cell Line and its Delay by Insulin,” The Journal of Clinical Investigation, 1995, vol. 95 (4), pp. 1897-1905.
  • Xie H., et al., “In Vitro Invasiveness of Du-145 Human Prostate Carcinoma Cells is Modulated by EGF Receptor-Mediated Signals,” Clinical & experimental metastasis, 1995, vol. 13 (6), pp. 407-419.
  • Xiong H.Q., et al., “Cetuximab, A Monoclonal Antibody Targeting the Epidermal Growth Factor Receptor, In Combination With Gemcitabine for Advanced Pancreatic Cancer: A Multicenter Phase II Trial,” Journal of Clinical Oncology, 2004, vol. 22 (13), pp. 2610.
  • Xu F., et al., “Antibody-Induced Growth Inhibition is Mediated through Immunochemically and Functionally Distinct Epitopes on the Extracellular Domain of the c-ErbB-2 (HER-2/neu) Gene Product p185,” International Journal of Cancer, 1993, vol. 53, pp. 401-408.
  • Xu Y.H., et al., “Characterization of Epidermal Growth Factor Receptor Gene Expression in Malignant and Normal Human Cell Lines,” Proceedings of the National Academy of Sciences, 1984, vol. 81 (23), pp. 7308-7312.
  • Xu Y.H., et al., “Human Epidermal Growth Factor Receptor CDNAIs Homologous to a Variety of RNAS Overproduced in A431 Carcinoma Cells,” Nature, 1984, vol. 309 (5971), pp. 806-810.
  • Yamanaka Y., et al., “Coexpression of Epidermal Growth Factor Receptor and Ligands in Human Pancreatic Cancer is Associated with Enhanced Tumor Aggressiveness,” Anticancer Research, 1993, vol. 13 (3), pp. 565-569.
  • Yamazaki H., et al., “A Deletion Mutation within the Ligand Binding Domain is Responsible for Activation of Epidermal Growth Factor Receptor Gene in Human Brain Tumors,” Japanese Journal of Cancer, 1990, vol. 81, pp. 773-779.
  • Yamazaki H., et al., “Amplification of the Structurally and Functionally Altered Epidermal Growth Factor Receptor Gene (c-erbB) in Human Brain Tumors,” Molecular and Cellular Biology , 1988, vol. 8 (4), pp. 1816-1820.
  • Yamazaki H., et al., “Inhibition of Tumor Growth by Ribozyme-Mediated Suppression of Aberrant Epidermal Growth Factor Receptor Gene Expression,” Journal of the National Cancer Institute, 1998, vol. 90 (8), pp. 581-587.
  • Yang E.B., et al., “Genistein, a Tyrosine Kinase Inhibitor, Reduces EGF-Induced EGF Receptor Internalization and Degradation in Human Hepatoma HepG2 Cells,” Biochimica et Biophysica Acta, 1996, vol. 224, pp. 309-317.
  • Yang, et al., “Modification of Gemcitabine-Induced Radiosesitization by the Nitroxide Tempol,” Proceedings of the American Society of Clinical Oncology, 1999, vol. 457a (Abstract 1765), pp. 18.
  • Yang, et al., “Therapeutic Potential of ABX-EGF, A Fully Human Anti-EGF Receptor Monoclonal Antibody, for Cancer Treatment,” Proceedings of the American Society of Clinical Oncology, 2000, vol. 48a (Abstract 183), pp. 19.
  • Yang L., et al., “Identification and Characterization of Ch806 Mimotopes,” Cancer Immunology, Immunotherapy, 2010, vol. 59 (10), pp. 1481-1487.
  • Yang W., et al., “Development of a Syngeneic Rat Brain Tumor Model Expressing EGFRvIII and its Use for Molecular Targeting Studies with Monoclonal Antibody L8A4,” Clinical Cancer Research, 2005, vol. 11 (1), pp. 341-350.
  • Yang W., et al., “Molecular Targeting and Treatment of EGFRvIII-Positive Gliomas Using Boronated Monoclonal Antibody L8A4,” Clinical Cancer Research, 2006, vol. 12 (12), pp. 3792-3802.
  • Yang X.D., et al., “Development of ABX-EGF, A Fully Human Anti-EGF Receptor Monoclonal Antibody, for Cancer Therapy,” Critical Reviews in Oncology/Hematology, 2001, vol. 38, pp. 17-23.
  • Yang X.D., et al., “Eradication of Established Tumors by a Fully Human Monoclonal Antibody to the Epidermal Growth Factor Receptor without Concomitant Chemotherapy,” Cancer Research, 1999, vol. 59 (6), pp. 1236-1243.
  • Yang Z., et al., “The epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 (Iressa) suppresses c-Src and Pak1 pathways and invasiveness of human cancer cells,” Clin cancer res, 2004, vol. 10 (2), pp. 658-667.
  • Yao M., et al., “Enhanced Expression of C-Myc and Epidermal Growth Factor Receptor (C-Erbb-1) Genes in Primary Human Renal Cancer,” Cancer Research, 1988, vol. 48 (23), pp. 6753-6757.
  • Yarden Y., et al., “Self-Phosphorylation of Epidermal Growth Factor Receptor: Evidence for a Model of Intermolecular Allosteric Activation,” Biochemistry, 1987, vol. 26 (5), pp. 1434-1442.
  • Yarden Y., et al., “Epidermal Growth Factor Induces Rapid, Reversible Aggregation of the Purified Epidermal Growth Factor Receptor,” Biochemistry, 1987, vol. 26 (5), pp. 1443-1451.
  • Yarden Y., et al., “Untangling the ErbB Signalling Network,” Nature Reviews. Molecular Cell Biology, 2001, vol. 2 (2), pp. 127-137.
  • Ye D., et al., “Augmentation of a Humanized Anti-HER2 MAB 4D5 Induced Growth Inhibition by a Human-Mouse Chimeric Anti-EGF Receptor Mab C225,” Oncogene, 1999, vol. 18 (3), pp. 731-738.
  • Yeatman T.J., “A Renaissance for SRC,” Nature Reviews Cancer, 2004, vol. 4 (6), pp. 470-480.
  • Yen L., et al., “Differential Regulation of Tumor Angiogenesis by Distinct ErbB Homo- and Heterodimers,” Molecular Biology of the Cell, 2002, vol. 13, pp. 4029-4044.
  • Yip Y.L., et al., “Identification of Epitope Regions Recognized by Tumor Inhibitory and Stimulatory Anti-ErbB-2 Monoclonal Antibodies: Implications for Vaccine Design,” Journal of Immunology, 2001, vol. 166 (8), pp. 5271-5278.
  • Yip Y.L., et al., “Structural Analysis of the Erbb-2 Receptor using Monoclonal Antibodies: Implications for Receptor Signalling,” International Journal of Cancer, 2003, vol. 104 (3), pp. 303-309.
  • Ymer, et al., “Glioma Specific Extracellular Missense Mutations in the First Cysteine Rich Region f Epidermal Growth Factor Receptor (EGFR) Initiate Ligand Independent Activation,” Cancers, 2011, vol. 3, pp. 2032-2049.
  • Ymer S., et al., “Constitutive Synthesis of Interleukin-3 by Leukaemia Cell Line Wehi-3B is due to Retroviral Insertion near the Gene,” Nature, 1985, vol. 317 (6034), pp. 255-258.
  • Yoshida K., et al., “Egf and Tgf-A, the Ligands of Hyperproduced Egfr in Human Esophageal Carcinoma Cells, act as Autocrine Growth Factors,” International Journal of Cancer, 1990, vol. 45 (1), pp. 131-135.
  • Yoshida K., et al., “Studies of the Expression of Epidermal Growth Factor Receptor in Human Renal Cell Carcinoma: A Comparison of Immunohistochemical Method Versus Ligand Binding Assay,” Oncology, 1997, vol. 54 (3), pp. 220-225.
  • Yoshimoto K., et al., “Development of a Real-Time RT-PCR Assay for Detecting EGFRvIII in Glioblastoma Samples,” Clinical Cancer Research, 2008, vol. 14 (2), pp. 488-493.
  • Yoshitake S., et al., “Conjugation of Glucose Oxidase from Aspergillus Niger and Rabbit Antibodies Using N-Hydroxysuccinimide Ester of N-(4-Carboxycyclohexylmethyl)-Maleimide,” European Journal of Biochemistry, 1979, vol. 101 (2), pp. 395-399.
  • Yu H., et al., “Co-Expression of EGFRvIII with ErbB-2 Enhances Tumorigenesis: EGFRvIII Mediated Constitutively Activated and Sustained Signaling Pathways, whereas EGF-Induced a Transient Effect on EGFR-Mediated Signaling Pathways,” Cancer Biology & Therapy, 2008, vol. 7 (11), pp. 1818-1828.
  • Yu T.W., et al., “The Biosynthetic Gene Cluster of the Maytansinoid Antitumor Agent Ansamitocin from Actinosynnema Pretiosum,” Proceedings of the National Academy of Sciences, 2002, vol. 99 (12), pp. 7968-7973.
  • Yu X., et al. , “Ligand-independent Dimer Formation of Epidermal Growth Factor Receptor (EGFR) is a Step Separable from Ligand-induced EGFR Signaling,” Molecular Biology of the Cell, 2002, vol. 13 (7), pp. 2547-2557.
  • Zalutsky M.R., et al., “Growth Factor Receptors as Molecular Targets for Cancer Diagnosis and Therapy,” Journal of Nuclear Medicine, 1997, vol. 41 (2), pp. 71-77.
  • Zarcone R., et al., “Epidermal Growth Factor Receptor Expression: is it the Same in Normal and Malignant Endometria”, Clinical & Experimental Obstetrics & Gynecology, 1995, vol. 22 (4), pp. 298-300.
  • Zhang H.,et al., “Therapeutic Monoclonal Antibodies for the ERBB Family of Receptor Tyrosine Kinases,” Cancer Biology & Therapy, 2003, vol. 2 (4 Suppl. 1), pp. S122-S126.
  • Zhang W., et al., “Novel Approaches to Treatment of Advanced Colorectal Cancer with Anti-EGFR Monoclonal Antibodies,” Annals of Medicine, 2006, vol. 38 (8), pp. 545-551.
  • Zhang X., et al., “An Allosteric Mechanism for Activation of the Kinase Domain of Epidermal Growth Factor Receptor,” Cell, 2006, vol. 125 (6), pp. 1137-1149.
  • Zhen Y., et al. , “Characterization of Glycosylation Sites of the Epidermal Growth Factor Receptor,” Biochemistry, 2003, vol. 42 (18), pp. 5478-5492.
  • Zhu, et al., “Inhibition of tumor growth and metastasis by targeting tumor-associated angiogenesis with antagonists to the receptors of vascular endothelial growth factor,” Investigational New Drugs, 1999, vol. 17, pp. 195-212.
  • Zhu H.J., et al., “Epidermal Growth Factor Receptor: Association of Extracellular Domain Negatively Regulates Intracellular Kinase Activation in the Absence of Ligand,” Growth Factors, 2003, vol. 21 (1), pp. 15-30.
  • Zhu X.F., et al., “Egfr Tyrosine Kinase Inhibitor Ag1478 Inhibits Cell Proliferation and Arrests Cell Cycle in Nasopharyngeal Carcinoma Cells,” Cancer Letters, 2001, vol. 169 (1), pp. 27-32.
  • Zinner, et al., “A Phase 1 Clinical and Biomarker Study of the Novel Pan-ERBB Tyrosine Kinase Inhibitor, CI-1033, in Patients with Solid Tumors,” Clinical Cancer Research, 2001, vol. 3767s (Abstract 566), pp. 7.
  • U.S. Appl. No. 14/226,430, filed Mar. 26, 2014, Scott et al.
  • Decker M.W., et al., ABT-089 [2-Methyl-3-(2-(S)-Pyrrolidinylmethoxy) Pyridine Dihydrochloride]: II. A Novel Cholinergic Channel Modulator with Effects on Cognitive Performance in Rats and Monkeys. The Journal of Pharmacology and Experimental Therapeutics, 1997, 283:247-258.
  • Ounissi H., et al., Gene Homogeneity for Aminoglycoside-Modifying Enzymes in Gram-Positive Cocc, Antimicrobial Agents and Chemotherapy, 1990, 34(11):2164-2168.
  • Sakahara H., et al., Effect of DTPA conjugation on the antigen binding activity and biodistribution of monoclonal antibodies against alpha-fetoprotein, J Nucl Med, 1985, 26(7):750-755.
  • Sugimura, K., <Japanese Review Article> Human Antibody Engineering, Bio-ventures, (2002) vol. 2(4), p. 30-33.
  • Hermanson GT., et al., BioConjugate Techniques, Second Edition, 1996: p. 456.
Patent History
Patent number: 9260524
Type: Grant
Filed: Dec 11, 2012
Date of Patent: Feb 16, 2016
Patent Publication Number: 20140255410
Assignee: Ludwig Institute for Cancer Research (New York, NY)
Inventors: Lloyd J. Old (New York, NY), Gerd Ritter (New York, NY), Achim Jungbluth (New York, NY), Elisabeth Stockert (Vienna), Webster K. Cavenee (Del Mar, CA)
Primary Examiner: Stephen Rawlings
Application Number: 13/711,368
Classifications
Current U.S. Class: Immunoglobulin, Antibody, Or Fragment Thereof, Other Than Immunoglobulin Antibody, Or Fragment Thereof That Is Conjugated Or Adsorbed (530/387.1)
International Classification: C07K 16/30 (20060101); C12N 5/20 (20060101); C07K 16/28 (20060101); A61K 39/395 (20060101); A61K 45/06 (20060101); A61K 39/00 (20060101);