Specific binding proteins and their uses

Abstract

We provide antibodies and drugs that demonstrate efficacy against a wide range of tumors. [Solution] The present invention relates to specific binding members, particularly antibodies and fragments thereof, that bind to amplified epidermal growth factor receptor (EGFR) and to the de2-7 EGFR truncation of said EGFR. In particular, the epitopes recognized by the specific binding members, particularly antibodies and fragments thereof, are enhanced or clearly discernible based on abnormal post-translational modifications. These specific binding members are useful in the diagnosis and treatment of cancer. The binding members of the present invention can also be used in combination with chemotherapeutic agents or anticancer agents and / or other antibodies or fragments thereof during treatment.

Description

[Technical Field]

[0001] Related application data This international PCT patent application claims priority to U.S. Patent Application No. 12 / 388,504, filed on 18 February 2009, and the disclosure of said U.S. Patent Application is incorporated herein by reference in its entirety. This international PCT application is related to the following: U.S. Patent Application No. 10 / 145,598 filed on May 13, 2002 (U.S. Patent No. 7,589,180 published on September 15, 2009); U.S. Provisional Patent Application No. 60 / 290,410 filed on May 11, 2001; U.S. Provisional Patent Application No. 60 / 326,019 filed on September 28, 2001; U.S. Provisional Patent Application No. 60 / 342,258 filed on December 21, 2001; and International PCT Patent Application No. PCT / US02 / 15185 filed on May 13, 2002 (WO published on November 21, 2002). The disclosures of the International PCT Patent Application No. PCT / US2008 / 009771 (published on February 19, 2009 as WO 2009 / 023265), filed on 14 August 2008 (published as WO 2009 / 023265), and the U.S. Provisional Patent Application No. 60 / 964,715, filed on 14 August 2007, are also incorporated by reference to this whole.

[0002] Field of Invention The present invention relates to specific binding members, particularly antibodies and fragments thereof, that bind to amplified epidermal growth factor receptor (EGFR), and to in-frame deletions of exons 2-7 of EGFR that result in a truncated EGFR receptor (de2-7EGFR) lacking 267 amino acids from the extracellular domain. In particular, the epitopes recognized by the specific binding members, particularly antibodies and fragments thereof, are enhanced or clearly discernible based on abnormal post-translational modifications. These specific binding members are useful in the diagnosis and treatment of cancer. The binding members of the present invention can also be used in combination with chemotherapeutic agents or anticancer agents and / or other antibodies or fragments thereof during treatment. [Background technology]

[0003] Background of related technologies The treatment of proliferative disorders, particularly cancer, with chemotherapy often relies on exploiting the differences between target proliferative cells and other normal cells in the human and animal body. For example, many chemicals are designed to be taken up by rapidly replicating DNA, disrupting the DNA replication and cell division processes. Another approach is to identify antigens on the surface of tumor cells or other abnormal cells that 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, that can block or neutralize them. In addition, binding proteins, including antibodies and their fragments, can deliver toxins or other substances that can directly or indirectly activate these toxins at the tumor site.

[0004] EGFR is overexpressed in many types of epithelial tumors, making it an attractive target for tumor-targeted antibody therapy (Voldborg et al. (1997). Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials. Ann Oncol. 8, 1197-206; den Eynde, B. and Scott, AMTumor Antigens. In: PJ Delves and IMRoitt (eds.), Encyclopedia of Immunology, Second Edition, pp. 2424-31. London: Academic Press (1998)). Furthermore, EGFR expression has been associated with poor prognosis in numerous tumor types, including those of the stomach, colon, bladder, breast, prostate, endometrium, kidney, and brain (e.g., glioma). As a result, numerous EGFR antibodies have been reported in the literature, and some have undergone clinical evaluation (Baselga et al. (2000) Phase I Studies of Anti-Epidermal Growth Factor Receptor Chimeric Antibody C225 Alone and in Combination With Cisplatin. J. Clin. Oncol. 18, 904; Faillot et al. (1996): A phase I study of an anti-epidermal growth factor receptor monoclonal antibody for the treatment of malignant gliomas. Neurosurgery. 39, 478-83; Seymour, L. (1999) Novel anti-cancer agents in development: exciting prospects and new challenges. Cancer Treat. Rev. 25, 301-12).

[0005] The results from studies using EGFR mAbs in patients with head and neck cancer, squamous cell lung cancer, glioma, and malignant astrocytoma have been promising. The antitumor activity of most EGFR antibodies is enhanced by their ability to block ligand binding (Sturgis et al. (1994) 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; Goldstein et al. (1995) Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in a human tumor xenograft model. Clin. Cancer Res. 1, 1311-8). Such antibodies can mediate these effects by regulating both cell proliferation function and antibody-dependent immune function (e.g., complement activation). However, the use of these antibodies may be limited by uptake in organs with high endogenous EGFR levels, such as the liver and skin (Baselga et al., 2000; Faillot et al., 1996).

[0006] A significant proportion of tumors with EGFR gene amplification (i.e., multiple copies of the EGFR gene) also co-express truncated versions of the receptor known as de2-7EGFR, ΔEGFR, or Δ2-7 (these terms are used interchangeably herein) (Wikstrand et al. (1998) The class III variant of the epidermal growth factor receptor (EGFR): characterization and utilization as an immunotherapeutic target. J. Neurovirol. 4, 148-158) (Olapade-Olaopa et al. (2000) Evidence for the differential expression of a variant EGF receptor protein in human prostate cancer. Br. J. Cancer. 82, 186-94).The rearrangement observed in de2-7 EGFR results in in-frame mature mRNA lacking 801 nucleotides across exons 2-7 (Wong et al. (1992) Structural alterations of the epidermal growth factor receptor gene in human gliomas. Proc. Natl. Acad. Sci. USA 89, 2965-9; Yamazaki et al. (1990) 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; Yamazaki et al. (1988) Amplification of the structurally and functionally altered epidermal growth factor receptor gene (c-erbB) in human brain tumors. Mol. Cell Biol. 8, 1816-20; Sugawa et al. (1990) Identical splicing of aberrant epidermal growth factor receptor transcripts from Amplified rearranged genes in human glioblastomas. Proc. Natl. Acad. Sci. USA 87, 8602-6). The corresponding EGFR protein has a deletion of 267 amino acids, including residues 6-273 in the extracellular domain, and a novel glycine residue at the fusion junction (Sugawa et al., 1990). This deletion, along with the insertion of the glycine residue, generates a junction peptide specific to this deletion interface (Sugawa et al., 1990).

[0007] de2-7 EGFR has been reported in numerous tumor types, including gliomas, breast, lung, ovarian, and prostate cancers (Wikstrand et al. (1997) Cell surface localization and density of the tumor-associated variant of the epidermal growth factor receptor, EGFRvIII. Cancer Res. 57, 4130-40; Olapade-Olaopa et al. (2000) Evidence for the differential expression of a variant EGF receptor protein in human prostate cancer. Br.J. Cancer. 82, 186-94; Wikstrand, et al. (1995) Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas. Cancer Res. 55, 3140-8; Garcia de Palazzo et al. (1993) Expression of mutated epidermal growth factor receptor by non-small cell lung carcinomas. Cancer Res. 53, 3217-20).This truncated receptor does not bind to the ligand but has low constitutive activity and provides a significant growth advantage to glioma cells grown as tumor xenografts in nude mice (Nishikawa et al. (1994) A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc. Natl. Acad. Sci. USA 91, 7727-31), and can also transform NIH3T3 cells (Batra et al. (1995) Epidermal growth factor ligand independent, unregulated, cell-transforming potential of a naturally occurring human mutant EGFRvIII gene. Cell Growth Differ. 6, 1251-9) and MCF-7 cells. The cellular mechanisms by which de2-7 EGFR is used in glioma cells are not fully understood, but they have been reported to include a reduction in apoptosis (Nagane et al. (1996) 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) and a small increase in proliferation (Nagane et al., 1996).

[0008] Since the expression of this truncated receptor is limited to tumor cells, it becomes a highly specific target for antibody therapy. Therefore, numerous laboratories have developed polyclonal (Humphrey et al. (1990) 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) and monoclonal (Wikstrand et al. (1995) Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas; Okamoto et al. (1996) Monoclonal antibody against the fusion junction of a deletion-mutant epidermal growth factor receptor. Br. J. Cancer. 73, 1366-72; Hills et al. (1995) Specific targeting of a mutant, activated EGF receptor found in glioblastoma using a monoclonal (Antibody. Int. J. Cancer. 63, 537-43) Reports the production of both antibodies.A series of mouse mAbs isolated after immunization with this specific de2-7 peptide all exhibited selectivity and specificity for the truncated receptor and targeted de2-7 EGFR-positive xenografts grown in nude mice (Wikstrand et al. (1995); Reist et al. (1997) 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; Reist et al. (1995) 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).

[0009] However, one potential drawback of de2-7 EGFR antibodies is that only a subset of tumors exhibiting amplification of the EGFR gene also express de2-7 EGFR (Ekstrand et al. (1992) 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). The exact percentage of tumors containing de2-7 EGFR has not been fully established, as the use of different techniques (i.e., PCR vs. immunohistochemistry) and various antibodies has resulted in a wide range of reported values ​​regarding the frequency of this presence. Published data show that approximately 25-30% of gliomas express de2-7 EGFR, with the lowest expression in anaplastic astrocytoma and the highest in glioblastoma multiforme (Wong et al. (1992); Wikstrand et al. (1998) The class III variant of the epidermal growth factor receptor (EGFR): characterization and utilization as an immunotherapeutic target. J. Neurovirol. 4, 148-58; Moscatello et al. (1995) Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors. Cancer Res. 55, 5536-9). The percentage of positive cells in de2-7 EGFR-expressing gliomas has been reported to range from 37-86% (Wikstrand et al. (1997)).It has been found that 27% of breast cancers and 17% of lung cancers are positive for de2-7 EGFR (Wikstrand et al. (1997); Wikstrand et al. (1995); Wikstrand et al. (1998); Hills et al., 1995). Therefore, it is expected that de2-7 EGFR-specific antibodies will be useful only for a certain proportion of EGFR-positive tumors. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Voldborg et al. (1997). Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials. Ann Oncol. 8, 1197-206 [Non-Patent Document 2] den Eynde, B. and Scott, AMTumor Antigens. In: PJ Delves and IMRoitt (eds.), Encyclopedia of Immunology, Second Edition, pp. 2424-31. London: Academic Press (1998) [Non-Patent Document 3] Baselga et al. (2000) Phase I Studies of Anti-Epidermal Growth Factor Receptor Chimeric Antibody C225 Alone and in Combination With Cisplatin.J.Clin.Oncol.18,904 [Non-Patent Document 4] Faillot et al. (1996): A phase I study of an anti-epidermal growth factor receptor monoclonal antibody for the treatment of malignant gliomas. Neurosurgery. 39, 478-83 [Non-Patent Document 5] Seymour, L. (1999) Novel anti-cancer agents in development: exciting prospects and new challenges. Cancer Treat. Rev. 25, 301-12) [Non-Patent Document 6] Sturgis et al. (1994) 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 [Non-Patent Document 7] Goldstein et al. (1995) Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in a human tumor xenograft model. Clin. Cancer Res. 1, 1311-8 [Non-Patent Document 8] Wikstrand et al. (1998) The class III variant of the epidermal growth factor receptor (EGFR): characterization and utilization as an immunotherapeutic target. J. Neurovirol. 4, 148-158 [Non-Patent Document 9] Olapade-Olaopa et al.(2000)Evidence for the differential expression of a variant EGF receptor protein in human prostate cancer.Br.J.Cancer.82,186-94 [Non-Patent Document 10] Wong et al.(1992)Structural alterations of the epidermal growth factor receptor gene in human gliomas.Proc.Natl.Acad.Sci.U.S.A.89,2965-9 [Non-Patent Document 11] Yamazaki et al.(1990)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 [Non-Patent Document 12] Yamazaki et al.(1988)Amplification of the structurally and functionally altered epidermal growth factor receptor gene(c-erbB)in human brain tumors.Mol.Cell Biol.8,1816-20 [Non-Patent Document 13] Sugawa et al.(1990)Identical splicing of aberrant epidermal growth factor receptor transcripts from amplified rearranged genes in human glioblastomas.Proc.Natl.Acad.Sci.U.S.A.87,8602-6 [Non-Patent Document 14] Wikstrand et al.(1997)Cell surface localization and density of the tumor-associated variant of the epidermal growth factor receptor,EGFRvIII.Cancer Res.57,4130-40 [Non-Patent Document 15] Olapade-Olaopa et al.(2000)Evidence for the differential expression of a variant EGF receptor protein in human prostate cancer.Br.J.Cancer.82,186-94 [Non-Patent Document 16] Wikstrand,et al.(1995)Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas.Cancer Res.55,3140-8 [Non-Patent Document 17] Garcia de Palazzo et al.(1993)Expression of mutated epidermal growth factor receptor by non-small cell lung carcinomas.Cancer Res.53,3217-20 [Non-Patent Document 18] Nishikawa et al.(1994)A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity.Proc.Natl.Acad.Sci.U.S.A.91,7727-31 [Non-Patent Document 19] Batra et al. (1995) Epidermal growth factor ligand independent, unregulated, cell-transforming potential of a naturally occurring human mutant EGFRvIII gene. Cell Growth Differ. 6, 1251-9 [Non-Patent Document 20] Nagane et al. (1996) 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 [Non-Patent Document 21] Humphrey et al. (1990) Anti-synthetic peptide antibody reacting at the fusion junction of deletion mutant epidermal growth factor receptors in human glioblastoma. Proc. Natl. Acad. Sci. U.S.A. 87, 4207-11 [Non-Patent Document 22] Wikstrand et al. (1995) Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas [Non-Patent Document 23] Okamoto et al.(1996)Monoclonal antibody against the fusion junction of a deletion-mutant epidermal growth factor receptor.Br.J.Cancer.73,1366-72 [Non-Patent Document 24] Hills et al.(1995)Specific targeting of a mutant, activated EGF receptor found in glioblastoma using a monoclonal antibody.Int.J.Cancer.63,537-43 [Non-Patent Document 25] Reist et al.(1997)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 [Non-Patent Document 26] Reist et al.(1995)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 [Non-Patent Document 27] Ekstrand et al. (1992) 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.USA89,4309-13 [Non-Patent Document 28] Moscatello et al.(1995)Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors.Cancer Res.55,5536-9 [Overview of the project] [Problems that the invention aims to solve]

[0011] Thus, while the existing evidence for the activity of EGFR antibodies is promising, limitations on their applicability and range of efficacy remain, reflecting the above. Therefore, the development of antibodies and similar agents that demonstrate efficacy against a wide range of tumors would be desirable, and this invention relates to achieving this goal.

[0012] The references cited herein should not be construed as an acknowledgment that they constitute prior art to the present invention. [Means for solving the problem]

[0013] Summary of the Invention The present invention provides isolated specific binding members, particularly antibodies or fragments thereof, that do not explicitly involve any amino acid sequence modifications or substitutions from wild-type EGFR, and that are found in tumorigenic, hyperproliferative, or abnormal cells but are generally undetectable in normal or wild-type cells (as used herein, the term “wild-type cells” refers to cells that express endogenous EGFR but not de2-7 EGFR, and this term excludes cells that overexpress the EGFR gene in particular; the term “wild-type” refers to a genotype, phenotype, or other characteristic present in normal cells but not in abnormal or tumorigenic cells). In a further embodiment, the present invention provides specific binding members, particularly antibodies or fragments thereof, that recognize EGFR epitopes found in tumorigenic, hyperproliferative, or abnormal cells but are generally undetectable in normal or wild-type cells, and that are enhanced or clearly discernible based on abnormal post-translational modifications or abnormal expression. In certain non-limiting examples provided herein, EGFR epitopes with incomplete or insufficient post-translational modifications, or those not as readily apparent as those seen in normal EGFR expression in wild-type cells, are enhanced or clearly defined. In one embodiment, EGFR epitopes are enhanced or clearly defined based on early or simple carbohydrate modifications or early glycosylation, particularly high mannose modifications, and reduced or unclear in the presence of complex carbohydrate modifications.

[0014] The specific binding members, which may be antibodies or fragments thereof, such as these immunogenic fragments, substantially do not bind to, and do not recognize, normal or wild-type cells containing normal or wild-type EGFR epitopes in the absence of abnormal expression and in the presence of normal EGFR post-translational modifications.

[0015] More specifically, the specific binding member of the present invention may be an antibody or fragment thereof that recognizes an EGFR epitope present in cells that overexpress EGFR (e.g., the EGFR gene is amplified) or de2-7 EGFR, particularly in the presence of abnormal post-translational modifications, which is generally undetectable in cells expressing EGFR under normal conditions, particularly in the presence of normal post-translational modifications.

[0016] The inventors have disclosed novel monoclonal antibodies, exemplified herein by antibodies referred to as mAb806, ch806, hu806, mAbl75, mAbl24, and mAb1133, that specifically recognize abnormally expressed EGFR. In particular, the antibodies of the present invention recognize EGFR epitopes that are found in tumorigenic, hyperproliferative, or abnormal cells but are not generally detectable in normal or wild-type cells and are enhanced or clearly discernible based on abnormal post-translational modifications. The novel antibodies of the present invention also recognize amplified wild-type EGFR and de2-7 EGFR, but bind to epitopes different from the specific junction peptide of this de2-7 EGFR mutation. The antibodies of the present invention specifically recognize abnormally expressed EGFR, including amplified EGFR and mutant EGFR (exemplified herein by the de2-7 mutation), particularly based on abnormal post-translational modifications. In addition, these antibodies do not recognize EGFR when expressed on the cell surface of glioma cell lines expressing normal amounts of EGFR, but they bind to the extracellular domain of EGFR (sEGFR) immobilized on the surface of the ELISA plate. This indicates recognition of a conformational epitope. These antibodies bind to the surface of A431 cells that have EGFR gene amplification but do not express de2-7 EGFR. Importantly, these antibodies did not significantly bind to normal tissues such as liver and skin, which express higher levels of endogenous wild-type (wt) EGFR than most other normal tissues but do not abnormally express or amplify EGFR.

[0017] The antibodies of the present invention can specifically classify the properties of EGFR tumors or tumorigenic cells by staining or otherwise recognizing tumors or cells that contain EGFR amplification and / or EGFR mutations, particularly abnormal EGFR expression including de2-7 EGFR. Furthermore, the antibodies of the present invention demonstrate significant in vivo antitumor activity against tumors containing amplified EGFR and against de2-7 EGFR-positive xenografts.

[0018] The unique specificity of these antibodies, which bind to de2-7 EGFR and amplified EGFR but not to normal, wild-type EGFR, leads to diagnostic and therapeutic applications that identify, characterize, and target numerous tumor types, such as head and neck, breast, or prostate tumors and gliomas, without the problems associated with normal tissue uptake sometimes seen with previously known EGFR antibodies.

[0019] Accordingly, the present invention provides a specific binding protein, such as an antibody, that binds to de2-7 EGFR at an epitope different from the junction peptide, but substantially does not bind to EGFR in normal cells in the absence of amplification of the EGFR gene. Amplification shall include the presence of multiple copies of EGFR in the cell.

[0020] Preferably, the epitope recognized by the antibody of the present invention is located within a region containing residues 273-501 of the mature normal or wild-type EGFR sequence, and preferably contains residues 287-302 (SEQ ID NO: 14) of the mature normal or wild-type EGFR sequence. Accordingly, we also provide a specific binding protein, such as an antibody, that binds to de2-7 EGFR at an epitope located within a region containing residues 273-501 and / or 287-302 (SEQ ID NO: 14) of the EGFR sequence. The epitope can be determined by any conventional epitope mapping technique known to those skilled in the art. Alternatively, the DNA sequence encoding residues 273-501 and / or 287-302 (SEQ ID NO: 14) can be digested, and the resulting fragment can be expressed in a suitable host. Antibody binding can be determined as described above.

[0021] In a preferred embodiment, the antibody has the characteristics of an antibody identified and characterized by the inventors, in particular, the ability to recognize abnormally expressed EGFR, such as that found in amplified EGFR and de2-7 EGFR.

[0022] In another embodiment, the present invention provides antibodies that can compete with the antibodies of the present invention, provided that at least 10% of the antibodies having the VH and VL chain sequences of the antibodies of the present invention are blocked from binding to de2-7 EGFR by competition with such antibodies in an ELISA assay. In particular, anti-idiotype antibodies are possible and are exemplified herein. Anti-idiotype antibodies LMH-11, LMH-12, and LMH-13 are provided herein.

[0023] The binding of the antibody to this target antigen is mediated by the complementarity-determining regions (CDRs) of the heavy and light chains, with the role of CDR3 being particularly important. Therefore, specific binding members based on the CDR3 region of the heavy or light chain, preferably both, of the antibody of the present invention would be useful specific binding members for in vivo therapy.

[0024] Therefore, the identified CDRs of the present invention, particularly antibodies based on the CDR3 region, and other specific binding proteins, would be useful for targeting tumors with amplified EGFR regardless of their de2-7 EGFR status. Since the antibodies of the present invention do not significantly bind to normal, wild-type receptors, they would not be subject to the limitations of currently developed EGFR antibodies in terms of significant uptake in normal cells.

[0025] In another embodiment, the present invention provides an isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain multiple copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, wherein the antibody does not bind to the de2-7 junction peptide consisting of the amino acid sequence of SEQ ID NO: 13; binds to an epitope in the sequence of residues 287-302 of human wild-type EGFR (SEQ ID NO: 14); and does not contain a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 2, nor does it contain a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4.

[0026] In another embodiment, the present invention provides an isolated antibody comprising a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence shown in SEQ ID NO: 42 and the light chain has the amino acid sequence shown in SEQ ID NO: 47.

[0027] In another embodiment, the present invention provides an isolated antibody comprising a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence shown in SEQ ID NO: 129 and the light chain has the amino acid sequence shown in SEQ ID NO: 134.

[0028] In another embodiment, the present invention provides an isolated antibody comprising a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence shown in SEQ ID NO: 22 and the light chain has the amino acid sequence shown in SEQ ID NO: 27.

[0029] In another embodiment, the present invention provides an isolated antibody comprising a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence shown in SEQ ID NO: 32 and the light chain has the amino acid sequence shown in SEQ ID NO: 37.

[0030] In another embodiment, the present invention provides an isolated antibody comprising a heavy chain and a light chain, wherein the variable region of the heavy chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequences shown in SEQ ID NOs: 44, 45, and 45.

[0031] In another embodiment, the present invention provides an isolated antibody comprising a heavy chain and a light chain, wherein the variable region of the light chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequences shown in SEQ ID NOs: 49, 50, and 51.

[0032] In another embodiment, the present invention provides an isolated antibody comprising a heavy chain and a light chain, wherein the variable region of the heavy chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequences shown in SEQ ID NOs: 130, 131, and 132.

[0033] In another embodiment, the present invention provides an isolated antibody comprising a heavy chain and a light chain, wherein the variable region of the light chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequences shown in SEQ ID NOs: 135, 136, and 137.

[0034] In another embodiment, the present invention provides an isolated antibody comprising a heavy chain and a light chain, wherein the variable region of the heavy chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequences shown in SEQ ID NOs: 23, 24, and 25.

[0035] In another embodiment, the present invention provides an isolated antibody comprising a heavy chain and a light chain, wherein the variable region of the light chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequences shown in SEQ ID NOs: 28, 29, and 30.

[0036] In another embodiment, the present invention provides an isolated antibody comprising a heavy chain and a light chain, wherein the variable region of the heavy chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequences shown in SEQ ID NOs: 33, 34, and 35.

[0037] In another embodiment, the present invention provides an isolated antibody comprising a heavy chain and a light chain, wherein the variable region of the light chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequences shown in SEQ ID NOs: 38, 39, and 40.

[0038] In another embodiment, the present invention provides an isolated antibody that is in the form of antibody F(ab')2, scFv fragment, diabody, triabody, or tetrabody.

[0039] In another embodiment, isolated antibodies further comprising detectable or functional labels are provided.

[0040] In another embodiment, the detectable or functional label is a drug that is covalently attached.

[0041] In another embodiment, the marker is a radial marker.

[0042] In another aspect, the present invention provides an isolated antibody that has been PEGylated.

[0043] In another embodiment, an isolated nucleic acid is provided, comprising a sequence encoding an isolated antibody as detailed herein.

[0044] In another embodiment, a method is provided for preparing an isolated antibody, which includes expressing a nucleic acid as detailed above and herein under conditions that induce the expression of the antibody, and recovering the antibody.

[0045] In another embodiment, a method for treating a tumor in a human patient is provided, the method comprising administering to the patient an effective amount of an isolated antibody as detailed herein.

[0046] In another embodiment, a kit is provided for the diagnosis of tumors in which EGFR is abnormally expressed or expressed in a truncated protein form, comprising an isolated antibody as detailed herein.

[0047] In another embodiment, the kit further includes reagents and / or instructions for use.

[0048] In another embodiment, a pharmaceutical composition comprising an isolated antibody as detailed herein is provided.

[0049] In another embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable vehicle, carrier, or diluent.

[0050] In another embodiment, the pharmaceutical composition further comprises an anticancer agent selected from the group consisting of chemotherapeutic agents, anti-EGFR antibodies, radioimmunotherapy agents, and combinations thereof.

[0051] In another embodiment, the chemotherapeutic agent is selected from the group consisting of tyrosine kinase inhibitors, phosphorylation cascade inhibitors, posttranslational regulators, cell growth or division inhibitors (e.g., antimitotic agents), signaling inhibitors, and combinations thereof.

[0052] In another embodiment, the tyrosine kinase inhibitor is selected from the group consisting of AG1478, ZD1839, STI571, OSI-774, SU-6668, and combinations thereof.

[0053] In another embodiment, the anti-EGFR antibody is selected from the group consisting of anti-EGFR antibodies 528, 225, SC-03, DR8.3, L8A4, Y10, ICR62, ABX-EGF, and combinations thereof.

[0054] In another embodiment, a method is provided for preventing and / or treating cancer in mammals, the method comprising administering a therapeutically effective amount of a pharmaceutical composition as detailed herein to a mammal.

[0055] In another embodiment, a method is provided for treating endogenous brain cancers that produce abnormally expressed EGFR in mammals, the method comprising administering a therapeutically effective amount of a pharmaceutical composition as detailed herein to a mammal.

[0056] In another embodiment, the endogenous brain cancer is selected from the group consisting of glioblastoma, medulloblastoma, meningioma, neoplastic astrocytoma, and neoplastic arteriovenous malformation.

[0057] In another embodiment, a single-celled host transformed with a recombinant DNA molecule encoding an isolated antibody, as detailed herein, is provided.

[0058] In another embodiment, the single-celled host is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NSO, SP2 / 0, R1.1, BW, LM, COS 1, COS 7, BSC1, BSC40, and BMT10 cells, plant cells, insect cells, and human cells (in tissue culture).

[0059] In another embodiment, a method is provided for detecting the presence of amplified EGFR, de2-7EGFR, or EGFR having high mannose glycosylation, the method comprising (a) contacting a biological sample from a mammal suspected to contain amplified EGFR, de2-7EGFR, or EGFR having high mannose glycosylation with the isolated antibody described in claim 1 under conditions that allow the EGFR to bind to the isolated antibody; and (b) measuring the EGFR by detecting whether binding has occurred between the EGFR from the sample and the isolated antibody, the detection of binding indicating the presence or activity of the EGFR in the sample.

[0060] In another embodiment of a method for detecting the presence of amplified EGFR, de2-7EGFR, or EGFR having high mannose glycosylation, the detection of the presence of said EGFR indicates the presence of a tumor or cancer in the mammal.

[0061] In another embodiment, the present invention provides an isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain multiple copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, wherein the antibody comprises a heavy chain and a light chain, the heavy chain having an amino acid sequence substantially homologous to the amino acid sequence shown in SEQ ID NO: 42, and the light chain having an amino acid sequence substantially homologous to the amino acid sequence shown in SEQ ID NO: 47.

[0062] In another embodiment, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 42, and in this case, the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 47.

[0063] In another embodiment, the present invention provides an isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain multiple copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, comprising a heavy chain and a light chain, wherein the variable region of the heavy chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NOs: 44, 45, and 46, and the variable region of the light chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NOs: 49, 50, and 51.

[0064] In another embodiment, the present invention provides an isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain numerous copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, comprising a heavy chain and a light chain, wherein the heavy chain has an amino acid sequence substantially homologous to the amino acid sequence shown in SEQ ID NO: 129, and the light chain has an amino acid sequence substantially homologous to the amino acid sequence shown in SEQ ID NO: 134.

[0065] In another embodiment, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 129, and in this case, the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 134.

[0066] In another embodiment, the present invention provides an isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain multiple copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, comprising a heavy chain and a light chain, wherein the variable region of the heavy chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NOs: 130, 131, and 132, and the variable region of the light chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NOs: 135, 136, and 137.

[0067] In another embodiment, the present invention provides an isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain multiple copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, comprising a heavy chain and a light chain, wherein the heavy chain has an amino acid sequence substantially homologous to the amino acid sequence shown in SEQ ID NO: 22, and the light chain has an amino acid sequence substantially homologous to the amino acid sequence shown in SEQ ID NO: 27.

[0068] In another embodiment, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 22, and in this case, the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 27.

[0069] In another embodiment, the present invention provides an isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain multiple copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, comprising a heavy chain and a light chain, wherein the variable region of the heavy chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequences shown in SEQ ID NOs: 23, 24, and 25, and the variable region of the light chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequences shown in SEQ ID NOs: 28, 29, and 30.

[0070] In another embodiment, the present invention provides an isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain numerous copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, comprising a heavy chain and a light chain, wherein the heavy chain has an amino acid sequence substantially homologous to the amino acid sequence shown in SEQ ID NO: 32, and the light chain has an amino acid sequence substantially homologous to the amino acid sequence shown in SEQ ID NO: 37.

[0071] In another embodiment, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 32, and in this case, the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 37.

[0072] In another embodiment, the present invention provides an isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain multiple copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, comprising a heavy chain and a light chain, wherein the variable region of the heavy chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NOs: 33, 34, and 35, and the variable region of the light chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NOs: 38, 39, and 40.

[0073] In another embodiment, an isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain multiple copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, It does not bind to the de2-7EGFR junction peptide, which consists of the amino acid sequence number 13; It binds to an epitope within the sequence of residues 287-302 of human wild-type EGFR; Including a light chain and a heavy chain, the variable region of the light chain is The amino acid sequence shown in formula I: HSSQDIXaa1SNIG (I) (In the formula, Xaa1 is an amino acid residue having a non-charged R group.) (Sequence ID: 151) A first polypeptide-binding domain region having the corresponding amino acid sequence, The amino acid sequence shown in formula II: HGTNLXaa2D (II) (In the formula, Xaa2 is an amino acid residue having a charged R group.) (Sequence ID: 152) A second polypeptide-binding domain region having the corresponding amino acid sequence, The amino acid sequence shown in formula III: VQYXaa3QFPWT (III) (In the formula, Xaa3 is selected from the group consisting of A, G, and amino acid residues in which A or G is conservatively substituted.) (Sequence ID: 153) It comprises a third polypeptide-binding domain region having an amino acid sequence corresponding to the heavy chain, and the variable region of the heavy chain is The amino acid sequence shown in formula VI: SDXaa4AWN (IV) (In the formula, Xaa4 is selected from the group consisting of F, Y, and amino acid residues in which F or Y is conservedly substituted.) (Sequence ID: 154) A first polypeptide-binding domain region having the corresponding amino acid sequence, The amino acid sequence shown in formula V, formula VI, or formula VII: YISYSGNTRYXaa5PSLKS (V) (In the formula, Xaa5 is an amino acid residue having a non-charged R group.) (Sequence ID: 155) YISYSXaa6NTRYNPSLKS (VI) (In the formula, Xaa6 is selected from the group consisting of G, A, and amino acid residues in which G or A is conservatively substituted.) (Sequence ID: 156) YISYSGNTRYNPSLXaa7S (VII) (In the formula, Xaa7 is a basic amino acid residue.) (Sequence ID: 157) A second polypeptide-binding region having the corresponding amino acid sequence, The amino acid sequence shown in formula VIII: Xaa8TAGRGFPY (VIII) (In the formula, Xaa8 is selected from the group consisting of V, A, and amino acid residues in which V or A is conservatively substituted.) (Sequence ID: 158) A third polypeptide-binding domain region having the corresponding amino acid sequence and It includes; and The present invention provides an antibody that does not contain a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 2, and does not contain a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 4.

[0074] In another aspect, X aa1 is N; X aa2 is D; X aa3 is A; X aa4 is F; X aa5 is an amino acid residue having an uncharged polar R group; X aa6 is G; X aa7 is K; and X aa8 is V.

[0075] In another aspect, X aa5 is N or Q.

[0076] In another aspect, X aa1 is N or S.

[0077] In another aspect, X aa2 is D or E.

[0078] In another aspect, X aa3 is A or G.

[0079] In another aspect, X aa4 is F or Y.

[0080] In another aspect, X aa5 is N or Q.

[0081] In another aspect, X aa6 is G or A, and X aa7 is independently K or R.

[0082] In another aspect, X aa8 is V or A.

[0083] In another embodiment, an isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain multiple copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, wherein it does not bind to the de2-7 junction peptide consisting of the amino acid sequence of SEQ ID NO: 13; it binds to an epitope in the sequence of residues 273-501 of human wild-type EGFR; it comprises a light chain and a heavy chain, wherein the variable region of the light chain comprises a first polypeptide-binding domain region having the amino acid sequence HSSQDINSNIG (SEQ ID NO: 18), a second polypeptide-binding domain region having the amino acid sequence HGTNLDD (SEQ ID NO: 19), and a third polypeptide-binding domain region having the amino acid sequence VQYAQFPWT (SEQ ID NO: 20). The variable region of the heavy chain comprises a first polypeptide-binding domain region having the amino acid sequence SDFAWN (SEQ ID NO: 15) and the amino acid sequence shown in formula IX: YISYSGNTRYX aa9 PSLKS (IX) (In the formula, X aa9 This is an amino acid residue having a non-charged R group. The present invention provides an antibody comprising a second polypeptide-binding domain region having the corresponding amino acid sequence and a third polypeptide-binding domain region having the amino acid sequence VTAGRGFPY (SEQ ID NO: 17).

[0084] In another embodiment, the antibody binds to an epitope within the sequence of residues 287-302 of human wild-type EGFR (SEQ ID NO: 14).

[0085] In another embodiment, X aa9 It is either N or Q.

[0086] In another embodiment, the binding domain region is held by a human antibody framework.

[0087] In another embodiment, the human antibody framework is a human IgG1 antibody framework.

[0088] In another embodiment, the present invention provides an isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain numerous copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, comprising a heavy chain and a light chain, wherein the heavy chain has an amino acid sequence substantially homologous to the amino acid sequence shown in SEQ ID NO: 2, and the light chain has an amino acid sequence substantially homologous to the amino acid sequence shown in SEQ ID NO: 4.

[0089] In another embodiment, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 2, and in this case, the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 4.

[0090] In another embodiment, the present invention provides an isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain multiple copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, comprising a heavy chain and a light chain, wherein the variable region of the heavy chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NOs: 15, 16, and 17, and the variable region of the light chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NOs: 18, 19, and 20.

[0091] Other purposes and advantages will become apparent to those skilled in the art from a closer examination of the subsequent “Detailed Description,” which is made with reference to the following illustrative drawings and the attached claims. [Brief explanation of the drawing]

[0092] [Figure 1]Figure 1 shows the results of flow cytometry analysis of glioma cell lines. U87MG (light gray histogram) and U87MG.Δ2-7 (dark gray histogram) cells were stained as shown with unrelated IgG2b antibody (white (open) histogram), DH8.3 (specific to de2-7 EGFR), or mAb806 or 528 (which binds both wild-type and de2-7 EGFR). [Figure 2-1] Figures 2A-D show the ELISA results for mAb806, mAbDH8.3, and mAb528. (A) Binding of mAb806 (▲), DH8.3 (●), or 528 (■) at increasing concentrations to sEGFR-coated ELISA plates. (B) Inhibition of binding of mAb806 and mAb528 to sEGFR-coated ELISA plates by soluble EGFR (sEGFR) at increasing concentrations in solution. (C) Binding of DH8.3 at increasing concentrations to the de2-7 junction peptide explains the binding curves (D) for mAb806 and mAb528 to immobilized wild-type sEGFR. [Figure 2-2] Figures 2E and 2F graphically present the results of BIAcore binding studies using a C-terminal biotinylated peptide and including the monoclonal antibody of the present invention together with other known antibodies (including the L8A4 antibody that recognizes the junction peptide of the de2-7 EGFR mutant) and controls. [Figure 3] Figure 3 illustrates the internalization of mAb806 and DH8.3 antibodies. U87MG.Δ2-7 cells were pre-incubated at 4°C with either mAb806 (▲) or DH8.3 (●), then transferred to 37°C, and internalization was determined by FACS. The data represent the mean internalization ± SE at each time point in three (DH8.3) or four (mAb806) independent experiments. [Figure 4] Figures 4A and 4B illustrate the in vivo distribution (%ID / g tumor tissue) of radiolabeled (a) 125I-mAb806 and (b) 131I-DH8.3 in nude mice carrying U87MG and U87MG.Δ2-7 xenografts. Each point represents the mean ± SE of 5 mice, excluding the 1-hour interval (n=4). [Figure 5] Figures 5A and 5B illustrate the in vivo distribution of radiolabeled 125I-mAb806 (white bars) and 131I-DH8.3 (black bars) antibodies in nude mice carrying U87MG.Δ2-7 xenografts, expressed as (a) tumor:blood or (b) tumor:liver ratios. Each bar represents the mean ± SE of 5 mice, excluding 1 hour (n=4). [Figure 6] Figures 6A-C illustrate flow cytometry analysis of cell lines with EGFR gene amplification. A431 cells were stained with either mAb806, DH8.3, or 528 (black histogram) and compared with unrelated IgG2b antibody (white histogram). [Figure 7] Figures 7A and 7B illustrate the in vivo distribution (%ID / g tumor tissue) of radiolabeled (a) 125I-mAb806 and (b) 131I-528 in nude mice carrying U87MG.Δ2-7 and A431 xenografts. [Figure 8] Figures 8A-D illustrate the in vivo distribution of radiolabeled 125I-mAb806 (white bars) and 131I-528 (black bars), as well as antibodies, in nude mice carrying (A,C) U87MG.Δ2-7 and (B,D) A431 xenografts, expressed as (A,B) tumor:blood or (C,D) tumor:liver ratios. [Figure 9] Figures 9A and 9B illustrate the antitumor effect of mAb806 on (A) U87MG and (B) U87MG.Δ2-7 xenograft growth rates in a prophylactic model. Four-to-six-week-old BALB / c nude mice (n=5) were sc-injected into both flanks on day 0 with 3 × 10⁶ U87MG or U87MG.Δ2-7 cells. One day prior to tumor cell inoculation, mice were intravenously injected with either 1 mg of mAb806 (●), 0.1 mg of mAb806 (▲), or a vehicle (○). Injections were administered three times a week for two weeks, as indicated by the arrows. Data are presented as mean tumor volume ± SE. [Figure 10]Figures 10A, 10B, and 10C illustrate the antitumor effects of mAb806 against (A) U87MG, (B) U87MG.Δ2-7, and (C) U87MG.wtEGFR xenografts in a stochastic model. Four-to-six-week-old BALB / c nude mice (n=5) were sc-injected with 3 × 10⁶ U87MG, U87MG.Δ2-7, or U87MG.wtEGFR cells into both flanks. When the tumor reached a mean tumor volume of 65–80 mm³, mice were ip-injected with either 1 mg of mAb806 (●), 0.1 mg of mAb806 (▲), or a vehicle (○). Injections were administered three times a week for two weeks, as indicated by the arrows. Data are presented as mean tumor volume ± SE. [Figure 11] Figures 11A and 11B illustrate the antitumor effect of mAb806 on A431 xenografts in (A) a prophylactic model and (B) a stochastic model. 4-6 week old BALB / c nude mice (n=5) were sc-injected with 3 × 10⁶ A431 cells into both flanks. In the prophylactic model, mice were intravenously injected with either 1 mg of mAb806 (●) or a vehicle (○) starting one day before tumor cell inoculation, or when the tumor reached a mean tumor volume of 200 mm³. Injections were administered three times a week for two weeks, as indicated by the arrows. Data are presented as mean tumor volume ± SE. [Figure 12] Figure 12 illustrates the antitumor effect of mAb806 treatment combined with AG1478 treatment for A431 xenografts in a prophylactic model. Data are shown as mean tumor volume ± SE. [Figure 13] Figure 13 depicts the binding of mAb806 to A431 cells in the presence of gradually increasing concentrations (0.5 μM and 5 μM) of AG1478. [Figure 14] Figures 14A and 14B illustrate (A) the nucleic acid sequence and (B) its amino acid translation (SEQ ID NO: 1 and SEQ ID NO: 2, respectively) of the 806VH chain gene. [Figure 15]Figures 15A and 15B illustrate (A) the nucleic acid sequence and (B) its amino acid translation (SEQ ID NO: 3 and SEQ ID NO: 4, respectively) of the 806VL chain gene. [Figure 16] Figure 16 shows the VH chain sequence (SEQ ID NO: 2) numbered according to Kabat, with CDRs (SEQ ID NOs: 15, 16, and 17) underlined. The key residues of the VH chain sequence (SEQ ID NO: 2) are 24, 37, 48, 67, and 78. [Figure 17] Figure 17 shows the VL chain sequence (SEQ ID NO: 4) numbered according to Kabat, with CDRs (SEQ ID NOs: 18, 19, and 20) underlined. The key residues of the VL chain sequence (SEQ ID NO: 4) are 36, 46, 57, and 71. [Figure 18] Figures 18A–18D show the results of in vivo studies designed to evaluate the therapeutic effects of combination antibody therapy, particularly mAb806 and 528 antibodies. Mice were inoculated with U87MG.D2-7 (A and B), U87MG.DK (C), or A431 (D) cells. [Figure 19] Figures 19A-D show the analysis of internalization by electron microscopy. U87MG.Δ2-7 cells were pre-conserved at 4°C with mAb806 or DH8.3, followed by gold-conjugated anti-mouse IgG, then transferred to 37°C, and internalization was examined at various time points by electron microscopy. (A) Localization of DH8.3 antibody to covering pits (arrows) at 5 minutes; (B) Internalization of mAb806 by macropinocytosis (arrows) at 2 minutes; (C) Localization of DH8.3 to lysosomes (arrows) at 20 minutes; (D) Localization of mAb806 to lysosomes (arrows) at 30 minutes. The original magnification for all images is 30,000x. [Figure 20] Figure 20 shows autoradiography of U87MG.Δ2-7 xenograft sections collected 8 hours after injection of 125I-mAb806. [Figure 21]Figure 21 shows flow cytometry analysis of cell lines with EGFR gene amplification. HM5 and MDA-468 cells were stained with unrelated IgG2b antibody (white histogram with dashed line), mAb806 (black histogram), or 528 (white histogram with closed line). DH8.3 antibody was completely negative for both cell lines (data not shown). [Figure 22] Figure 22 shows immunoprecipitation of EGFR from cell lines. EGFR was immunoprecipitated from 35S-labeled U87MG.Δ2-7 or A431 cells with mAb806, sc-03 antibody, or IgG2b isotype control. Side arrows indicate the locations of de2-7 and wtEGFR. Identical banding patterns were obtained in three independent experiments. [Figure 23] Figure 23 shows an autoradiography of an A431 xenograft section collected 24 hours after injection of 125I-mAb806, highlighting the localization area in living tissue (arrow). [Figure 24-1] Figures 24A and 24B show the extended survival of nude mice with intracranial U87MG.ΔEGFR (A) and LN-Z308.ΔEGFR (B) xenografts treated systemically with mAb806. U87MG.EGFR cells (1 × 10⁵) or LN-Z308.ΔEGFR cells (5 × 10⁵) were implanted in the brains of nude mice, and these animals were treated with either mAb806, PBS, or isotyped IgG from day 0 to day 14 post-implantation. Figure 24E shows the extended survival of nude mice with intracranial U87MG.ΔEGFR xenografts treated intratumorally with mAb806. U87MG.ΔEGFR was implanted as described. Starting on day 1, 10 mg of mAb806 or isotyped IgG control was injected into the tumor injection site in a volume of 5 μL every other day for 5 doses. [Figure 24-2]Figures 24C and 24D show the inhibition of intracranial tumor growth by mAb806 treatment. Nude mice (5 per group) treated with either mAb806 or an isotype IgG control were euthanized on day 9 for U87MG.EGFR(C) and on day 15 for LN-Z308.ΔEGFR(D). Their brains were collected, fixed, and dissected. Data were calculated by considering the control tumor volume as 100%. Values ​​are mean ± SD. ***, P<0.001; control vs. mAb806. Arrowhead, tumor tissue. [Figure 25] Figures 25A, 25B, and 25C show that mAb806 extends the survival of mice with U87MG.wtEGFR brain tumors, but not mice with U87MG.DK or U87MG brain tumors. U87MG(A), U87MG.DK(B), or U87MG.wtEGFR(C) cells (5 × 10⁵) were implanted in the brains of nude mice, treated with mAb806 from day 0 to day 14 post-implantation, and then observed after discontinuation of treatment. [Figure 26]Figure 26A shows the FACS analysis of the reactivity of mAb806 in the U87MG cell line. U87MG, U87MG.ΔEGFR, U87MG.DK, and U87MG.wtEGFR cells were stained with anti-EGFR mAb528, EGFR.1, and anti-ΔEGFR antibodies, and mAb806. The monoclonal EGFR.1 antibody exclusively recognized wtEGFR, while the monoclonal 528 antibody reacted with both wtEGFR and ΔEGFR. mAb806 reacted strongly with U87MG.ΔEGFR and U87MG.DK, and weakly with U87MG.wtEGFR. Bars on the x-axis represent maximum staining of cells in the absence of the primary antibody. The results were reproduced in three different experiments. Figure 26B shows the immunoprecipitation of mAb806 in EGFR morphology. Mutants and wtEGFR were immunoisolated from (lane 1) U87MG, (lane 2) U87Δ.EGFR, (lane 3) U87MG.DK, and (lane 4) U87MG.wtEGFR cells using anti-EGFR antibody 528, EGFR.1, or anti-ΔEGFR antibody mAb806, and then detected by Western blotting with anti-pan EGFR antibody C13. [Figure 27] Figures 27A and 27B show that systemic treatment with mAb806 reduces ΔEDFR phosphorylation and Bcl-XL expression in U87MG.ΔEGFR brain tumors. U87MG.ΔEGFR tumors were resected on day 9 of mAb806 treatment, immediately frozen in liquid nitrogen, and stored at -80°C until tumor lysate preparation. (A) Western blot analysis of ΔEGFR expression and autophosphorylation. 30 μg of tumor lysate was exposed to an SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, probed with an anti-phosphotyrosine mAb, then stripped and re-probed with anti-EGFR antibody, C13. (B) Western blotting for Bcl-XL using the same tumor lysate as in (A). The membrane was probed with anti-human Bcl-X polyclonal antibody. Lanes 1 and 2: U87MG.ΔEGFR brain tumors treated with isotype controls; Lanes 3 and 4: U87MG.ΔEGFR brain tumors treated with mAb806. [Figure 28] Figure 28 shows that mAb806 treatment results in decreased growth and vascularization, as well as increased apoptosis and macrophage accumulation in U87MG.ΔEGFR tumors. Tumor sections were stained for Ki-67. The cell proliferation index was assessed by the percentage of Ki-67-positive total cells from four randomly selected high-magnification fields (400x) of intracranial tumors from four mice in each group. Data are mean ± SE. Apoptotic cells were detected by the TUNEL assay. The apoptosis index was assessed by the ratio of TUNEL-positive cells to total cells from four randomly selected high-magnification fields (400x) of intracranial tumors from four mice in each group. Data are mean ± SE. Tumor sections were immunostained with anti-CD31 antibody. MVA was analyzed by computer image analysis from four randomly selected fields (200x) of intracranial tumors from four mice in each group. Macrophage peritumoral infiltration in mAb806-treated U87MG.ΔEGFR tumors. Tumor sections were stained with anti-F4 / 80 antibody. [Figure 29] Figure 29 shows flow cytometry analysis of parental U87MG glioma cell lines and transfected U87MG glioma cell lines. As shown, cells were stained with either unrelated IgG2b antibody (white histogram) or 528 antibody or mAb806 (black histogram). [Figure 30] Figure 30 shows immunoprecipitation of EGFR from cell lines. EGFR was immunoprecipitated from 35S-labeled U87MG.wtEGFR, U87MG.Δ2-7, and A431 cells with mAb806 (806), sc-03 antibody (c-term), or IgG2b isotype control (con). Arrows indicate the locations of de2-7 and wtEGFR. [Figure 31]Figure 31 shows representative H&E-stained paraffin sections of U87MG.Δ2-7 and U87MG.wtEGFR xenografts. Xenografts of U87MG.Δ2-7 (collected 24 days after tumor inoculation) and U87MG.wtEGFR (collected 42 days after tumor inoculation) were excised from mice treated as described in Figure 10 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 little necrosis (left panel), but extensive necrosis (arrows) was observed in both U87MG.Δ2-7 and U87MG.wtEGFR treated with mAb806 (right panel). [Figure 32] Figure 32 shows immunohistochemical analysis of EGFR expression in frozen sections collected from U87MG, U87MG.Δ2-7, and U87MG.wtEGFR xenografts. Sections were collected at the time described in Figure 31 above. Xenograft sections were immunostained with 528 antibody (left panel) and mAb806 (right panel). No decrease in immunoreactivity to wtEGFR, amplified EGFR, or de2-7 EGFR was observed in xenografts treated with mAb806. Consistent with in vitro data, parental U87MG xenografts were positive for 528 antibody but negative for mAb806 staining. [Figure 33] Figure 33 shows a schematic diagram of the bisistronic expression construct produced. Transcription of the chimeric antibody chain is initiated by the elongation factor-1 promoter and terminated by a strong artificial termination sequence. IRES sequences were introduced between the coding region of the light chain and the coding region of NeoR, and between the coding region of the heavy chain and the coding region of the dhfr gene. [Figure 34]Figures 34A and 34B show the in vivo distribution analysis of ch806 radiolabeled with either (A) 125I or (B) 111In in BALB / c nude mice carrying U87MG-de2-7 xenograft tumors. 5 μg of radiolabeled antibody was injected into the mice, and four mice were sacrificed at each time point at 8, 28, 48, or 74 hours. Organs were collected, weighed, and their radioactivity was measured using a gamma counter. [Figure 35] Figures 35A and 35B depict (A) % ID / Gram tissue and (B) tumor-to-blood ratio. Indium-111 antibody shows approximately 30% ID / Gram tissue and a tumor-to-blood ratio of 4.0. [Figure 36] Figure 36 depicts the therapeutic efficacy of the chimeric antibody ch806 in a stochastic tumor model. Female nude mice aged 4–6 weeks were sc-inoculated bilaterally with 3 × 10⁶ U87MG.Δ2-7 cells in 100 μL of PBS. mAb806 was included as a positive control. Treatment was initiated when the tumor reached a mean volume of 50 mm³, and this treatment consisted of 1 mg of ch806 or mAb806 administered intravenously for a total of 5 injections over the indicated days. Data are presented for each treatment group as mean tumor volume ± SE. [Figure 37] Figure 37 shows the CDC activity of the anti-EGFR chimeric IgGI antibody ch806 and the control cG250 against target (A) U87MG.de2-7 and (B) A431 cells. The mean (bar; ±SD) percentage of cytotoxicity based on triple repeat judgments is presented. [Figure 38] Figure 38 shows ADCC against target (A)U87MG.de2-7 and (B)A431 cells at a 50:1 effector:target cell ratio mediated by ch806 and isotype control cG250 (0-10 μg / mL). Results are presented as mean (bar; ±SD) percent cytotoxicity in triple repeat judgments. [Figure 39]Figure 39 shows ADCC mediated by 1 μg / mL parental mAb806 and ch806 against target U87MG.de2-7 cells for a range of effector:target ratios. The mean (bar; ±SD) of triple repeat judgments is presented. [Figure 40] Figure 40 shows the initial selection of 25 hybridomas that produce antibodies that bind to ch806 but not to huIgG. Subsequently, four of these anti-ch806 hybridomas with high affinity binding (clones 3E3, 5B8, 9D6, and 4D8) were tracked for clonal proliferation from single cells by restriction dilution and named Ludwig Institute for Cancer Research Melbourne Hybridoma (LMH)-11, -12, -13, and -14, respectively. In addition, two hybridomas producing huIgG-specific mAbs were also cloned and further characterized: clones 2C10 (LMH-15) and 2B8 (LMH-16). [Figure 41] Figures 41A, 41B, and 41C show that after clonal proliferation, the hybridoma culture supernatant was examined by triple replicate ELISA for its ability to neutralize the ch806 or mAb806 antigen-binding activity with sEGFR621. The mean (±SD) results clearly demonstrated the antagonist activity of anti-idiotype mAbs LMH-11, -12, -13, and -14 by blocking the binding of both ch806 and mouse mAb806 to sEGFR-coated plates in a lysed state (LMH-14 was not shown). [Figure 42-1]Figures 42A and 42B show macrotiter plates coated with purified (A) LMH-11, (B) LMH-12, and (C) LMH-13 at 10 μg / mL. These three purified clones were compared for their ability to capture ch806 or mAb806 in serum or 1% FCS / medium. The bound ch806 or mAb806 was then detected. In addition to controls for secondary conjugate avidin-HRP and ABTS substrates, isotype control antibodies hu3S193 and m3S193 in serum and 1% FCS / medium were included. Results are presented as the mean (±SD) of triple-repeat samples using biotinylated LMH-12 (10 μg / mL) for detection. These results indicate that LMH-12, used for capture and detection, exhibited the best sensitivity to serum ch806 (3 ng / mL) and negligible background binding. [Figure 42-2] 42C shows macrotiter plates coated with purified (A)LMH-11, (B)LMH-12, and (C)LMH-13 at 10 μg / mL. These three purified clones were compared for their ability to capture ch806 or mAb806 in serum or 1% FCS / medium. The bound ch806 or mAb806 was then detected. In addition to controls for secondary conjugate avidin-HRP and ABTS substrates, isotype control antibodies hu3S193 and m3S193 in serum and 1% FCS / medium were included. Results are presented as the mean (±SD) of triple-repeat test samples using biotinylated LMH-12 (10 μg / mL) for detection. These results indicate that LMH-12 used for capture and detection had the best sensitivity to ch806 (3 ng / mL) in serum and negligible background binding. [Figure 43]Figure 43 shows the validation of 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 independent ELISAs were performed in quadruple replicates to measure ch806 (●) in donor serum or ch806 (■) in 1% BSA / medium from three healthy donors, together with the isotype control hu3S193 (▲) in serum or the isotype control hu3S193 (▼) in 1% BSA / medium. A control for secondary conjugate avidin-HRP (◆) and a control for ABTS substrates (hexagon) were also included in each ELISA. The mean (±SD) results clearly demonstrate highly reproducible binding curves for the measurement of ch806 (2 μg / mL–1.6 ng / mL) in serum at a detection limit of 3 ng / mL (n=12; 1–100 ng / mL, coefficient of variation <25%; 100 ng / mL–5 μg / mL, coefficient of variation <15%). No clearly discernible background binding was observed for any of the three serum samples tested, and negligible binding was observed for the isotype control hu3S193. [Figure 44] Figure 44 depicts the immunoblot of recombinant sEGFR expressed in CHO cells, blotted with mAb806. Recombinant sEGFR was treated with PNGaseF to remove N-linked glycosylation (deglycosylation) or untreated (untreated) proteins, subjected to SDS-PAGE, transferred to a membrane, and immunoblotted with mAb806. [Figure 45] Figure 45 depicts the immunoprecipitation of EGFR from 35S-labeled cell lines (U87MG.Δ2-7, U87MG-wtEGFR, and A431) with different antibodies (SC-03, 806, and 528 antibodies). [Figure 46] Figure 46 depicts immunoprecipitation of EGFR from different cells (A431 and U87MG.Δ2-7) at different time points (time 0 to 240 min) after pulse labeling with 35S methionine / cysteine. Antibodies 528 and 806 were used for immunoprecipitation. [Figure 47]Figure 47 depicts immunoprecipitation of EGFR from various cell lines (U87MG.Δ2-7, U87MG-wtEGFR, and A431) with various antibodies (SC-03, 806, and 528) in the absence of Endo H digestion (-) and after Endo H digestion (+) to remove high-mannose carbohydrates. [Figure 48] Figure 48 depicts immunoprecipitation with mAb806 antibody after cell surface iodization of A431 and U87MG.Δ2-7 cell lines, with or without Endo H digestion. This figure confirms that EGFR bound to the cell surface of A431 cells by mAb806 is in an Endo H-sensitive form. [Figure 49-1] Figure 49-1 shows the pREN ch806 LC Neo Vector (Sequence ID: 7). [Figure 49-2] Figure 49-2 shows the pREN ch806 LC Neo Vector (Sequence ID: 7). [Figure 49-3] Figure 49-3 shows the pREN ch806 LC Neo Vector (SEQ ID NO: 7). [Figure 49-4] Figure 49-4 shows the pREN ch806 LC Neo Vector (SEQ ID NO: 7). [Figure 49-5] Figure 49-5 shows the pREN ch806 LC Neo Vector (SEQ ID NO: 7). [Figure 49-6] Figure 49-6 shows the pREN ch806 LC Neo Vector (Sequence ID: 7). [Figure 50-1] Figure 50-1 shows the pREN ch806 HC DHFR Vector (Sequence ID: 8). [Figure 50-2] Figure 50-2 shows the pREN ch806 HC DHFR Vector (Sequence ID: 8). [Figure 50-3] Figure 50-3 shows the pREN ch806 HC DHFR Vector (Sequence ID: 8). [Figure 50-4] FIG. 50-4 shows pREN ch806 HC DHFR Vector (SEQ ID NO: 8). [Figure 50-5] FIG. 50-5 shows pREN ch806 HC DHFR Vector (SEQ ID NO: 8). [Figure 50-6] FIG. 50-6 shows pREN ch806 HC DHFR Vector (SEQ ID NO: 8). [Figure 50-7] FIG. 50-7 shows pREN ch806 HC DHFR Vector (SEQ ID NO: 8). [Figure 51-1] FIGS. 51A-B show the nucleic acid sequences (SEQ ID NO: 21 and 26, respectively) and amino acid sequences (SEQ ID NO: 22 and 27, respectively) of mAb124 VH and VL chains. [Figure 51-2] FIGS. 51C-D show the nucleic acid sequences (SEQ ID NO: 21 and 26, respectively) and amino acid sequences (SEQ ID NO: 22 and 27, respectively) of mAb124 VH and VL chains. [Figure 52-1] FIGS. 52A-B show the nucleic acid sequences (SEQ ID NO: 31 and 36, respectively) and amino acid sequences (SEQ ID NO: 32 and 37, respectively) of mAb1133 VH and VL chains. [Figure 52-2] FIGS. 52C-D show the nucleic acid sequences (SEQ ID NO: 31 and 36, respectively) and amino acid sequences (SEQ ID NO: 32 and 37, respectively) of mAb1133 VH and VL chains. [Figure 53] FIG. 53 shows a DNA plasmid map of a composite two-gene Lonza plasmid comprising pEE12.4 containing the hu806H (VH+CH) expression cassette and pEE6.4 containing the hu806L (VL+CL) expression cassette. [Figure 54-1]Figure 54-1 shows the DNA sequence (SEQ ID NO: 41; complement SEQ ID NO: 162) of the composite Lonza plasmid described in Figure 53. This sequence also shows all translations associated with the hu806 antibody (SEQ ID NOs: 42-51 and 163-166). This plasmid was sequence-validated to confirm the coding sequence and translations. Sections of the sequence are shaded to identify regions of interest; the shaded regions correspond to the actual splice junctions. The color coding is as follows: (gray): signal region, first coding sequence found in both the heavy chain variable region and the light chain variable region; (lavender): hu806 VH chain, veneered heavy chain variable region; (pink): hu806 CH chain, codon-optimized heavy chain constant region; (green): hu806 VL chain, veneered light chain variable region; and (yellow): hu806 CL chain, codon-optimized light chain constant region. [Figure 54-2] Figure 54-2 shows the DNA sequence (SEQ ID NO: 41; complement SEQ ID NO: 162) of the composite Lonza plasmid described in Figure 53. This sequence also shows all translations associated with the hu806 antibody (SEQ ID NOs: 42-51 and 163-166). This plasmid was sequence-validated to confirm the coding sequence and translations. Sections of the sequence are shaded to identify regions of interest; the shaded regions correspond to the actual splice junctions. The color coding is as follows: (gray): signal region, first coding sequence found in both the heavy chain variable region and the light chain variable region; (lavender): hu806 VH chain, veneered heavy chain variable region; (pink): hu806 CH chain, codon-optimized heavy chain constant region; (green): hu806 VL chain, veneered light chain variable region; and (yellow): hu806 CL chain, codon-optimized light chain constant region. [Figure 54-3]Figure 54-3 shows the DNA sequence (SEQ ID NO: 41; complement SEQ ID NO: 162) of the composite Lonza plasmid described in Figure 53. This sequence also shows all translations associated with the hu806 antibody (SEQ ID NOs: 42-51 and 163-166). This plasmid was sequence-validated to confirm the coding sequence and translations. Sections of the sequence are shaded to identify regions of interest; the shaded regions correspond to the actual splice junctions. The color coding is as follows: (gray): signal region, first coding sequence found in both the heavy chain variable region and the light chain variable region; (lavender): hu806 VH chain, veneered heavy chain variable region; (pink): hu806 CH chain, codon-optimized heavy chain constant region; (green): hu806 VL chain, veneered light chain variable region; and (yellow): hu806 CL chain, codon-optimized light chain constant region. [Figure 54-4] Figure 54-4 shows the DNA sequence (SEQ ID NO: 41; complement SEQ ID NO: 162) of the composite Lonza plasmid described in Figure 53. This sequence also shows all translations associated with the hu806 antibody (SEQ ID NOs: 42-51 and 163-166). This plasmid was sequence-validated to confirm the coding sequence and translations. Sections of the sequence are shaded to identify regions of interest; the shaded regions correspond to the actual splice junctions. The color coding is as follows: (gray): signal region, first coding sequence found in both the heavy chain variable region and the light chain variable region; (lavender): hu806 VH chain, veneered heavy chain variable region; (pink): hu806 CH chain, codon-optimized heavy chain constant region; (green): hu806 VL chain, veneered light chain variable region; and (yellow): hu806 CL chain, codon-optimized light chain constant region. [Figure 54-5]Figure 54-5 shows the DNA sequence (SEQ ID NO: 41; complement SEQ ID NO: 162) of the composite Lonza plasmid described in Figure 53. This sequence also shows all translations associated with the hu806 antibody (SEQ ID NOs: 42-51 and 163-166). This plasmid was sequence-validated to confirm the coding sequence and translations. Sections of the sequence are shaded to identify regions of interest; the shaded regions correspond to the actual splice junctions. The color coding is as follows: (gray): signal region, first coding sequence found in both the heavy chain variable region and the light chain variable region; (lavender): hu806 VH chain, veneered heavy chain variable region; (pink): hu806 CH chain, codon-optimized heavy chain constant region; (green): hu806 VL chain, veneered light chain variable region; and (yellow): hu806 CL chain, codon-optimized light chain constant region. [Figure 54-6] Figure 54-6 shows the DNA sequence (SEQ ID NO: 41; complement SEQ ID NO: 162) of the composite Lonza plasmid described in Figure 53. This sequence also shows all translations associated with the hu806 antibody (SEQ ID NOs: 42-51 and 163-166). This plasmid was sequence-validated to confirm the coding sequence and translations. Sections of the sequence are shaded to identify regions of interest; the shaded regions correspond to the actual splice junctions. The color coding is as follows: (gray): signal region, first coding sequence found in both the heavy chain variable region and the light chain variable region; (lavender): hu806 VH chain, veneered heavy chain variable region; (pink): hu806 CH chain, codon-optimized heavy chain constant region; (green): hu806 VL chain, veneered light chain variable region; and (yellow): hu806 CL chain, codon-optimized light chain constant region. [Figure 54-7]Figure 54-7 shows the DNA sequence (SEQ ID NO: 41; complement SEQ ID NO: 162) of the composite Lonza plasmid described in Figure 53. This sequence also shows all translations associated with the hu806 antibody (SEQ ID NOs: 42-51 and 163-166). This plasmid was sequence-validated to confirm the coding sequence and translations. Sections of the sequence are shaded to identify regions of interest; the shaded regions correspond to the actual splice junctions. The color coding is as follows: (gray): signal region, first coding sequence found in both the heavy chain variable region and the light chain variable region; (lavender): hu806 VH chain, veneered heavy chain variable region; (pink): hu806 CH chain, codon-optimized heavy chain constant region; (green): hu806 VL chain, veneered light chain variable region; and (yellow): hu806 CL chain, codon-optimized light chain constant region. [Figure 54-8] Figure 54-7 shows the DNA sequence (SEQ ID NO: 41; complement SEQ ID NO: 162) of the composite Lonza plasmid described in Figure 53. This sequence also shows all translations associated with the hu806 antibody (SEQ ID NOs: 42-51 and 163-166). This plasmid was sequence-validated to confirm the coding sequence and translations. Sections of the sequence are shaded to identify regions of interest; the shaded regions correspond to the actual splice junctions. The color coding is as follows: (gray): signal region, first coding sequence found in both the heavy chain variable region and the light chain variable region; (lavender): hu806 VH chain, veneered heavy chain variable region; (pink): hu806 CH chain, codon-optimized heavy chain constant region; (green): hu806 VL chain, veneered light chain variable region; and (yellow): hu806 CL chain, codon-optimized light chain constant region. [Figure 54-9]Figure 54 shows the DNA sequence (SEQ ID NO: 41; complement SEQ ID NO: 162) of the composite Lonza plasmid described in Figure 53. This sequence also shows all translations associated with the hu806 antibody (SEQ ID NOs: 42-51 and 163-166). This plasmid was sequence-validated to confirm the coding sequence and translations. Sections of the sequence are shaded to identify regions of interest; the shaded regions correspond to the actual splice junctions. The color coding is as follows: (gray): signal region, first coding sequence found in both the heavy chain variable region and the light chain variable region; (lavender): hu806 VH chain, veneered heavy chain variable region; (pink): hu806 CH chain, codon-optimized heavy chain constant region; (green): hu806 VL chain, veneered light chain variable region; and (yellow): hu806 CL chain, codon-optimized light chain constant region. [Figure 55] Figures 55A and 55B show the translated amino acid sequences of hu806 (VH and VL chains of SEQ ID NOs: 164 and 166 and their respective signal peptides of SEQ ID NOs: 163 and 165; CH and CL chains of SEQ ID NOs: 43 and 48), and provide the Kabat numbers for the VH and VL chains (SEQ ID NOs: 164 and 165, respectively), with CDRs (SEQ ID NOs: 44-46 and 49-51) underlined. [Figure 56-1] Figures 56A and 56B show the first stage in the veneering design, grading of amino acid residues within the mAb806 sequences (VH chain of SEQ ID NO: 167 and VL chain of SEQ ID NO: 12) for surface exposure. The grade is given by the number of asterisks (*) above each residue, with the maximum exposed residue having three asterisks. These figures include blueprints showing how the first oligonucleotides (VH chain: Figure 56C and SEQ ID NOs: 52 and 169-177; VL chain: Figure 57C and SEQ ID NOs: 62, 66, 68 and 181-187) overlap to form the first veneered product (VH chain of SEQ ID NO: 168 and VL chain of SEQ ID NO: 180). [Figure 56-2]Figure 56C shows the first stage in the veneer design, grading of amino acid residues within the mAb806 sequences (VH chain of SEQ ID NO: 167 and VL chain of SEQ ID NO: 12) for surface exposure. The grade is given by the number of asterisks (*) above each residue, with the most exposed residue having three asterisks. These figures include blueprints showing how the first oligonucleotides (VH chain: Figure 56C and SEQ ID NOs: 52 and 169-177; VL chain: Figure 57C and SEQ ID NOs: 62, 66, 68 and 181-187) overlap to form the first veneered product (VH chain of SEQ ID NO: 168 and VL chain of SEQ ID NO: 180). [Figure 57-1] Figures 57A and 57B show the first stage in the veneering design, grading of amino acid residues within the mAb806 sequences (VH chain of SEQ ID NO: 167 and VL chain of SEQ ID NO: 12) for surface exposure. The grade is given by the number of asterisks (*) above each residue, with the most exposed residue having three asterisks. These figures include blueprints showing how the first oligonucleotides (VH chain: Figure 56C and SEQ ID NOs: 52 and 169-177; VL chain: Figure 57C and SEQ ID NOs: 62, 66, 68 and 181-187) overlap to form the first veneered product (VH chain of SEQ ID NO: 168 and VL chain of SEQ ID NO: 180). [Figure 57-2] Figure 57C shows the first stage in the veneer design, grading of amino acid residues within the mAb806 sequences (VH chain of SEQ ID NO: 167 and VL chain of SEQ ID NO: 12) for surface exposure. The grade is given by the number of asterisks (*) above each residue, with the most exposed residue having three asterisks. These figures include a blueprint showing how the first oligonucleotides (VH chain: Figure 56C and SEQ ID NOs: 52 and 169-177; VL chain: Figure 57C and SEQ ID NOs: 62, 66, 68 and 181-187) overlap to form the first veneered product (VH chain of SEQ ID NO: 168 and VL chain of SEQ ID NO: 180). [Figure 58-1]Figure 58-1 shows maps of the codon-optimized huIgG1 heavy-chain DNA sequence (SEQ ID NO: 80; complement SEQ ID NO: 178) and amino acid translation (SEQ ID NO: 43). [Figure 58-2] Figure 58-2 shows maps of the codon-optimized huIgG1 heavy-strand DNA sequence (SEQ ID NO: 80; complement SEQ ID NO: 178) and amino acid translation (SEQ ID NO: 43). [Figure 59] Figure 59 shows a protein alignment comparing the original reference file for the hu806 VH+CH amino acid sequence (8C65AAG hu806 VH+CH; SEQ ID NO: 81) and the mAb806 VH chain (SEQ ID NO: 167). The highlighted region indicates the conserved amino acid sequence within the VH chain. The CDR is underlined. Asterisks indicate changes planned and implemented in the initial veneering method. Numbered sites are references to later modifications. [Figure 60] Figure 60 shows the corresponding alignment of the hu806 VL+CL amino acid sequence (8C65AAG hu806 signal+VL+CL; SEQ ID NO: 83) to the original reference file for the mAb806 VL chain (SEQ ID NO: 179). This includes an additional file (r2vk1 hu806 signal+VL+CL; SEQ ID NO: 82) and a precursor construct, included to illustrate the modification made at modification number 7. [Figure 61] Figure 61 shows the nucleotide and amino acid alignments of the hu806 signal + VL and CL sequences (8C65AAG hu806 Vl+Cl; SEQ ID NOs: 190 and 188) and the corresponding ch806 sequence (pREN ch806 LC Nco; LICR; SEQ ID NO: 189). These are modified and annotated as described in Figure 62. [Figure 62]Figure 62 shows the nucleotide alignment of the hu806 signal + VH sequence (8C65AAG hu806 VH chain; SEQ ID NO: 192) and the corresponding mAb806 sequence [mAb806 VH chain before codon change (cc) and veneering (ven); SEQ ID NO: 191]. Nucleotide changes after the amino acid changes in Figures 59 and 60 are illustrated, as well as conserved nucleic acid changes that resulted in no amino acid changes. For easier inspection, the signal and inter-VH chain in hu806 introns have been removed. The signal sequence and CDR are underlined. The corresponding amino acid sequence (SEQ ID NO: 42) is superimposed on the alignment. [Figure 63] Figure 63 shows the binding of purified hu806 antibody obtained from transient transfectant 293 cells to recombinant EGFR-ECD as determined by Biacore. No binding to EGFR-ECD was observed with the purified control human IgG1 antibody. [Figure 64-1] This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-2] This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-3] This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-4] This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-5] This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-6]Shows a GenBank-format text document of the sequence (SEQ ID NO: 41) and annotation of plasmid 8C65AAG encoding IgG1 hu806. [Figure 64-7] Shows a GenBank-format text document of the sequence (SEQ ID NO: 41) and annotation of plasmid 8C65AAG encoding IgG1 hu806. [Figure 64-8] Shows a GenBank-format text document of the sequence (SEQ ID NO: 41) and annotation of plasmid 8C65AAG encoding IgG1 hu806. [Figure 64-9] Shows a GenBank-format text document of the sequence (SEQ ID NO: 41) and annotation of plasmid 8C65AAG encoding IgG1 hu806. [Figure 64-10] Shows a GenBank-format text document of the sequence (SEQ ID NO: 41) and annotation of plasmid 8C65AAG encoding IgG1 hu806. [Figure 64-11] Shows a GenBank-format text document of the sequence (SEQ ID NO: 41) and annotation of plasmid 8C65AAG encoding IgG1 hu806. [Figure 64-12] Shows a GenBank-format text document of the sequence (SEQ ID NO: 41) and annotation of plasmid 8C65AAG encoding IgG1 hu806. [Figure 64-13] Shows a GenBank-format text document of the sequence (SEQ ID NO: 41) and annotation of plasmid 8C65AAG encoding IgG1 hu806. [Figure 64-14] Shows a GenBank-format text document of the sequence (SEQ ID NO: 41) and annotation of plasmid 8C65AAG encoding IgG1 hu806. [Figure 64-15]This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-16] This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-17] This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-18] This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-19] This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-20] This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-21] This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-22] This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-23] This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-24]This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-25] This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-26] This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-27] This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-28] This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-29] This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-30] This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 64-31] This shows the sequence (SEQ ID NO: 41) and annotation text document in GenBank format for plasmid 8C65AAG, which encodes IgG1 hu806. [Figure 65] Figure 65 shows the amino acid sequence alignment for CDRs from mAb806 (SEQ ID NOs: 15-18, 20, and 193) and mAb175 (SEQ ID NOs: 130-132, 135, and 194-195). Sequence differences between the two antibodies are shown in bold. [Figure 66]Figures 66A and 66B show immunohistochemical staining of cell lines and normal human liver with mAb175. (A) Sections prepared from blocks containing A431 cells (overexpressing wtEGFR), U87MG.Δ2-7 cells (expressing Δ2-7EGFR), and U87MG cells (moderately expressing wtEGFR) were stained with biotinylated mAb175. (B) Staining of normal human liver with mAb175 (left panel), isotype control (center panel), and secondary antibody control (right panel) (400x). No specific sinusoidal capillary or hepatocyte staining was observed. [Figure 67] Figures 67A, 67B, and 67C show the reactivity of EGFR fragments presented in yeast with mAb806 and mAb175. (A) Representative flow cytometry histograms depicting the mean fluorescence signals of mAb175 and mAb806 labeling of yeast-presented EGFR fragments. In yeast displays, a certain percentage of cells do not express proteins on these surfaces, resulting in two histogram peaks. Since all fragments contain a linear C-terminal c-myc tag, the 9E10 antibody is used as a positive control. (B) Overview of antibodies binding to various EGFR fragments. (C) EGFR fragments were denatured by heating the yeast pellet at 800°C for 30 minutes. In all cases, the c-myc tag was still recognized by the 9E10 anti-myc antibody. This demonstrates that heat treatment does not impair yeast surface-presented proteins. Denaturation was confirmed using the higher-order structure-sensitive EGFR antibody mAb225. [Figure 68]Figures 68A, 68B, 68C, and 68D show the antitumor effects of mAb175 on brain and prostate cancer xenografts. (A) Mice (n=5) with U87MG.Δ2-7 xenografts were administered via ip injection three times a week for two weeks with PBS, 1 mg of mAb175 or mAb806 (positive control) on days 6, 8, 10, 13, 15, and 17 when the starting tumor volume was 100 mm3. Data are shown as mean tumor volume ± SE. (B) Cells were stained with two unrelated antibodies (blue, filled, and green, hollow), mAb 528 (pink, filled), mAb806 (light blue, hollow) and mAb175 (orange, hollow) for total EGFR, and then analyzed by FACS. (C) DU145 cells were lysed and subjected to IP with mAb 528, mAb 806, mAb 175, or two independent, unrelated antibodies, followed by immunoblotting for EGFR. (D) Mice carrying DU145 xenografts (n=5) were administered IP once daily with PBS, 1 mg of mAb 175, or mAb 806 on days 18-22, 25-29, and 39-43 when the starting tumor volume was 85 mm3. Data are presented as mean tumor volume ± SE. [Figure 69]Figures 69A, 69B, 69C, 69D, 69E, and 69F show the crystal structures of EGFR peptide 287-302 bound to the Fab fragment. (A) Computer printout image of Fab 806, light chain, red; heavy chain, blue; bound peptide, yellow; and superimposed EGFR287-302 from EGFR, purple. (B) Computer printout image of Fab 175, light chain, yellow; heavy chain, green; bound peptide, lilac; and EGFR287-302 from EGFR(DI-3), purple. (C) Detail of (B) showing the similarity of EGFR287-302 in the receptor to the peptide bound to Fab 175. The peptide backbone is shown as a Cα trace and the interacting side chains as sticks. With respect to the main chain, O atoms are colored red; N, blue; S, orange; and C. (D) Superposition of the Fab175:peptide complex and EGFR showing spatial overlap. The surface of EGFR187-286 is colored blue-green, as in (C). (E) Orthogonal view to (D) with EGFR187-286 shown in opaque blue and the transparent light chain surface (orange) and heavy chain surface (green). (F) Detailed three-dimensional view of the 175Fab complex examining the antigen binding site. Colored as in (C), with hydrogen bonds shown as black dots. Water molecules hidden within the complex are shown as red spheres. [Figure 70]Figures 70A, 70B, 70C, and 70D show the effect of 271-283 cysteine ​​binding on mAb806 to EGFR. (A) Cells transfected with wtEGFR, EGFR-C271A, EGFR-C283A, or C271A / C283A mutants were stained with mAb528 (filled pink histogram), mAb806 (blue line), or secondary antibody only (purple), and then analyzed by FACS. Gain was set using class-matched, unrelated antibodies. (B) BaF3 cells expressing EGFR-C271A or C271 / 283A EGFR were examined for their response to EGF in the MTT assay described. EC50S was derived using Bolzman fit of data points. Data are presented as mean and standard deviation of triple repeated measures. (C) BaF3 cells expressing wild-type or EGFR-C271A / C283A were starved with IL-3 and serum, and then exposed to EGF or vehicle control. Whole cell lysates were isolated by SDS-PAGE and immunoblotted with anti-phosphotyrosine antibody (upper panel) or anti-EGFR antibody (lower panel). (D) BaF3 cells expressing wild-type (left panel) or C271A / C283A (right panel) EGFR were stimulated with increasing concentrations of EGF in the absence of antibody (white circle), in the presence of mAb 528 (gray circle), or mAb 806 (black triangle) (both 10 μg / mL). Data are presented as mean and standard deviation of triple repeated measures. [Figure 71] Figures 71A, 71B, and 71C show: (A) Whole-body gamma camera images of the in vivo distribution of 111In ch806 in a patient with metastatic squamous cell carcinoma of the vocal cords [showing quantitatively high uptake into the tumor in the right neck (arrow). Blood pool activity and small catabolism of free 111In in the liver are also observed]. (B) Single-photon computed tomography (SPECT) images of the neck of the same patient [showing 111In uptake in the viable tumor (arrow) and central decreased uptake indicating necrosis]. (C) Corresponding neck CT scan [clearly showing a large right neck tumor volume (arrow) and central necrosis]. [Figure 72] Figures 72A and 72B show a three-dimensional model of the untethered EGFR1-621 structure. The receptor skeleton is depicted in blue, and the ligand TGF-α in red. The mAb806 / 175 epitope is depicted in blue-green, and the disulfide bond in yellow. The atoms of the disulfide bond linking the epitope to the receptor are shown in a space-filling configuration. This model was constructed by docking the EGFR-ECD CR2 domain from the tethered higher-order structure to the structure of the untethered EGFR monomer in the presence of this ligand. [Figure 73] Figure 73 shows the reactivity of EGFR fragments with mAb806. Lysates from 293T cells transfected with vectors expressing soluble 1-501 EGFR fragments or CH / EGFR fragment fusion proteins (GH-274-501, GH-282-501, GH-290-501, and GH-298-501) were split by SDS-PAGE, transferred to membranes, and immunoblotted with mAb806 (left panel) or anti-myc antibody 9B11 (right panel). [Figure 74] Figures 74A and 74B show the mAb175 VH chain nucleic acid sequence (sequence number: 128) and amino acid sequence (single or multiple) (sequence number: 129), respectively. [Figure 75] Figures 75A and 75B show the mAb175 VL chain nucleic acid sequence (SEQ ID NO: 133) and the amino acid sequence (single or multiple) (SEQ ID NO: 134), respectively. [Figure 76-1]Figures 76A and 76B show (A) volume product concentration and (B) viable cell concentration of GS-CHO (14D8, 15B2, and 40A10) and GS-NS0(36)hu806 transfectants in small-scale (100 mL) shaking flask cultures [product concentration was estimated by ELISA using 806 anti-idiotype as the coating antibody and ch806 Clinical Lot:J06024 as the standard substance]; and (C) cell growth and volume production of GS-CHO 40A10 transfectant in a 15 L stirred tank bioreactor [viable cell density (◆x10⁵ cells / mL), cell viability (■), and production (▲ mg / L)]. [Figure 76-2] 76C shows (A) volume product concentration and (B) viable cell concentration of GS-CHO (14D8, 15B2, and 40A10) and GS-NS0(36)hu806 transfectants in small-scale (100 mL) shaking flask cultures [product concentration was estimated by ELISA using 806 anti-idiotype as the coating antibody and ch806 Clinical Lot:J06024 as the standard substance]; (C) cell growth and volume production of GS-CHO 40A10 transfectant in a 15 L stirred tank bioreactor [viable cell density (◆x10⁵ cells / mL), cell viability (■), and production (▲ mg / L)]. [Figure 77-1] Figures 77A, 77B, and 77C show size exclusion chromatography (Biosep SEC-S3000) analyses of purified protein A hu806 antibody constructs and control ch806 and mAb806 produced by small-scale culture. The upper panel of each figure shows the chromatogram at A214nm, and the lower panel shows the chromatogram at A280nm. [Figure 77-2] Figures 77D and 77E show size exclusion chromatography (Biosep SEC-S3000) analysis of purified protein A hu806 antibody constructs and control ch806 and mAb806 produced by small-scale culture. The upper panel of each figure shows the chromatogram at A214nm, and the lower panel shows the chromatogram at A280nm. [Figure 78] Figure 78 shows the size exclusion chromatography (Biosep SEC-S3000) analysis of the purified protein A hu806 antibody construct 40A10 after large-scale production and protein A purification. The chromatogram at 214 nm shows a purity of 98.8% with 1.2% aggregates present. [Figure 79] Figure 79 shows the analysis of purified transfectant hu806 standard (5 μg) GS CHO (14D8, 15B2, and 40A10) and GS-NS0(36)hu806 under reducing conditions using a precast 4-20% Tris / glycine gel from Novex, USA, under standard SDS-PAGE conditions. Protein detected by Coomassie blue stain. [Figure 80] Figure 80 shows the analysis of purified transfectant hu806 standard (5 μg) GS CHO (14D8, 15B2, and 40A10) and GS-NS0 (36) under non-reducing conditions using a precast 4-20% Tris / glycine gel under standard SDS-PAGE conditions. Proteins detected by Coomassie blue stain. [Figure 81] Figure 81 shows the analysis of purified transfectant hu806 GS CHO 40A10 (5 μg) after large-scale production using a precast 4-20% Tris / glycine gel under standard SDS-PAGE conditions. The protein was detected by Coomassie blue stain. [Figure 82] Figure 82 shows the isoelectric focusing gel analysis of purified transfectant hu806 GS CHO 40A10 (5 μg) after 15 L production. Protein detected by Coomassie blue staining. Lane 1, pI marker; Lane 2, hu806 (3 isoforms, pI 8.66 to 8.82); Lane 3, pI marker. [Figure 83]Figure 83 shows the binding to A431 cells: flow cytometry analysis of purified protein A hu806 antibody preparation (20 μg / mL) and isotype control huA33 (20 μg / mL). Controls include secondary antibody alone (green) and ch806 (red). Hu806 constructs were produced by small-scale culture. [Figure 84] Figure 84 shows the binding to A431 cells: flow cytometry analysis of purified mAb806, ch806, and hu806 40A10 antibody preparations (20 μg / mL), 528 (which binds both wild-type EGFR and de2-7 EGFR), and an unrelated control antibody (20 μg / mL), as shown, which bind ~10% of wild-type EGFR on the cell surface. [Figure 85] Figure 85 shows the binding to U87MG.de2-7 glioma cells. Flow cytometry analysis of purified mAb806, ch806, and hu806 40A10 antibody preparations (20 μg / mL) and 528 anti-EGFR and unrelated control antibodies (20 μg / mL). [Figure 86] Figure 86 shows the specific binding of the 125I radiolabeled 806 antibody construct to (A) U87MG.de2-7 glioma cells and (B) A431 carcinoma cells. [Figure 87] Figure 87 shows a scatchard analysis: binding of 125I-labeled (A)ch806 and (B)hu806 antibody constructs to U87MG.de2-7 cells. [Figure 88] Figure 88 shows a scatchard analysis: binding of 125I-labeled (A)ch806 and (B)hu806 antibody constructs to A431 cells. [Figure 89] Figure 89 shows the BIAcore analysis of the binding of (A)hu806 and (B)ch806 to the immobilized peptide at progressively increasing concentrations of 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, and 300 nM to the 287-302 EGFR 806 peptide epitope. [Figure 90]Figure 90 shows the ch806-mediated and hu806-mediated antibody-dependent cytotoxicity against target A431 cells, as determined by (A) each antibody at 1 μg / mL over the effector-to-target cell ratio range (E:T = 0.78:1 to 100:1) and (B) each antibody at E:T = 50:1 over the concentration range (3.15 ng / mL to 10 μg / mL) against target A431. [Figure 91] Figure 91 shows established A431 xenograft treatment in BALB / c nude mice. A group of five mice received antibody therapy at a dose of 6 × 1 mg for two weeks, as shown (arrows). Mean ± SEM tumor volume up to the end of the study is presented. [Figure 92] Figure 92 shows established treatment with U87Mg.de2-7 xenografts in BALB / c nude mice. A group of five mice received antibody therapy at a dose of 6 × 1 mg for two weeks, as shown (arrows). Mean ± SEM tumor volume up to the end of the study is presented. [Figure 93] Figure 93 shows the deviations from random coil chemical shift values ​​for mAb806 peptides (A)N, (B)HN, and (C)HA. The peptides were prepared in an H2O solution containing 5% 2H2O, 70 mM NaCl, and 50 mM NaPO4 at pH 6.8. All spectra used for sequential assignment were acquired at 298 K using a Bruker Avance 500. [Figure 94] Figures 94A, 94B, 94C, 94D, 94E, and 94F show forward gamma camera images of patient 7, A) ventral and B) dorsal, 5 days after 111In-ch806 injection. The high uptake of 111In-ch806 in metastatic lesions in the lungs (arrows) is clearly visible. C) and D) show metastatic lesions on CT scans. E) 3D SPECT image of the chest, and F) SPECT and CT co-registered tomographic images showing specific uptake of 111In-ch806 in metastatic lesions (arrows). [Figure 95]Figures 95A, 95B, 95C, 95D, 95E, and 95F show planar images of the head and neck of Patient 8 obtained on A) day 0, B) day 3, and C) day 7 after injection of 111In-ch806. Initial blood pool activity is observed on day 0, and uptake of 111In-ch806 in the anaplastic astrocytoma in the right frontal lobe is clearly visible on day 3 (arrow) and increases until day 7. Specific uptake of 111In-ch806 is confirmed in D) SPECT images of the brain (arrow), and is clearly visible in E) 18F-FDG PET and F) MRI at the tumor site (arrow). [Figure 96] Figures 96A, 96B, 96C, and 96D clearly show similar uptake of 111In-ch806 in tumors in patient 3 compared to patient 4, despite differences in 806 antigen expression in the selected tumor samples. A) Localization of 111In-ch806 in lung metastases (arrows) in SPECT tomography of patient 4 with clearly visible cardiac blood pooling activity (B). B) Corresponding CT scan. Conserved tumors were found to have a positive rate of <10% for 806 expression. C) Localization of 111In-ch806 in lung metastases (arrows) of patient 3 with clearly visible cardiac blood pooling activity (B). D) Corresponding CT scan. Conserved tumors were found to have a positive rate of 50-75% for 806 expression. [Figure 97] Figure 97 shows the pharmacokinetics of a pooled population of ch806 protein as measured by ELISA. Measured and predicted ch806 (%ID / L) as time (hours) after injection. [Figure 98] Figures 98A and 98B show individual patient results for A) normalized systemic clearance and B) hepatic clearance of 111In-ch806 at dose levels of 5 mg / m2 (■), 10 mg / m2 (△), 20 mg / m2 (▽), and 40 mg / m2 (◆). Linear regression [A) r² = 0.9595; B) r² = 0.9415] for the datasets shown in each panel. [Modes for carrying out the invention]

[0093] Detailed description In accordance with the present invention, conventional molecular biology, microbiology, and recombinant DNA techniques within the scope of the skills in the art can be utilized. Such techniques are well described in the literature. For example, Sambrook et al., "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology" Volumes IE [Ausubel, RM, ed. (1994)]; "Cell Biology: A Laboratory Handbook" Volumes I-III [JECelis, ed. (1994))]; "Current Protocols in Immunology" Volumes I-III[Coligan,JE,ed.(1994)];"Oligonucleotide Synthesis"(MJGait ed.1984);"Nucleic Acid Hybridization"[BDHames & SJHiggins eds.(1985)];"Transcription And Translation"[BDHames & SJHiggins,eds.(1984)];"Animal Cell Culture” [RIFreshney, ed. (1986)]; “Immobilized Cells And Enzymes” [IRL [Press, (1986)]; see B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

[0094] In this specification, the following terms are considered to have definitions, but are not limited to them.

[0095] The term "specific binding member" describes a member of a molecular pair that has binding specificity to one another. Members of a specific binding pair may be naturally occurring or may be entirely or partially synthesized. One member of the molecular pair has an area or cavity on its surface that specifically binds to a particular spatial and polar mechanism of the other member of the molecular pair, and is therefore complementary. Thus, the members of the pair have the property of specifically binding to one another. Examples of types of specific binding pairs include antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, and enzyme-substrate. This application relates to antigen-antibody type reactions.

[0096] In these various grammatical forms, the term “abnormal expression” means and may include any increase in the expression, modification, or overexpression of a protein in a tissue, such as an increase in the amount of protein, caused by any means including increased expression or translation, modification of a protein promoter or regulator, amplification of a gene for the protein, or improvement of half-life or stability, in contrast to a non-overexpression state, resulting in more protein being present or detectable at any given time. Abnormal expression is considered to include and includes any scenario or modification in which a protein expression or post-translational modification mechanism in a cell is overworked or disrupted due to increased protein expression or increased levels or amounts, including scenarios or modifications in which a modified protein, such as a mutant or variant resulting from sequence alteration, deletion, or insertion, or a modified folding is expressed.

[0097] It is important to understand that the term “abnormal expression” has been specifically chosen herein to encompass the presence of an abnormal (usually increased) amount / level of protein, regardless of the cause of this abnormal amount or level. For example, abnormal amounts of protein may result from overexpression of a protein in the absence of gene amplification, as is the case in many cell / tissue samples taken from the head and neck of subjects with cancer, while other samples may present abnormal protein levels attributable to gene amplification.

[0098] In relation to the latter, some of the inventors' studies presented herein to illustrate the present invention involve the analysis of samples, some of which exhibit abnormal protein levels due to amplification of EFGR. This explains the presentation herein of experimental findings that refer to amplification, as well as the use of the terms “amplified” and similar terms when describing abnormal levels of EFGR. However, this is an observation of abnormal amounts or levels of the protein that define the environment or circumstances under which clinical intervention, such as relying on the binding member of the present invention, may be considered, and for this reason, herein we consider the term “abnormal expression” to be more broadly applicable to the environment that causes the corresponding abnormality of EFGR levels.

[0099] Therefore, although the terms “overexpression” and “amplification” in these various grammatical forms are understood to have different technical meanings, they are considered equivalent to each other insofar as they describe a state in which abnormal EFGR protein levels are present in relation to the present invention. Accordingly, the term “abnormal expression” has been chosen to encompass the terms “overexpression” and “amplification” within this scope for the purposes herein, and therefore, when used herein, all terms can be considered equivalent to each other.

[0100] The term "antibody" describes immunoglobulins, whether naturally occurring or partially or completely synthetically produced. This term also encompasses any polypeptide or protein that has an antibody-binding domain, or a binding domain homologous to an antibody-binding domain. CDR-grafted antibodies are also considered under this term.

[0101] While antibodies can be modified in numerous ways, the term “antibody” should be interpreted to encompass any binding member or substrate having a binding domain with the desired specificity. Therefore, the term includes antibody fragments, antibody derivatives, functional equivalents, and homologs, whether natural or entirely or partially synthetic, containing any polypeptide with an immunoglobulin-binding domain. This also includes chimeric molecules or equivalents containing an immunoglobulin-binding domain fused to another polypeptide. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023, and U.S. Patent Nos. 4,816,397 and 4,816,567.

[0102] It has been demonstrated that fragments of all antibodies can sometimes function as binding antigens. Examples of binding fragments include: (i) Fab fragments consisting of VL, VH, CL, and CH1 domains; (ii) Fd fragments consisting of VH and CH1 domains; (iii) Fv fragments consisting of VL and VH domains of a single antibody; (iv) dAb fragments consisting of a VH domain (Ward, E et al. (1989) Nature 341, 544-546); (v) isolated CDR regions; (vi) F(ab')2 fragments, bivalent fragments containing two linked Fab fragments; (vii) single-stranded Fv molecules (scFv) in which the VH and VL domains are linked by a peptide linker that can associate the two domains to form an antigen-binding site (Bird et al. (1988) Science. 242, 423-426; Huston et al. (1988) PNAS) USA.85,5879-5883); (viii) Multivalent antibody fragments (scFv dimers, trimers and / or tetramers (Power and Hudson (2000) J. Immunol. Methods 242,193-204)); (ix) Bispecific single-stranded Fv dimers (PCT / US92 / 09965) and (x) "diabodies," multivalent or multispecific fragments constructed by gene fusion (WO94 / 13804; P. Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90,6444-6448).

[0103] The "antibody binding site" is a structural part of an antibody molecule that consists of a light chain or heavy chain, as well as a light chain variable region and a hypervariable region, which specifically bind to an antigen.

[0104] In this specification, the phrase "antibody molecule" in these various grammatical forms refers to both intact immunoglobulin molecules and the immunoactive portion of immunoglobulin molecules.

[0105] Exemplary antibody molecules include intact immunoglobulin molecules, substantially intact immunoglobulin molecules, and portions of immunoglobulin molecules containing paratopes, the portions of which include those known in the art as Fab, Fab', F(ab')Z, and F(v), and these portions are preferred for use in the therapeutic methods described herein.

[0106] Antibodies may also be bispecific, in which case one binding domain of the antibody is the specific binding member of the present invention, and the other binding domain has a different specificity, for example, to supplement effector function or similar functions. The bispecific antibody of the present invention has one binding domain that is the specific binding member of the present invention [including this fragment], and the other binding domain that is a different antibody or this fragment [different anti-EGFR antibodies, e.g., antibody 528 (US Patent No. 4,943,533), chimeric and humanized 225 antibody (US Patent No. 4,943,533 and WO / 9640210), anti-de2-7 antibody, e.g., 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 CF et al. (2000) Cancer Res. 60(16):44534460), ICR62 (Modjtahedi H et al. This includes bispecific antibodies, such as those described in al. (1993) Cell Biophys. Jan-Jun; 22(1-3):129-46; Modjtahedi et al. (2002) PAACR 55(14):3140-3148, or the antibody of Wikstrand et al. (Wikstrand C. et al (1995) Cancer Res. 55(14):3140-3148). Other binding domains may be antibodies that recognize or target specific cell types, such as nerve or glial cell-specific antibodies.In the case of the bispecific antibody of the present invention, one binding domain of the antibody of the present invention may be bound to another binding domain or molecule that recognizes a specific cell receptor and / or modulates a cell in a specific manner, such as an immunomodulatory component (e.g., one or more interleukins), a growth regulatory component or cytokine (e.g., tumor necrosis factor (TNF), and in particular the type of TNF bispecificity demonstrated in U.S. Patent Application No. 60 / 355,838 filed on February 13, 2002, which is incorporated herein in whole), a toxin (e.g., lysine), or an antimitotic or apoptotic agent or factor.

[0107] The Fab and F(ab')2 portions of the antibody molecule can be prepared by proteolytic reactions of papain and pepsin, respectively, to substantially intact antibody molecules by known methods. See, for example, Theofilopolous et al., U.S. Patent No. 4,342,566. The Fab' antibody molecule portion is also known and is produced from the F(ab')2 portion, then the disulfide bond linking the two heavy chain portions is reduced with mercaptoethanol or the like, and the resulting protein mercaptan is subsequently alkylated with a reagent such as iodoacetamide. Antibodies containing intact antibody molecules are preferred herein.

[0108] In these various grammatical forms, the phrase "monoclonal antibody" refers to an antibody that has only one type of antibody-binding site capable of immunely reacting with a specific antigen. Therefore, monoclonal antibodies generally exhibit only a single binding affinity to any antigen they immunely react with. Monoclonal antibodies can also include antibody molecules that have multiple antibody-binding sites, each immune-specific to different antigens, such as bispecific (chimeric) monoclonal antibodies.

[0109] The term "antigen-binding domain" describes a portion of an antibody that contains areas that specifically bind to some or all of an antigen and are complementary to some or all of the antigen. When the antigen is large, the antibody can only bind to specific portions of the antigen, which are called epitopes. The antigen-binding domain may be provided by one or more antibody variable domains. Preferably, the antigen-binding domain includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0110] "Post-translational modifications" can encompass any one or more modifications (one or more) that a protein experiences after translation is complete and after co-translational release from a ribosome or onto an incomplete polypeptide, including covalent modifications. Examples of post-translational modifications include, but are not limited to, phosphorylation, myristylation, ubiquitination, glycosylation, coenzyme attachment, methylation, and acetylation. Post-translational modifications may modulate or affect the activity of a protein, its intracellular or extracellular destination, its stability or half-life, and / or its recognition by a ligand, receptor, or other protein. Post-translational modifications may occur in organelles, in the nucleus or cytoplasm, or extracellularly.

[0111] The term "specific" can be used to describe a situation in which one member of a specific binding pair will not exhibit any significant binding to any molecule other than its specific binding partner(s). This term also applies, for example, when an antigen-binding domain is specific to a particular epitope held by multiple antigens, in which case the specific binding member holding the antigen-binding domain will be able to bind to various antigens holding this epitope.

[0112] The term "comprise" is generally used in the sense of "include," that is, to allow the presence of one or more features or components.

[0113] The term "essentially consisting of" refers to a product of a defined number of residues that are not covalently attached to a larger product, particularly a peptide sequence. However, in the case of the peptides of the present invention mentioned above, it will be understood by those skilled in the art that small modifications to the N or C terminus of the peptide are possible, such as terminal chemical modifications to add a protecting group or similar, such as C-terminal amidation.

[0114] The term “isolated” refers to the state in which, in accordance with the present invention, a specific binding member of the present invention, or a nucleic acid encoding such a binding member, will be. The members and nucleic acids will be free from, or substantially free from, naturally associated materials, such as other polypeptides or nucleic acids, that are found in their natural environment or in the environment in which they are prepared (e.g., cell culture) (when such preparation is carried out by recombinant DNA technology performed in vitro or in vivo). The members and nucleic acids may be prepared with diluents or adjuvants and still be, in practice, isolated—for example, the members will typically be mixed with gelatin or other carriers when used to coat microtiter plates for use in immunoassays, or with pharmaceutically acceptable carriers or diluents when used in diagnostics or therapy. Specific binding members may be glycosylated naturally or by heterologous eukaryotic cell systems, or they may not be glycosylated (e.g., when produced by expression in eukaryotic cells).

[0115] Furthermore, as used herein, the terms "glycosylation" and "glycosylated" include and encompass post-translational modifications of proteins called glycoproteins by the addition of oligosaccharides. In particular, oligosaccharides, including N-linked and O-linked oligosaccharides, are added to glycosylation sites in glycoproteins. N-linked oligosaccharides are the most common found in glycoproteins, particularly when Asn residues are present in the sequence NXS / T (where X cannot be Pro or Asp). In the biosynthesis of N-linked glycoproteins, high-mannose oligosaccharides (generally consisting of dolichol, N-acetylglucosamine, mannose, and glucose) are initially formed in the endoplasmic reticulum (ER). These high-mannose glycoproteins are then transported from the ER to the Golgi apparatus, where further processing and modification of the oligosaccharides occur. O-linked oligosaccharides are attached to the hydroxyl group of a Ser or Thr residue. In the case of O-linked oligosaccharides, N-acetylglucosamine is initially transported to the Ser or Thr residue by acetylglucosaminyltransferase in the ER. This protein then moves to the Golgi apparatus, where further modification and chain extension occur. O-linked modifications may occur as simple addition of an OGlcNAc monosaccharide alone to the Ser or Thr site, and under different conditions, this modification may be phosphorylation rather than glycosylation.

[0116] In this specification, "pg" means picogram, "ng" means nanogram, "ug" or "μg" means microgram, "mg" means milligram, "uL" or "μL" means microliter, "mL" means milliliter, and "L" means liter.

[0117] The terms “806 antibody,” “mAb806,” “ch806,” and any variants not specifically listed herein may be used interchangeably and, as used throughout this application and claims, refer to proteinaceous materials comprising one or more proteins, and extend to the chimeric antibody ch806 incorporated in and constituting parts of SEQ ID NOs: 7 and 8, as well as the amino acid sequence data described herein and presented in SEQ ID NOs: 2 and 4, and SEQ ID NOs: 7 and 8, and proteins having the activity profiles described herein and in claims. Proteins exhibiting substantially equivalent or modified activity are therefore considered similarly. These modifications may be planned, such as modifications obtained by site-directed mutagenesis, or accidental, such as those obtained by mutations in the host that produces this complex or as this designated subunit. Furthermore, the terms “806 antibody,” “mAb806,” and “ch806” are intended to include, within their scope, the proteins specifically listed herein, as well as substantially homologous analogs and allelic variants.

[0118] The terms “humanized 806 antibody,” “[hu806],” and “venierated 806 antibody,” as well as any variants not specifically listed herein, may be used interchangeably herein and, as used throughout this application and claims, refer to proteinaceous materials comprising one or more proteins, and extend to proteins having the amino acid sequence data described herein and presented in SEQ ID NO: 42 and SEQ ID NO: 47, as well as the activity profiles described herein and in claims. Proteins exhibiting substantially equivalent or modified activity are thus similarly considered. These modifications may be planned, such as modifications obtained by site-directed mutagenesis, or accidental, such as those obtained by mutations in the host that produces this complex or such as this designated subunit. Furthermore, the terms “humanized 806 antibody,” “[hu806],” and “venierated 806 antibody” are intended to include, within these scopes, the proteins specifically listed herein, as well as substantially homologous analogs and allelic variants.

[0119] The terms “175 antibody” and “mAb175,” as well as any variants not specifically enumerated, may be used interchangeably herein and, as used throughout this application and claims, refer to proteinaceous materials comprising one or more proteins, and extend to proteins having the amino acid sequence data described herein and presented in SEQ ID NO: 129 and SEQ ID NO: 134, as well as the activity profiles described herein and in claims. Proteins exhibiting substantially equivalent or modified activity are therefore similarly considered. These modifications may be planned, such as modifications obtained by site-directed mutagenesis, or accidental, such as those resulting from mutations in the host that produces this complex or as this designated subunit. Furthermore, the terms “175 antibody” and “mAb175” are intended to include, within their scope, the proteins specifically enumerated herein, as well as substantially homologous analogs and allelic variants.

[0120] The terms “124 antibody” and “mAb124,” as well as any variants not specifically enumerated herein, may be used interchangeably herein and, as used throughout this application and claims, refer to proteinaceous materials comprising one or more proteins, and extend to proteins having the amino acid sequence data described herein and presented in SEQ ID NO: 22 and SEQ ID NO: 27, as well as the activity profiles described herein and in claims. Proteins exhibiting substantially equivalent or modified activity are therefore considered similarly. These modifications may be planned, such as modifications obtained by site-directed mutagenesis, or they may be accidental, such as those obtained by mutations in the host that produces this complex or this designated subunit. Furthermore, the terms “124 antibody” and “mAb124,” as well as substantially homologous analogs and allelic variants, are intended to be included within these scopes.

[0121] The terms “1133 antibody” and “mAb1133,” as well as any variants not specifically enumerated, may be used interchangeably herein and, as used throughout this application and claims, refer to proteinaceous materials comprising one or more proteins, and extend to proteins having the amino acid sequence data described herein and presented in SEQ ID NO: 32 and SEQ ID NO: 37, as well as the activity profiles described herein and in claims. Proteins exhibiting substantially equivalent or modified activity are therefore considered similarly. These modifications may be planned, such as modifications obtained by site-directed mutagenesis, or accidental, such as those obtained by mutations in the host that produces this complex or such as this designated subunit. Furthermore, the terms “11133 antibody” and “mAb1133” are intended to include, within these scopes, the proteins specifically enumerated herein, as well as substantially homologous analogs and allelic variants.

[0122] The amino acid residues described herein are preferably in the "L" isomer form. However, any L-amino acid residue can be substituted with a "D" isomer residue, as long as the desired functional properties of immunoglobulin binding are maintained by this polypeptide. NH2 refers to the free amino group at the amino terminus of the polypeptide. COOH refers to the free carboxyl group at the carboxy terminus of the polypeptide. Following standard polypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969), the abbreviations for amino acid residues are shown in the following correspondence table:

[0123] [Table 1] It should be noted that all amino acid residue sequences are represented herein by formulas where the left-right orientation is the conventional direction from the amino terminus to the carboxyl terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide formed by the attachment of one or more amino acid residues to a further sequence. The table above is provided to correlate the three-letter and one-letter notations that may alternately appear herein.

[0124] A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an independent unit of DNA replication in vivo, meaning it can replicate under its own control.

[0125] A "vector" is a replicon, such as a plasmid, phage, or cosmid, that can be fitted with another DNA segment and cause replication of that fitted segment.

[0126] "DNA molecule" refers to a polymerized form of deoxyribonucleotide (adenine, guanine, thymidine, or cytosine) in either a single-stranded or double-stranded helix. This term refers only to the primary and secondary structures of the molecule and is not limited to any particular tertiary form. Accordingly, this term includes, in particular, double-stranded DNA found in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. When discussing the structure of a particular double-stranded DNA molecule, sequences may be described herein according to the usual practice of giving the sequence exclusively in the 5' to 3' direction along the non-transcribed DNA strand (i.e., the strand with a sequence homologous to mRNA).

[0127] A "replication origin" refers to a DNA sequence involved in DNA synthesis.

[0128] A DNA "coding sequence" is a double-stranded DNA sequence that, when controlled by appropriate regulatory sequences, is transcribed and translated into polypeptides in vivo. The boundaries of a coding sequence are determined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. Coding sequences include, but are not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. Polyadenylation signals and transcription termination sequences are typically located 3' relative to the coding sequence.

[0129] Transcriptional and translational regulatory sequences are DNA regulatory sequences that prepare for the expression of coding sequences in host cells, such as promoters, enhancers, polyadenylation signals, terminators, and similar elements.

[0130] A "promoter sequence" is a DNA regulatory region that can bind to RNA polymerase within a cell and initiate the transcription of a downstream (3' direction) coding sequence. For the purposes of defining this invention, a promoter sequence is bound at its 3' end by a transcription start site and extends upstream (5' direction) to contain the minimum number of bases or elements necessary to initiate transcription at a detectable level above the background. Within the promoter sequence, there will be a transcription start site (traditionally defined by mapping at nuclease S1), as well as a protein-binding domain (consensus sequence) responsible for RNA polymerase binding. Eukaryotic promoters often, though not always, contain "TATA" and "CAT" boxes. Prokaryotic promoters contain the Shinc Dalgarno sequence in addition to the -10 and -35 consensus sequences.

[0131] A “regulatory 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 intracellular transcription and translation regulatory sequences when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.

[0132] A signal sequence can be included before the coding sequence. This sequence codes for a signal peptide, which communicates to the host cell to direct the polypeptide towards the cell surface or to secrete the polypeptide into the culture medium. The signal peptide is clipped by the host cell before the protein leaves the cell. Signal sequences can be found in relation to a variety of proteins from prokaryotes and eukaryotes.

[0133] As used herein in reference to the probes of the present invention, the term "oligonucleotide" is defined as a molecule composed of two or more, preferably three or more, ribonucleotides. This exact size depends on many factors, which in turn will depend on the ultimate function and application of the oligonucleotide.

[0134] As used herein, the term "primer" refers to an oligonucleotide, whether spontaneously occurring in purified restriction digestion or synthetically produced, that can act as a starting point for synthesis when subjected to conditions that induce the synthesis of a primer extension product complementary to a nucleic acid chain, i.e., in the presence of a nucleotide and an inducer, such as DNA polymerase, and at a suitable temperature and pH. Primers may be single-stranded or double-stranded and must be long enough to initiate the synthesis of the desired extension product in the presence of an inducer. The exact length of the primer will depend on many factors, including temperature, primer source, and the use of this method. For example, for diagnostic applications, depending on the complexity of the target sequence, oligonucleotide primers generally contain 15-25 or more nucleotides, but may contain fewer.

[0135] The primers described 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 each of these strands in order to hybridize. Therefore, the primer sequence does not need to reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment can be attached to the 5' end of the primer, and the remainder of this primer sequence may be complementary to the strand. Alternatively, non-complementary bases or longer sequences may be scattered on the primer, provided that this primer sequence has sufficient complementarity to the strand's sequence to hybridize with the strand's sequence and thereby form a template for the synthesis of the extension product.

[0136] As used herein, the terms “restriction endonuclease” and “restriction enzyme” refer to bacterial enzymes that cleave double-stranded DNA at or near a specific nucleotide sequence.

[0137] Cells are “transformed” when exogenous or heterologous DNA is introduced into them. The transformed DNA may or may not be integrated (covalently linked) into the chromosomal DNA that constitutes the cell’s genome. For example, in prokaryotic cells, yeast, and mammalian cells, transformed DNA may be maintained on episomal elements such as plasmids. With respect to eukaryotic cells, stably transformed cells are those in which the transformed DNA is integrated into the chromosome and inherited by daughter cells through chromosomal replication. This stability is demonstrated by the ability of eukaryotic cells to establish cell lines or clones consisting of a population of daughter cells containing transformed DNA. A “clone” is a population of cells induced by mitosis from a single cell or a common ancestor. A “cell line” is a clone of primary cells that can grow stably in vitro over many generations.

[0138] 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 their nucleotides match over the defined length of these nucleic acid sequences. Substantially homologous sequences can be identified by comparing the sequences using standard software available in sequence databases, or by Southern hybridization experiments under stringent conditions as defined for this particular system. Defining appropriate hybridization conditions is within the scope of the skills of the art. See, for example, Maniatis et al., cited above; DNA Cloning, Vols. I & II, cited above; Nucleic Acid Hybridization, cited above.

[0139] While the disclosed sequence encodes an antibody, it should be understood that DNA sequences encoding a specific binding member (antibody) of the present invention that are degenerate to such a sequence are also within the scope of the present invention. "Degenerate to" means that specific amino acids are specified using different three-letter codons. It is well known in the art that the following codons can be used interchangeably to encode each specific amino acid.

[0140] Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or L) UUA or UUG or CUU or CUC or CUA or CUG Isoleucine (He or I) AUU or AUC or AUA Methionine (Met or M) AUG Valine (Val or V), GUU or GUA, GUC 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), UAG (amber), or UGA (opal) It should be understood that the codons listed above refer to RNA sequences. The corresponding codons for DNA have a T instead of a U.

[0141] For example, the disclosed sequence of the antibody of the present invention can be mutated so that a specific codon is changed to a codon encoding a different amino acid. Such mutations can generally be made by enabling the fewest nucleotide changes. This type of substitutional mutation can be performed non-conservatively (i.e., by changing the codon from an amino acid belonging to a particular classification of amino acids of a specific size or characteristic to an amino acid belonging to a different classification) or conservatively (i.e., by changing the codon from an amino acid belonging to a particular classification of amino acids of a specific size or characteristic to an amino acid belonging to the same classification) to change the amino acids in the resulting protein. Such conservative changes generally result in little to no change in the structure and function of the resulting protein. Non-conservative changes are 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 that do not significantly alter the activity or binding properties of the resulting protein.

[0142] The following is an example of various classifications of amino acids: amino acids containing a nonpolar R group Alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine amino acids having a non-charged R group Glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine Amino acids containing a charged R group (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)

[0143] Another classification might be amino acids that contain a phenyl group: Phenylalanine, tryptophan, tyrosine

[0144] Another classification method could be based on molecular weight (i.e., the size of the R group): Glycine 75 Alanine 89 Serine 105 Proline 115 Valin 117 Threonine 119 Cysteine ​​121 Leucine 131 Isoleucine 131 Asparagine 132 Aspartic acid 133 Glutamine 146 Ricin 146 Glutamic acid 147 Methionine 149 Histidine (at pH 6.0) 155 Phenylalanine 165 Arginine 174 Tyrosine 181 Tryptophan 204

[0145] Particularly preferred substitutions are: - To maintain a positive charge, use Lys instead of Arg, and vice versa; - To maintain a negative charge, use Glu instead of Asp and vice versa; - Ser instead of Thr to maintain free -OH; and - To maintain free NH2, use Gin instead of Asn That is the case.

[0146] Amino acid substitutions can also be introduced to replace amino acids with particularly desirable properties. For example, Cys can be introduced at potential sites for disulfide crosslinking with another Cys. His can be introduced specifically as a "catalytic" site (i.e., His can act as an acid or base, and is the most common amino acid in biochemical catalysts). Pro can be introduced to induce this particularly planar structure (three turns within the protein structure).

[0147] Two amino acid sequences are "substantially homologous" if at least about 70% (preferably at least about 80%, and most preferably at least 90 or 95%) of the amino acid residues are identical or represent conservative substitutions.

[0148] A “non-homologous” region of a DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not naturally found associated with this larger molecule. Therefore, when a non-homologous region codes for a mammalian gene, this gene would typically be adjacent to DNA that is not adjacent to the mammalian genomic DNA within the genome of the source organism. Another example of a homologous coding sequence is a coding sequence that is itself a construct not naturally found (e.g., a cDNA containing introns, or a synthetic sequence with codons different from those of the native gene). Allele mutations or spontaneous mutation events do not result in non-homologous regions of DNA as defined herein.

[0149] The phrase "medically acceptable" refers to molecular entities and compositions that are physiologically acceptable and do not generally cause allergic or similar adverse reactions, such as acute gastric motility, dizziness, and similar reactions, when administered to humans.

[0150] The phrase “therapeutic dose” is used herein to mean an amount sufficient to prevent a clinically significant change in a target cell population, cancer cell population, or tumor growth or progression or mitotic activity, or other characteristics of the disease state, and preferably by at least about 30 percent, preferably at least 50 percent, preferably at least 70 percent, preferably at least 80 percent, and preferably at least 90 percent. For example, it may reduce EGFR activation, or the activity, quantity, or number of EGFR-positive cells, particularly antibody or binding member activity or positive cells.

[0151] A DNA sequence is “operatably linked” to an expression regulatory sequence when the expression regulatory sequence controls and regulates the transcription and translation of the DNA sequence. The term “operatably linked” includes having an appropriate start signal (e.g., ATG) before the expressed DNA sequence and maintaining the correct reading frame in order to enable the expression of the DNA sequence under the control of the expression regulatory sequence and the production of the desired product encoded by the DNA sequence. If a gene that you wish to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal may be inserted before this gene.

[0152] The term “standard hybridization conditions” refers to salt and temperature conditions substantially equivalent to 5 × SSC and 65°C for both hybridization and washing. However, it will be understood by those skilled in the art that such “standard hybridization conditions” depend on individual conditions, including the concentrations of sodium and magnesium in the buffer, nucleotide sequence lengths and concentrations, mismatch rates, formamide percentages, and similar factors. Whether the two sequences to be hybridized are RNA-RNA, DNA-DNA, or RNA-DNA is also important in determining the “standard hybridization conditions.” Such standard hybridization conditions can be readily determined by those skilled in the art according to well-known principles, where hybridization is generally 10-20°C below the predicted or confirmed Tm, and accompanied by washing with higher stringency if desired.

[0153] The present invention provides novel specific binding members, particularly antibodies or fragments thereof (including immunogenic fragments), that recognize EGFR epitopes found in tumorigenic, hyperproliferative, or abnormal cells, which are enhanced or clearly discernible in said cells based on abnormal post-translational modifications and are undetectable in normal or wild-type cells. In specific, but non-limiting, embodiments, the binding member, e.g., an antibody, recognizes EGFR epitopes that are enhanced or clearly discernible based on simple carbohydrate modifications or early glycosylation and are reduced or undistinguishable in the presence of complex carbohydrate modifications or glycosylation. The specific binding member, e.g., an antibody or fragment thereof, does not bind to or recognize normal or wild-type cells containing normal or wild-type EGFR epitopes in the absence of overexpression and in the presence of normal EGFR post-translational modifications.

[0154] The present invention relates to EGFR epitopes exhibited in tumorigenic, hyperproliferative, or abnormal cells, which are enhanced, exposed, or clearly discernible in such cells and undetectable in normal or wild-type cells, particularly EGFR peptides. 287CGADSYEMEEDGVRKC 302 The present invention provides novel antibodies 806, 175, 124, 1133, ch806, and hu806, as well as fragments thereof (including immunogenic fragments), that recognize (SEQ ID NO: 14). In a particular, but non-limiting, embodiment, the antibodies recognize EGFR epitopes that are enhanced or clearly recognized based on simple carbohydrate modification or early glycosylation, and reduced or unclear in the presence of complex carbohydrate modification or glycosylation. The antibodies or fragments thereof do not bind to or recognize normal or wild-type cells containing normal or wild-type EGFR epitopes in the absence of overexpression, amplification, or oncogenic events.

[0155] In a special aspect of the present invention, and as described above, we have discovered novel monoclonal antibodies 806, 175, 124, 1133, ch806, and hu806 that specifically recognize amplified wild-type EGFR and de2-7 EGFR, and moreover, bind to an epitope different from the characteristic junction peptide of the de2-7 EGFR mutation. In addition, mAb806, mAb175, mAb124, mAb1133, and hu806 do not recognize normal, wild-type EGFR expressed on the cell surface of glioma cells, but bind to the extracellular domain of EGFR immobilized on the surface of the ELISA plate, indicating a higher-order structural epitope having a polypeptide configuration.

[0156] Importantly, mAb806, mAb175, mAb124, mAb1133, ch806, and hu806 express higher levels of endogenous wtEGFR in most other normal tissues, but do not significantly bind to normal tissues such as liver and skin where EGFR is not overexpressed or amplified. Therefore, mAb806, mAb175, mAb124, mAb1133, and hu806 demonstrate novel and useful specificity by recognizing de2-7 EGFR and amplified EGFR, but not recognizing the specific junction peptide that is unique to normal, wild-type EGFR or de2-7 EGFR. In a preferred embodiment, the mAb806, mAb175, mAb124, mAb1133, and hu806 of the present invention comprise the VH and VL chain CDR domain amino acid sequences shown in Figures 14B and 15B; 74B and 75B; 51B and 51D; 52B and 52D; and 55A and 55B, respectively (SEQ ID NOs: 2 and 4, 129 and 134, 22 and 27, 32 and 37, and 42 and 47; SEQ ID NO: 42, which contains the hu806 VH chain signal peptide and VH chain sequence of SEQ ID NOs: 163 and 164, respectively; and SEQ ID NOs: 47, which contains the hu806 VL chain signal peptide and VL chain sequence of SEQ ID NOs: 165 and 166, respectively).

[0157] In another embodiment, the present invention provides an antibody that can compete with the 175 antibody under conditions in which at least 10% of antibodies having the VH and VL chain sequences of the 175 antibody (sequence numbers: 129 and 134, respectively) are blocked from binding to de2-7 EGFR by competition with such antibodies in an ELISA assay. As shown above, an anti-idiotype antibody is considered here.

[0158] The present invention relates to specific binding members, particularly antibodies or fragments thereof, that are present in cells expressing amplified EGFR or de2-7 EGFR and recognize EGFR epitopes that are undetectable in cells expressing normal or wild-type EGFR, especially in the presence of normal post-translational modifications.

[0159] An additional non-limiting observation or feature of the antibodies of the present invention is the recognition of these epitopes in the presence of high mannose groups, which is specific to early glycosylation or simple carbohydrate modification, as further stated and demonstrated herein. For example, modified or abnormal glycosylation promotes the presence and / or recognition of antibody epitopes, or includes a portion of antibody epitopes.

[0160] Glycosylation includes and encompasses post-translational modifications of proteins called glycoproteins, which involve the addition of oligosaccharides. Oligosaccharides, particularly N-linked and O-linked oligosaccharides, are added to glycosylation sites in glycoproteins. N-linked oligosaccharides are the most common type found in glycoproteins, especially when Asn residues are present in the sequence NXS / T (where X cannot be Pro or Asp). In the biosynthesis of N-linked glycoproteins, high-mannose oligosaccharides (generally consisting of dolichol, N-acetylglucosamine, mannose, and glucose) are initially formed in the endoplasmic reticulum (ER). These high-mannose glycoproteins are then transported from the ER to the Golgi apparatus, where further processing and modification of the oligosaccharide typically occur. O-linked oligosaccharides are attached to the hydroxyl group of a Ser or Thr residue. In the case of O-linked oligosaccharides, N-acetylglucosamine is initially transported to the Ser or Thr residue by acetylglucosaminyltransferase in the ER. This protein then moves to the Golgi apparatus, where further modification and chain extension occur.

[0161] In a special aspect of the present invention, and as described above, the inventors have discovered novel monoclonal antibodies, exemplified herein by antibodies called mAb806 (and its chimeric ch806), mAb175, mAb124, mAb1133, and hu806, that specifically recognize amplified wild-type EGFR and de2-7 EGFR but bind to an epitope different from the characteristic junction peptide of this 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 based on abnormal post-translational modifications. In addition, these antibodies do not recognize normal, wild-type EGFR expressed on the cell surface of glioma cells, but bind to the extracellular domain of EGFR immobilized on the surface of an ELISA plate, which indicates a higher-order structural epitope having a polypeptide configuration. Importantly, these antibodies express higher levels of endogenous wtEGFR in most other normal tissues, but do not significantly bind to normal tissues such as liver and skin that do not overexpress or amplify EGFR. Therefore, these antibodies demonstrate novel and useful specificity, recognizing de2-7 EGFR and amplified EGFR, but not recognizing the specific junction peptide that characterizes normal, wild-type EGFR, or de2-7 EGFR.

[0162] In a preferred embodiment, the antibody has the characteristics of an antibody identified and characterized by the inventors, particularly the characteristic of recognizing amplified EGFR and de2-7EGFR. In a particularly preferred embodiment, the antibody is mAb806, mAb175, mAb124, mAb1133, and hu806, or active fragments thereof. In a further preferred embodiment, the antibody of the present invention comprises VH and VL chain amino acid sequences as illustrated in Figures 16 and 17; 74B and 75B; 51B and 51D; 52B and 52D; and 55A and 55B, respectively.

[0163] Preferably, the specific binding member or antibody epitope is located within a region containing residues 273-501 of the mature normal or wild-type EGFR sequence, and preferably, the epitope contains residues 287-302 (SEQ ID NO: 14) of the mature normal or wild-type EGFR sequence. Thus, we also provide a specific binding protein, such as an antibody, that binds to de2-7 EGFR with an epitope located within a region containing residues 273-501 and residues 287-302 (SEQ ID NO: 14) of the EGFR sequence. The epitope can be determined by any conventional epitope mapping technique known to those skilled in the art. Alternatively, the DNA sequence encoding residues 273-501 and 287-302 (SEQ ID NO: 14) can be digested, and the resulting fragment can be expressed in a suitable host. Antibody binding can then be determined as described above.

[0164] In particular, the member will bind to epitopes containing residues 273-501 of mature normal or wild-type EGFR, and more specifically, residues 287-302 (SEQ ID NO: 14). However, other antibodies exhibiting the same or substantially similar reactivity patterns also constitute embodiments of the present invention. This can be determined by comparing such members with antibodies containing the VH and VL chain domains shown in SEQ ID NOs: 2 and 4; 129 and 134; 22 and 27; 32 and 37; and 42 and 47, respectively. Generally, this comparison will be performed using Western blotting, where the binding member is bound to a double-repeated blot prepared from a nuclear sample of cells to directly compare the binding patterns.

[0165] In another embodiment, the present invention provides antibodies that can compete with mAb806 and at least 10% of antibodies having VH and VL chain sequences for one such antibody, under conditions that such antibodies are blocked from binding to de2-7EGFR by competition with such antibodies in an ELISA assay. As described above, anti-idiotype antibodies are conceivable and are illustrated herein.

[0166] In another embodiment, the present invention provides antibodies that can compete with mAb175, mAb124, and / or mAb1133, and at least 10% of antibodies having VH and VL chain sequences for one of such antibodies, under conditions that they are blocked from binding to de2-7EGFR by competition with such antibodies in an ELISA assay. As described above, anti-idiotype antibodies are conceivable and are illustrated herein.

[0167] In another embodiment, the present invention provides antibodies that can compete with mAb806, mAb175, mAb124, mAb1133 and / or hu806, and at least 10% of antibodies having VH and VL chain sequences for one of such antibodies, under conditions that such antibodies are blocked from binding to de2-7EGFR by competition with such antibodies in an ELISA assay. As described above, anti-idiotype antibodies are conceivable and are illustrated herein.

[0168] Another aspect of the present invention comprises an isolated polypeptide essentially consisting of an epitope containing mature wild-type EGFR residues 273-501 and more specifically residues 287-302 (SEQ ID NO: 14). The peptides of the present invention are particularly useful therapeutically or prophylactically, including in diagnostic assays or kits and in antitumor or anticancer vaccines. Accordingly, the peptide compositions of the present invention include pharmaceutical compositions and immunogenic compositions.

[0169] Diagnostic and therapeutic applications The specific properties of the specific binding members of the present invention, particularly antibodies or fragments thereof, that enable the binding member(s) to recognize EGFR epitopes found in tumorigenic, hyperproliferative, or abnormal cells and undetectable in normal or wild-type cells, and that these epitopes are enhanced or clearly discernible based on abnormal post-translational modifications, and that the member(s) bind to de2-7 EGFR and amplified EGFR but not to wtEGFR, result in diagnostic and therapeutic applications for identifying, characterizing, targeting, and treating, reducing or eliminating numerous tumorigenic cell types and tumor types, such as head and neck, breast, lung, bladder, or prostate tumors and gliomas, without the problems associated with normal tissue uptake sometimes seen with previously known EGFR antibodies. Accordingly, cells that overexpress EGFR (for example, by amplification or expression of mutant or variant EGFR), particularly those exhibiting abnormal post-translational modifications, can be recognized, isolated, characterized, targeted, and treated or eliminated using the binding members(s) of the present invention, especially antibodies(s) or fragments thereof.

[0170] In a further embodiment of the present invention, a method is provided for treating any condition associated with or resulting from tumors, cancerous conditions, precancerous conditions, and hyperproliferative cell growth, comprising the administration of mAb806, mAb175, mAb124, mAb1133, and / or hu806.

[0171] Accordingly, the antibodies of the present invention can specifically classify the properties of EGFR tumors or tumorigenic cells by staining or otherwise recognizing tumors or cells in which EGFR overexpression, particularly amplification, and / or EGFR mutations, particularly de2-7EGFR, are present. Furthermore, the antibodies of the present invention, as exemplified by mAb806 (and chimeric antibody ch806), mAb175, mAb124, mAb1133, and hu806, demonstrate significant in vivo antitumor activity against tumors containing amplified EGFR and against de2-7EGFR-positive xenografts.

[0172] As briefly described above, the inventors have discovered that the specific binding member of the present invention recognizes tumor-associated forms of EGFR (de2-7 EGFR and amplified EGFR) when expressed in normal cells, but does not recognize normal, wild-type receptors. Antibody recognition is thought to depend on abnormal post-translational modifications of EGFR expressed in cells exhibiting EGFR gene overexpression (e.g., specific glycosylation, acetylation, or phosphorylation variants).

[0173] As described below, the antibodies of the present invention have been used in therapeutic studies and have been shown to inhibit the growth of overexpressing (e.g., amplified) human tumor EGFR xenografts and human de2-7 EGFR-expressing xenografts, as well as to induce significant necrosis within such tumors.

[0174] Furthermore, the antibody of the present invention inhibits the growth of intracranial tumors in a prophylactic model. This model involves injecting glioma cells expressing de2-7 EGFR into nude mice, and then intracranially injecting the antibody on the same day or within 1 to 3 days, optionally in repeated doses. The antibody dose is appropriately about 10 μg. When mice injected with the antibody were compared to controls, a significant increase in the survival rate of treated mice was found.

[0175] Accordingly, a further embodiment of the present invention provides a method for treating any condition associated with or resulting from hyperproliferative cell growth, including the administration of a specific binding member of the present invention.

[0176] The antibodies of the present invention are designed for use in methods for the diagnosis and treatment of tumors, particularly epithelial tumors, in human or animal subjects. These tumors may be any type of primary or secondary solid tumor, including but not limited to gliomas, breast, lung, prostate, head, or neck tumors.

[0177] Binding members and antibody production The general methodology for producing monoclonal antibodies using hybridomas is well known. Other techniques, such as direct transformation of B lymphocytes with oncogenic DNA or transfection with Epstein-Barr virus, can also be used to create immortal, antibody-producing cell lines. See, for example, M. Schreier et al., "Hybridoma Techniques" (1980); Hammering et al., "Monoclonal Antibodies And T cell Hybridomas" (1981); Kennett et al., "Monoclonal Antibodies" (1980); U.S. Patent 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; and 4,493,890.

[0178] A panel of monoclonal antibodies produced against EFGR can be screened for various properties, such as isotype, epitope, and affinity. Monoclonal antibodies that mimic the activity of EFGR or its subunits are of particular interest. Such monoclonal antibodies can be readily identified by specific binding member activity assays. High-affinity antibodies are also useful when immunoaffinity purification of natural or recombinant specific binding members is possible.

[0179] Methods for producing polyclonal anti-EFGR antibodies are well known in the art. See Nestor et al., U.S. Patent No. 4,493,795. Monoclonal antibodies generally containing the Fab and / or F(ab')2 portions of a useful antibody molecule can be prepared using the hybridoma technique described in Antibodies—A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, New York (1988) (which is incorporated herein by reference). Briefly, lymphocytes obtained from the spleen of a mammal immunized with appropriate EGFR are fused with melanoma or other autoimmune cell lines to form a hybridoma from which a monoclonal antibody composition is produced.

[0180] Typically, polyethylene glycol (PEG) 6000 is used to fuse spleen cells with melanoma cells. The fused hybrids are selected based on their sensitivity to HAT. Hybridomas that produce monoclonal antibodies useful for carrying out the present invention are identified by their ability to immune-react with the antibodies or binding members of the present invention, and by their ability to inhibit specified tumorigenic or hyperproliferative activity in target cells.

[0181] Monoclonal antibodies useful for carrying out the present invention can be produced by initiating a monoclonal antibody hybridoma culture containing a nutrient medium that contains hybridomas that secrete antibody molecules with appropriate antigen specificity. The culture is maintained under conditions and for a period of time sufficient for the hybridomas to secrete antibody molecules into the medium. The antibody-containing medium is then collected. The antibody molecules can then be further isolated by known techniques.

[0182] Culture media useful for preparing these compositions are well known and commercially available in the art, as well as synthetic media, inbred mice, and similar materials. An exemplary synthetic medium is Dulbecco's Minimum Essential Medium (DMEM; Dulbecco et al., Virol. 8:396 (1959)) supplemented with 4.5 g m / L glucose, 20 mg glutamine, and 20% fetal bovine serum. An exemplary inbred mouse strain is Balb / c.

[0183] Methods for producing monoclonal anti-EGFR antibodies are also well known in the art. See Niman et al., Proc. Natl. Acad. Sci. USA, 80:4949-4953 (1983). Typically, previously described procedures for producing anti-EGFR monoclonal antibodies use EGFR or a peptide analog alone, or conjugate it to an immunogenic carrier as an immunogen. These hybridomas are screened for their ability to produce antibodies that immunely respond to EGFR present in tumorigenic, abnormal, or hyperproliferative cells. Other anti-EGFR antibodies include, but are not limited to, HuMAX-EGFr antibody from Genmab / Medarex, antibody 108 (ATCC HB9764) and U.S. Patent No. 6,217,866, and antibody 14E1 from Schering AG (U.S. Patent No. 5,942,602).

[0184] Recombinant binding members, chimeras, bispecificity, and fragments In general, CDR1 regions containing amino acid sequences substantially represented as the CDR1 regions of SEQ ID NOs: 2 and 4; 129 and 134; 22 and 27; 32 and 37; and 42 and 47, respectively, will be retained within a structure that enables the binding of the CDR1 region to a tumor antigen. For example, in the case of the CDR1 region of SEQ ID NO: 4, this CDR1 region is preferably retained by the VL chain region of SEQ ID NO: 4 (and similarly for the other listed sequences).

[0185] In general, CDR2 regions containing amino acid sequences substantially represented as the CDR2 regions of SEQ ID NOs: 2 and 4; 129 and 134; 22 and 27; 32 and 37; and 42 and 47, respectively, will be retained within a structure that enables the binding of the CDR2 region to a tumor antigen. For example, in the case of the CDR2 region of SEQ ID NO: 4, this CDR2 region is preferably retained by the VL chain region of SEQ ID NO: 4 (and similarly for the other listed sequences).

[0186] In general, CDR3 regions containing amino acid sequences substantially represented as the CDR3 regions of SEQ ID NOs: 2 and 4; 129 and 134; 22 and 27; 32 and 37; and 42 and 47, respectively, will be retained within a structure that enables the binding of the CDR3 region to a tumor antigen. For example, in the case of the CDR3 region of SEQ ID NO: 4, this CDR3 region is preferably retained by the VL chain region of SEQ ID NO: 4 (and similarly for the other listed sequences).

[0187] "Substantially representative" means that the CDR region of the present invention, for example, the CDR3 region, will be identical or highly homologous to the designated regions of SEQ ID NOs: 2 and 4; 129 and 134; 22 and 27; 32 and 37; and 42 and 47, respectively. "Highly homologous" may mean that one or more of the CDRs may have only a few substitutions, preferably 1 to 8, preferably 1 to 5, preferably 1 to 4, or 1 to 3 or 1 or 2 substitutions. Such terminology may also include truncation of the CDR, insofar as the resulting antibody exhibits the characteristic properties of the antibody class discussed herein, such as those represented by mAb806, mAb175, mAb124, mAb1133, and hu806.

[0188] The structure for holding the CDR, particularly CDR3, of the present invention is generally the structure of an antibody heavy or light chain sequence or substantial portion thereof, where the CDR region is located in a position corresponding to the CDR region of the naturally occurring VH and VL chain antibody variable domains encoded by a rearranged immunoglobulin gene. The structure and position of the immunoglobulin variable domain can be determined by referring to Kabat, EA et al, Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and this latest edition, now available on the internet (http: / / immu_number_bme.nwu.edu). Furthermore, as is known to those skilled in the art, CDR determination can be performed in various ways. For example, Kabat, Chothia, and combined domain determination analyses can be used. For this, see, for example, http: / / www.bioinf.org.uk / abs / #cdrid.

[0189] Preferably, the amino acid sequence substantially represented as a VH chain CDR residue in the antibody of the present invention is in the human heavy chain variable domain or a substantial portion thereof, and the amino acid sequence substantially represented as a VL chain CDR residue in the antibody of the present invention is in the human light chain variable domain or a substantial portion thereof.

[0190] The aforementioned variable domain may be derived from any germline or rearranged human variable domain, or it may be a synthetic variable domain based on a consensus sequence of a known human variable domain. For example, the CDR3-derived sequence of the present invention, as defined in the above paragraph, can be introduced into a repertoire of variable domains lacking the CDR3 region using recombinant DNA technology.

[0191] For example, Marks et al. (Bio / Technology, 1992, 10:779-783) describe a method for producing a repertoire of antibody variable domains by using a consensus primer directed to or adjacent to the 5' end of the variable domain area, together with a consensus primer for the third framework region of the human VH gene, to generate a repertoire of VH variable domains lacking CDR3. Marks et al. further describe a method for combining this repertoire with the CDR3 of a specific antibody. Using a similar technique, the CDR3-derived sequence of the present invention can be shuffled with a repertoire of VH or VL domains lacking CDR3, and the shuffled complete VH or VL domains can be combined with cognate VL or VH domains to generate specific binding members of the present invention. Subsequently, this repertoire can be presented in a suitable host system, such as a phage display of WO92 / 01047, to select a suitable specific binding member. The repertoire is 10 4 From the above individual members, for example, 10 6 from 10 8 or 10 10 It can consist of any of the members.

[0192] Similar shuffling or combinatorial techniques have also been disclosed by Stemmer (Nature, 1994, 370:389-391), who describes a technique for p-lactamase genes and states that this approach can be used for antibody production.

[0193] A further option is to induce mutations within the entire variable domain using, for example, random mutagenesis of the mAb806 VH or VL gene to generate a novel VH or VL region having the CDR3-derived sequence of the present invention. Such a technique was described by Gram et al. (1992, Proc. Natl. Acad. Sci., USA, 89:3576-3580), who used error-prone PCR.

[0194] Another method that can be used is to induce mutagenesis in the CDR region of the VH or VL gene. Such techniques have been 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).

[0195] All the techniques described above are publicly known and do not constitute part of the present invention. Those skilled in the art may use such techniques to produce the specific bonding members of the present invention using methodologies that are common in the art.

[0196] A substantial portion of the immunoglobulin variable domain will include at least three CDRs together with these 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% is the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Additional residues at the N-terminus or C-terminus of a substantial portion of the variable domain may be those not typically associated with naturally occurring variable domain regions. For example, the construction of the specific binding member of the present invention by recombinant DNA technology may result in the introduction of N or C-terminal residues encoded by a linker introduced to facilitate cloning or other manipulation steps. Other manipulation steps include the introduction of a linker to conjugate the variable domain of the present invention to further protein sequences including immunoglobulin heavy chains, other variable domains (e.g., in the production of diabodies), or protein labels as discussed in more detail below.

[0197] In preferred embodiments of the present invention, specific binding members comprising pairs of binding domains based on sequences substantially shown in SEQ ID NOs: 2 and 4; 129 and 134; 22 and 27; 32 and 37; and 42 and 47, respectively, are preferred, but single binding domains based on these sequences constitute further embodiments of the present invention. In the case of binding domains based on sequences substantially shown in the VH chain, such binding domains can be used as targeting agents for tumor antigens, since it is known that immunoglobulin VH domains can bind to target antigens in a specific manner.

[0198] In the case of any single-strand specific binding domain, these domains can be used to screen for complementary domains that can form two-domain specific binding members having good or equal in vivo properties to the mAb806, ch806, mAb175, mAb124, mAb1133, and hu806 antibodies disclosed herein.

[0199] This use can be achieved by phage display screening methods employing a so-called hierarchical dual combinatorial approach, such as that disclosed in U.S. Patent No. 5,969,108, in which individual colonies containing either H or L strand clones are used to infect a complete library of clones encoding the other strand (L or H), and the resulting double-strand specific binding members are selected according to phage display techniques, such as those described in this reference. This technique is also disclosed in Marks et al., ibid.

[0200] The specific binding members of the present invention may further comprise an antibody constant region or a portion thereof. For example, a specific binding member based on a VL chain sequence can be attached at its C-terminus to an antibody light chain constant domain comprising a human Ck or Cλ chain, preferably a Cλ chain. Similarly, a specific binding member based on a VH chain sequence can be attached at its C-terminus to all or part of an immunoglobulin heavy chain derived from any antibody isotype, e.g., IgG, IgA, IgE, IgD, and IgM, and any subclass of said isotype (IgG1, IgG2b, and IgG4 are particularly preferred). IgG1 is preferred.

[0201] The emergence of monoclonal antibody (mAb) technology 25 years ago has brought about a vast repertoire of useful research reagents, creating opportunities to use antibodies as approved pharmaceutical reagents and antithrombotic agents in cancer therapy, autoimmune diseases, graft rejection, and antiviral prophylaxis (Glennie and Johnson, 2000). The application of molecular engineering to convert mouse mAbs into chimeric mAbs (mouse V region, human C region) and humanized reagents in which only the mAb complementarity-determining region (CDR) is mouse-derived has been crucial to the clinical success of mAb therapy. mAbs produced by this molecular engineering have significantly reduced immunogenicity or immunogenicity deficient, increased serum half-life, and the human Fc portion of this mAb increases its potential to recruit complement immune effectors and cytotoxic cells (Clark, 2000). Investigations of the in vivo distribution, pharmacokinetics, and any induction of immune responses to clinically administered mAbs require the development of analyses to distinguish between formulated proteins and endogenous proteins.

[0202] The aforementioned antibodies or any fragments thereof can also be conjugated or recombinantly fused with any cytotoxin, bacterium, or other substance, such as Pseudomonas endotoxin, lysine, or diphtheria toxin. The portion of the toxin used may be the whole toxin or any specific domain of the toxin. Such antibody-toxin molecules have been successfully used for targeting and treating different types of cancer. See, for example, 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 Jan;14(1):129-35; Ghetie et al., Mol.Biotechnol. 2001 Jul;18(3):251-68.

[0203] Bi- and trip-specific multimers can be formed by the association of different scFv molecules, and they are designed as crosslinking reagents for T cell replacement (immunotherapy) and viral retargeting (genetherapy) for tumors, and as hemagglutinating agents (immunodiagnostics). See, for example, Todorovska et al., J.Immunol.Methods. 2001 Feb 1;248(l-2):47-66; Tomlinson et al., Methods Enzymol. 2000;326:461-79; McCall et al., J.Immunol. 2001 May 15;166(10):6112-7.

[0204] Fully human antibodies can be prepared by immunizing transgenic mice that possess a large proportion of human immunoglobulin weight and light chain. These mice are well known in the art, and examples of such mice include Xenomouse® (Abgenix, Inc.) (U.S. Patent Nos. 6,075,181 and 6,150,584), HuMAb-Mouse® (Medarex, Inc. / GenPharm) (U.S. Patent Nos. 5,545,806 and 5,569,825), TransChromo Mouse (Kirin), and KM Mouse (Medarex / Kirin).

[0205] Next, antibodies can be prepared, for example, by standard hybridoma technology or by phage display. These antibodies will then contain only complete human amino acid sequences.

[0206] Fully human antibodies can also be produced using phage display from human libraries. Phage display can be performed using methods well known to those skilled in the art, such as those described in Hoogenboom et al. and Marks et al. (Hoogenboom HR and Winter G. (1992) J.Mol.Biol.227(2):381-8; Marks JD et al. (1991) J.Mol.Biol.222(3):581-97; and also U.S. Patent Nos. 5,885,793 and 5,969,108).

[0207] Therapeutic antibodies and their use The in vivo properties of the specific binding members of the present invention, particularly with respect to tumor:blood ratio and clearance rate, will be at least comparable to those of mAb806. When such specific binding members are administered to human or animal subjects, they will exhibit 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 greater than 1:1, preferably greater than 2:1, and even 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 distant from the tumor site. These ratios exclude catabolic and secretory organs of the administered specific binding member. Thus, in the case of scFv and Fab (as shown in the attached examples), the binding member is secreted by the kidney and is present in greater quantities there than in other organs. In the case of total IgG, clearance will be at least partially by the liver. The peak localization ratio of intact antibodies is typically achieved between 10 hours and 200 hours after administration of the specific binding member. More specifically, this ratio can be measured in a tumor xenograft of approximately 0.2–1.0 g subcutaneously formed in one flank of athymic nude mice.

[0208] The antibodies of the present invention can be labeled with detectable or functional labels. Detectable labels include radiolabeling, such as isotopes. 3 H, 14 C, 32 P,35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 121 I, 124 I, 125 I, 131 I, 111 In, 211 At, 198 Au, 67 CU, 225 Ac, 213 Bi, 99 Tc and 186 Examples of labels include, but are not limited to, Re, and these can be attached to the antibody of the present invention using conventional chemistry known in the field of antibody imaging technology. Examples of labels include fluorescent labels and labels conventionally used in the field of MRI-CT imaging technology. Furthermore, examples of labels include specific co-detectable moieties, such as chemical moieties that can be detected by binding to labeled avidin, such as biotin.

[0209] Functional labels include substances designed to target tumor sites to cause destruction of tumor tissue. Examples of such functional labels include cytotoxic drugs that can be converted from prodrugs to active drugs at the tumor site, such as 5-fluorouracil or lysine, and enzymes, such as bacterial carboxypeptidases or nitroreductases.

[0210] Furthermore, antibodies containing both polyclonal and monoclonal antibodies, as well as drugs that modulate the production or activity of the specific binding members, antibodies, and / or their subunits, can have certain diagnostic applications and can be used, for example, to detect and / or measure conditions such as cancer, precancerous lesions, conditions associated with or resulting from hyperproliferative cell growth, or similar conditions. For example, the specific binding members, antibodies, or their subunits can be used to produce both polyclonal and monoclonal antibodies against them in various culture media using known techniques, such as hybridoma technology utilizing fused mouse splenic lymphocytes and myeloma cells. Similarly, small molecules that mimic or antagonize the activity(s) of the specific binding members of the present invention can be discovered or synthesized and used in diagnostic and / or therapeutic protocols.

[0211] Radiolabeled specific binding members, particularly antibodies and their fragments, are useful in in vitro diagnostic techniques, in vivo radiographic imaging techniques, and in radioimmunotherapy. In the case of in vivo imaging, the specific binding members of the present invention may be conjugated not with radioisotopes (one or more), but with imaging agents, including (but not limited to) magnetic resonance imaging enhancers, in which case a larger number of paramagnetic ions are loaded onto the antibody molecule by, for example, a chelating group. Examples of chelating groups include EDTA, porphyrins, polyamines, crown ethers, and polyoximes. Examples of paramagnetic ions include gadolinium, iron, manganese, rhenium, europium, lanthanium, holmium, and erbium. In a further embodiment of the present invention, radiolabeled specific binding members, particularly antibodies and their fragments, particularly radioimmunoconjugates, are useful in radioimmunotherapy, especially as radiolabeled antibodies for cancer therapy. In a further embodiment, radiolabeled specific binding members, particularly antibodies and fragments thereof, are useful in radioimmunoguided surgical techniques, in which case they can identify and indicate the presence and / or location of cancer cells, precancerous cells, tumor cells and hyperproliferative cells before, during, or after surgery to remove such cells.

[0212] The specific binding members of the present invention, particularly antibodies and their fragments, which conjugate or attach to other molecules or drugs, as well as the immune complexes or antibody fusion proteins of the present invention, further include, but are not limited to, binding members conjugated to chemical ablation agents, toxins, immunomodulators, cytokines, cytotoxic agents, chemotherapeutic agents, or drugs.

[0213] Radioimmunotherapy (RAIT) is entering the clinical setting and demonstrating its efficacy using various antibody immune conjugates. 131The 1-labeled humanized anti-fetal cancer antigen (anti-CEA) antibody hMN-14 has been evaluated in colorectal cancer (Behr TM et al (2002) Cancer 94(4 Suppl):1373-81), and the same antibody with 90Y labeling has been evaluated in medullary thyroid carcinoma (Stein R et al (2002) Cancer 94(1):51-61). Radioimmunotherapy using monoclonal antibodies has also been evaluated and reported for non-Hodgkin lymphoma and pancreatic cancer (Goldenberg DM (2001) Crit. Rev. Oncol. Hematol. 39(1-2):195-201; Gold DV et al. (2001) Crit. Rev. Oncol. Hematol. 39(1-2)147-54). Methods of radioimmunotherapy using specific antibodies are also described in U.S. Patent Nos. 6,306,393 and 6,331,175. Radioimmunoguided surgery (RIGS), including the use of anti-CEA antibodies and antibodies against tumor-associated antigens, is also entering the clinical field and has demonstrated efficacy and usefulness (Kim JC et al (2002) Jut. 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 DG et al (1997) Cancer Biother. Radiopharm. 12(4):287-94).

[0214] The antibodies of the present invention can be administered to patients in need of treatment via any suitable route, usually by bloodstream injection or injection into the CSF, or directly to the tumor site. The exact dose will depend on numerous factors, including whether the antibody is for diagnostic or therapeutic purposes, the size and location of the tumor, the exact nature of the antibody (whole antibody, fragment, diabody, etc.), and the detectable or functional label attached to the antibody. When radionuclides are used in therapy, the maximum suitable single dose is approximately 45 mCi / m³ 2 It has a maximum capacity of approximately 250 mCi / m2 The preferred dosage range is 15 to 40 mCi, with a more preferred range of 20 to 30 mCi, or 10 to 30 mCi. Such therapies may require bone marrow or stem cell replacement. Typical antibody doses for either tumor imaging or tumor treatment would be in the range of 0.5 to 40 mg, preferably 1 to 4 mg, of antibody in F(ab')2 form. Naked antibodies are preferably administered in doses of 20 to 1000 mg of protein per dose, or 20 to 500 mg of protein per dose, or 20 to 100 mg of protein per dose. These doses are for single treatment of adult patients and can be adjusted proportionally for children and infants, as well as proportionally to other antibody forms in relation to molecular weight. Treatment can be repeated daily, twice weekly, or at weekly or monthly intervals, as directed by a physician.

[0215] These formulations may contain a second binding protein, such as the EGPR-binding protein described above. In a particularly preferred form, this second binding protein is a monoclonal antibody, such as 528 or 225, as discussed above.

[0216] Pharmaceutical compositions and therapeutic compositions The specific binding member of the present invention will typically be administered in the form of a pharmaceutical composition which may contain at least one component in addition to the specific binding member.

[0217] Accordingly, the pharmaceutical compositions of the present invention, and pharmaceutical compositions for use in accordance with the present invention, may contain, in addition to the active ingredient, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the potency of the active ingredient. The exact properties of the carrier or other materials will depend on the route of administration, which may be orally or by injection, for example, intravenously.

[0218] Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders, or liquids. Tablets may contain a solid carrier, such as gelatin, or an adjuvant. Liquid pharmaceutical compositions generally contain a liquid carrier, such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. They may also contain saline solution, dextrose or other sugar solution, or glycol, such as ethylene glycol, propylene glycol, or polyethylene glycol.

[0219] For intravenous or pain site injection, the active ingredient should be in the form of a parenterally acceptable aqueous solution that is pyrogenically free and has a suitable pH, isotonicity, and stability. Those skilled in the art can easily prepare a suitable solution using an isotonic vehicle such as sodium chloride injection, Ringer's solution, or lactated Ringer's solution. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0220] The aforementioned compositions may be administered alone or in combination with other treatments, therapeutic agents, or drugs, simultaneously or sequentially, depending on the condition to be treated. In addition, the present invention considers and includes compositions comprising the binding members described herein, in particular antibodies or fragments thereof, with other drugs or therapies, such as anticancer agents or therapeutic agents, hormones, anti-EGFR agents or antibodies or immunomodulatory components. More generally, these anticancer agents may be tyrosine kinase inhibitors or phosphorylation cascade inhibitors, posttranslational regulators, cell growth or division inhibitors (e.g., antimitotic agents), or signal transduction inhibitors. Other treatments or therapies include the administration of appropriate doses of pain relievers, such as nonsteroidal anti-inflammatory drugs (e.g., aspirin, paracetamol, ibuprofen, or ketoprofen), or opioates, such as morphine, or antiemetics. The composition may be administered in combination (sequentially (i.e., before or after) or simultaneously) with tyrosine kinase inhibitors (including, but not limited to, AG1478 and ZD1839, STI571, OSI-774, and SU-6668), doxorubicin, temozolomide, cisplatin, carboplatin, nitrosourea, procarbazine, vincristine, hydroxyurea, 5-fluorouracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, carmustine, lomustine, and / or other chemotherapeutic agents. For example, these agents may be anti-EGFR specific agents or tyrosine kinase inhibitors, such as AG1478, ZD1839, STI571, OSI-774, or SU-6668, or more common anticancer and antineoplastic agents, such as doxorubicin, cisplatin, temozolomide, nitrosourea, procarbazine, vincristine, hydroxyurea, 5-fluorouracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, carmustine, or lomustine. In addition, the composition may be administered together with hormones that stimulate an immune response and reduce or eliminate cancer cells or tumors, such as dexamethasone, immunomodulatory components, such as interleukins, tumor necrosis factor (TNF), or other growth factors or cytokines.

[0221] Immunomodulatory components, such as TNF, may be used in combination with members of the present invention in the form of bispecific antibodies that not only recognize the EGFR epitopes recognized by the antibodies of the present invention but also bind to the TNF receptor. The compositions may be administered together with other anti-EGFR antibodies, including (but not limited to) anti-EGFR antibodies 528, 225, SC-03, DR8.3, L8A4, Y10, ICR62, and ABX-EGF, or may include combinations with such other anti-EGFR antibodies.

[0222] Previously, the combination of drugs such as doxorubicin and cisplatin with anti-EGFR antibodies resulted in enhanced antitumor activity (Fan et al, 1993; Baselga et al, 1993). The combination of doxorubicin and mAb 528 resulted in established complete eradication of A431 xenografts, while treatment with either drug alone resulted only in transient in vivo growth inhibition (Baselga et al, 1993). Similarly, the combination of cisplatin with either mAb 528 or 225 also resulted in well-established eradication of A431 xenografts, which was not observed when treatment with either drug alone (Fan et al, 1993).

[0223] Conventional radiotherapy In addition, the present invention envisions and includes therapeutic compositions for the combined use of the binding member with conventional radiotherapy. It has been noted that treatment with antibodies targeting EGF receptors can improve the effectiveness of conventional radiotherapy (Milas et al., Clin. Cancer Res. 2000 Feb:6(2):701, Huang et al., Clin. Cancer Res. 2000 Jun:6(6):2166).

[0224] As demonstrated herein, the combination of the binding member of the present invention, in particular the antibody or fragment thereof, in particular mAb806, ch806, mAb175, mAb124, mAb1133, or hu806 or fragments thereof, with anticancer therapeutic agents, in particular anti-EGFR therapeutic agents (including other anti-EGFR antibodies), demonstrates an effective therapy for xenograft tumors, and especially for surgery. In the examples, for example, the combination of AG1478 and mAb806 is demonstrated to result in a significantly enhanced reduction in A431 xenograft tumor volume compared to treatment with either agent alone. AG1478 (4-(3-chloroanilino)-6,7-dimethoxyquinazoline) is a potent selective inhibitor of EGF receptor kinase, described in detail in U.S. Patent No. 5,457,105, which is incorporated herein by reference 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 examples herein further demonstrate the therapeutic synergy between the antibody of the present invention and other anti-EGFR antibodies, particularly the 528 anti-EGFR antibody.

[0225] The present invention further considers therapeutic compositions useful for carrying out the therapeutic methods of the present invention. The therapeutic composition comprises a mixture of a pharmaceutically acceptable excipient (carrier) and one or more of the specific binding member, polypeptide analogs thereof, or fragments 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 binding member / antibody to target cells.

[0226] The preparation of therapeutic compositions containing polypeptides, analogs, or active fragments as active ingredients is well understood in the art. Typically, such compositions are prepared as injections, either as solutions or suspensions. However, solid forms suitable for dissolution or suspension in liquid before injection can also be prepared. Preparations can also be emulsified. The active therapeutic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active agent. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, or similar substances, and combinations thereof. In addition, if desired, the composition may contain small amounts of auxiliary substances that enhance the efficacy of the active ingredient, such as wetting or emulsifying agents, pH buffers, etc.

[0227] Polypeptides, analogs, or active fragments can be formulated into therapeutic compositions in the form of neutralized, pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of polypeptides or antibody molecules) and those formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and similar substances. Salts formed from free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium, ammonium, or ferric acid, as well as organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and similar substances.

[0228] Compositions containing therapeutic polypeptides, analogs, or active fragments are conventionally administered intravenously, for example, by injection of a unit dose. As used in relation to the therapeutic compositions of the present invention, the term "unit dose" refers to a physically distinct unit suitable as a single dose for a human, each containing a predetermined amount of active material calculated to produce a desired therapeutic effect in conjunction with the necessary diluent, i.e., carrier, or vehicle.

[0229] The composition is administered in a manner compatible with the drug formulation and in a therapeutically effective dose. The dose depends on the subject being treated, the subject's ability to utilize the active ingredient, and the desired degree of EGFR binding capacity. The exact amount of active ingredient required for administration depends on the physician's judgment and is unique to each individual. However, a suitable dose will depend on the administration route and will range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably 1 to several milligrams of the active ingredient per kilogram of body weight per day. Suitable regimens for initial administration and booster inoculations also vary, but a typical example is an initial dose followed by repeated administrations at intervals of one hour or more by subsequent injections or other doses. Alternatively, continuous intravenous infusion sufficient to maintain a concentration of 10 nanomoles to 10 micromoles in the blood may be considered.

[0230] Pharmaceutical compositions for oral administration may be in tablet, capsule, powder, or liquid form. Tablets may contain a solid carrier, such as gelatin, or an adjuvant. Liquid pharmaceutical compositions generally contain a liquid carrier, such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. They may also contain saline solution, dextrose or other sugar solution, or glycol, such as ethylene glycol, propylene glycol, or polyethylene glycol.

[0231] For intravenous or pain site injection, the active ingredient should be in the form of a parenterally acceptable aqueous solution that is pyrogenically free and has a suitable pH, isotonicity, and stability. Those skilled in the art can easily prepare a suitable solution using an isotonic vehicle such as sodium chloride injection, Ringer's solution, or lactated Ringer's solution. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0232] Diagnostic assay The present invention also relates to various diagnostic applications, including a method for detecting the presence of stimuli such as abnormally expressed EGFR by referring to these abilities recognized by the specific binding members of the present invention. As previously mentioned, EGFR can be used to produce antibodies against it by various known techniques, and such antibodies can then be isolated and used, for example, in testing for the presence of specific EGFR activity in suspected target cells.

[0233] The diagnostic uses of the specific binding members of the present invention, particularly antibodies and their fragments, are well known and common to those skilled in the art and are based herein, including in vitro and in vivo uses. Diagnostic assays and kits for in vitro assessment and evaluation of EGFR status, particularly abnormal expression of EGFR, can be used to diagnose, evaluate, and monitor patient samples, including those from tumor samples, those known or suspected to have cancer, precancerous conditions, conditions associated with hyperproliferative cell growth, etc. Assessment and evaluation of EGFR status is also useful in determining a patient's suitability for clinical trials of drugs, or for the administration of specific chemotherapeutic agents or the specific binding members of the present invention, particularly antibodies (including combinations thereof), to different drugs or binding members. This type of diagnostic monitoring and assessment has already been put into practice in breast cancer with antibodies against the HER2 protein (Hercep Test, Dako Corporation), and the assay in this case is also used to evaluate patients for antibody therapy using Herceptin. In vivo uses include tumor imaging or assessment of an individual's cancer status (including radiographic imaging).

[0234] As previously proposed, the diagnostic method of the present invention comprises examining a cell sample or culture medium by an assay comprising an effective amount of an antagonist to the EFGR / protein, such as an anti-EFGR antibody, preferably an affinity-purified polyclonal antibody, and more preferably an mAb. In addition, the anti-EFGR antibody molecule used herein is preferably in the form of a Fab, Fab', F(ab')2 or F(v) moiety or the whole antibody molecule. As previously discussed, patients who may benefit from this method include those suffering from cancer, precancerous lesions, viral infections, conditions including or resulting from hyperproliferative cell growth, or other similar pathological disorders. Methods for isolating EFGR and inducing anti-EFGR antibodies, as well as methods for determining and optimizing the ability of anti-EFGR antibodies to assist in the examination of target cells, are all well known in the art.

[0235] Preferably, the anti-EFGR antibody used in the diagnostic method of the present invention is an affinity-purified polyclonal antibody. More preferably, the antibody is a monoclonal antibody (mAb). In addition, the anti-EFGR antibody molecule used herein may be in the form of the Fab, Fab', F(ab')2, or F(v) portion of the whole antibody molecule.

[0236] As described in detail above, antibodies against EGFR can be produced and isolated by standard methods, including well-known hybridoma techniques. For convenience, antibodies against EGFR are referred to as Ab1 in this specification, and antibodies produced in another species are referred to as Ab2.

[0237] The presence of EGFR within cells can be determined by standard in vitro or in vivo immunological procedures applicable to such determinations. Numerous useful procedures are publicly known. Three particularly useful such procedures utilize EGFR labeled with a detectable label, an antibody labeled with a detectable label (Ab), or an antibody labeled with a detectable label (Ab2). These procedures can be summarized by the following equation (where an asterisk indicates that the particle is labeled, and "R" represents EGFR): A. R * +Ab1=R * Ab1, B. R+Ab * =RAb1 * C. R+Ab1+Ab2 * =RAb1Ab2 *

[0238] All of the aforementioned procedures and their uses are well known to those skilled in the art and can therefore be used within the scope of the present invention. The “competing” procedure, Procedure A, is described in U.S. Patent Nos. 3,654,090 and 3,850,752. Procedure C, the “sandwich” procedure, is described in U.S. Patent Nos. RE31,006 and 4,016,043. Further procedures, such as the “double antibody” or “DASP” procedure, are also known.

[0239] In each of the above examples, EGFR forms a complex with one or more antibodies or binding partners, and one member of this complex is labeled with a detectable label. The formation of the complex and, if desired, the amount thereof can be determined by known methods applicable to the detection of the label.

[0240] From the above, it can be seen that a characteristic feature of Ab2 is its reaction with Ab1. This characteristic is due to the use of Ab1, produced in a mammalian species, as an antigen to produce the antibody Ab2 in another species. For example, rabbit antibodies can be used as an antigen to produce Ab2 in goats. Therefore, Ab2 would be an anti-rabbit antibody produced in a goat. For the purposes of this specification and claims, Ab1 would refer to a primary or anti-EGFR antibody, and Ab2 would refer to a secondary or anti-Ab1 antibody.

[0241] The most commonly used labels for these studies are radioactive elements, enzymes, chemicals that fluoresce when exposed to ultraviolet light, and others.

[0242] Numerous fluorescent materials are known and can be used as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA Blue, and Lucifer Yellow. A special detection material is an anti-rabbit antibody prepared in goats and conjugated to fluorescein with an isothiocyanate.

[0243] EGFR or its binding partner(s), such as this specific binding member, can also be labeled with a radioactive element or enzyme. This radiolabeling can be detected by any of the currently available counting procedures. Preferred isotopes are 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 121 I, 124 I, 125 I, 131 I, 111 In, 211 At, 198 Au, 67 CU,225 Ac, 213 Bi, 99 Tc and 186 You can choose from Re.

[0244] Enzyme labeling is equally useful and can be detected by any of the currently available colorimetric, spectrophotometric, fluorescence spectrophotometric, amperometric, or gas quantification techniques. Selected particles can be conjugated with enzymes by reaction with crosslinking molecules, such as carbodiimides, diisocyanates, glutaraldehyde, and similar substances. Many enzymes are known and can be utilized for these procedures. Peroxidases, β-glucuronidases, β-D-glucosidases, β-D-galactosidases, ureases, glucose oxidase + peroxidase, and alkaline phosphatases are preferred. U.S. Patent Nos. 3,654,090; 3,850,752; and 4,016,043 are referenced, for example, to these disclosures of alternative labeling materials and methods.

[0245] A specific assay system that can be advantageously utilized according to 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 both labeled and unlabeled materials are inoculated into a certain cell test colony. Subsequently, a binding study is performed to determine the degree to which the labeled material binds to the cell receptor. In this way, differences in affinity between the materials can be determined.

[0246] Therefore, the purified amount of the specific binding member can be labeled with, for example, an antibody or other inhibitor against it, or compounded with an antibody or other inhibitor against it, and then binding studies can be conducted. Next, solutions containing various amounts of labeled and unlabeled, uncompounded specific binding members are prepared, then inoculated into cell samples, and then incubated. Next, the resulting cell monolayer is washed and solubilized, and then counted with a gamma counter for a sufficient time to produce a standard error of <5%. Next, these data are subjected to scatchard analysis, and then observations and conclusions regarding material activity are drawn. The above is illustrative, but it shows that receptor assays can be performed and utilized when the cell binding ability of the assayed material can serve as a distinguishing characteristic.

[0247] A useful and conceivable assay according to the present invention is known as a "cis / trans" assay. Simply put, this assay utilizes two gene constructs, one of which is a plasmid that continuously expresses a specific receptor of interest when transfected into a suitable cell line, and the other is a plasmid that expresses a receptor, such as luciferase, under the control of a receptor / ligand complex. Therefore, for example, if it is desired to evaluate a compound as a ligand for a specific receptor, one of the plasmids would be a construct that results in the expression of this receptor in a selected cell line, and the other plasmid would have a promoter linked to the luciferase gene into which a response element for this specific receptor is inserted. If the compound being tested is an agonist of the receptor, this ligand will complex with the receptor, and the resulting complex will bind to the response element, initiating transcription of the luciferase gene. The resulting chemiluminescence is then measured photometrically to obtain a dose-response curve, which is then compared to that of a known ligand. The protocol described above is described in U.S. Patent No. 4,981,784 and PCT International Publication No. WO 88 / 03168, and is therefore referred to by those skilled in the art.

[0248] In further embodiments of the present invention, commercially available test kits suitable for use by medical professionals can be constructed to determine the presence or absence of abnormal EGFR expression (including, but not limited to, amplified EGFR and / or EGFR mutations) in suspected target cells. According to the test techniques discussed above, one class of such kits would contain at least labeled EGFR or its binding partner, such as an antibody specific to it, and instructions, but of course, it would depend on the method chosen, e.g., “competitive,” “sandwich,” “DASP,” and similar methods. The kits may also contain peripheral reagents, such as buffers and stabilizers.

[0249] Therefore, a test kit for demonstrating the presence or cellular capacity for abnormal expression or post-translational modification of EGFR, (a) A predetermined amount of at least one labeled immunochemically reactive component obtained by directly or indirectly attaching the specific binding member or its specific binding partner to a detectable label; (b) Other reagents; and (c) Instructions for use of the Kit A test kit containing [the specified element] can be prepared.

[0250] More specifically, the diagnostic test kit is, (a) Generally, a known amount of specific binding members (or binding partners) as described above, which bind to one of a number of such terminal products (or their binding partners), which bind to a solid phase to form an immunoadsorbent, or alternatively, a suitable tag, or a number of such terminal products (or their binding partners); (b) Other reagents if necessary; and (c) Instructions for use of the preliminary test kit Includes.

[0251] Further variations operate according to a predetermined protocol (e.g., "competitive," "sandwich," "double antibody," etc.), (a) A labeled component obtained by coupling the specific binding member to a detectable label; (b) One or more additional immunochemical reagents [at least one of these reagents is a ligand or immobilized ligand, and this ligand is (i) A ligand capable of binding to the labeled component (a); (ii) A ligand capable of binding to a binding partner of labeled component (a); (iii) A ligand capable of binding to at least one of the components (one or more) to be determined; and (iv) A ligand capable of binding to at least one binding partner of at least one component(s) to be determined. Selected from the group consisting of ] and; (c) Instructions for carrying out a protocol for detecting and / or determining one or more components of an immunochemical reaction between EGFR, the specific binding member, and its specific binding partner. A test kit containing the above can be prepared and used for the purposes described above.

[0252] According to the above, an assay system can be constructed for screening drugs that may be effective in modulating EGFR activity, abnormal expression or post-translational modification of EGFR, and / or the activity or binding of the specific binding member. The receptor or binding member is introduced into the test system, and the promising drug is also introduced into the resulting cell culture. The culture is then examined to observe any changes in the S-phase activity of these cells, either due to the addition of the promising drug alone or the amount of a known drug(s) added.

[0253] nucleic acid The present invention further provides isolated nucleic acids encoding specific binding members of the present invention. Nucleic acids include DNA and RNA. In a preferred embodiment, the present invention provides nucleic acids encoding polypeptides of the present invention as defined above, including polypeptides such as those shown as CDR residues of the VH and VL chains of the antibody of the present invention.

[0254] The present invention also provides constructs in the form of plasmids, vectors, transcripts, or expression cassettes that contain at least one polynucleotide as described above.

[0255] The present invention also provides recombinant host cells comprising one or more constructs as described above. A nucleic acid encoding any of the provided specific binding members constitutes an aspect of the present invention, and a method for producing the specific binding member, comprising expressing it from the nucleic acid encoding it, also constitutes an aspect of the present invention. Expression can be conveniently achieved by culturing the recombinant host cells containing the nucleic acid under appropriate conditions. After production by expression, the specific binding member can be isolated and / or purified using any suitable technique and then used as appropriate.

[0256] The present invention provides specific binding members and encoding nucleic acid molecules and vectors, which may be isolated and / or purified from their natural environments, in substantially pure or homogeneous form, or, in the case of nucleic acids, have no or substantially no nucleic acids or gene origins other than the sequence encoding the polypeptide having the required function. The nucleic acids according to the present invention may include DNA or RNA and may be entirely or partially synthetic.

[0257] Systems for cloning and expressing various different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, and baculovirus systems. Mammalian cell systems available in the art for the expression of heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, and many others. A common and preferred bacterial host is E. coli.

[0258] 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, Pluckthun, A. Bio / Technology 9:545-551 (1991). Expression in eukaryotic cells in culture can also be utilized by those skilled in the art as an option for producing specific binding members. For recent reviews, see, for example, Raff, ME (1993) Curr. Opinion Biotech. 4:573-576; Trill J Jet al. (1995) Curr. Opinion Biotech 6:553-560.

[0259] A suitable vector can be selected or constructed containing appropriate regulatory sequences, including, as appropriate, promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences. The vector may, as appropriate, be a plasmid, virus, such as a phage or phagemid. 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 the preparation of nucleic acid constructs, mutagenesis, sequencing, DNA introduction into cells, and manipulation of nucleic acids in gene expression, as well as protein analysis, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The aforementioned disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference.

[0260] Accordingly, further embodiments of the present invention provide host cells containing the nucleic acids disclosed herein. Further embodiments also provide methods for introducing such nucleic acids into host cells. This introduction can utilize any available technology. Suitable technologies for eukaryotic cells include calcium phosphate transfection, DEAE-dextran, electrophoresis, liposome-mediated transfection, and transduction, using retroviruses or other viruses, such as vaccinia, or, for insect cells, baculoviruses. Suitable technologies for bacterial cells include calcium chloride transformation, electrophoresis, and transfection, using bacteriophages.

[0261] Following this introduction, expression from the nucleic acid can be induced or made possible, for example, by culturing host cells under conditions for gene expression.

[0262] In one embodiment, the nucleic acid of the present invention is incorporated into the genome (e.g., chromosomes) of a host cell. This incorporation can be facilitated by including sequences that promote recombination with the genome, according to standard techniques.

[0263] The present invention also provides a method comprising using the constructs described above in an expression system to express the specific binding members or polypeptides described above.

[0264] As described above, the present invention also relates to specific binding members having amino acid sequences shown in SEQ ID NOs: 2 and 4; 129 and 134; 22 and 27; 32 and 37; and / or 42 and 47, particularly antibodies or recombinant DNA molecules or cloned genes encoding this fragment, or mutated variants thereof, preferably the nucleic acid molecule encoding the binding member or antibody, particularly recombinant DNA molecules or cloned genes, having a nucleotide sequence or being complementary to a DNA sequence encoding one of such sequences.

[0265] Another feature of the present invention is the expression of DNA sequences disclosed herein. As is well known in the art, DNA sequences can be expressed by ligating them to expression regulatory sequences in a suitable expression vector and by transforming a suitable single-cell host using this expression vector.

[0266] Such operational ligation of the DNA sequence of the present invention to an expression control sequence, of course, includes providing a start codon, ATG, within the correct reading frame upstream of the DNA sequence if it is not already part of the DNA sequence.

[0267] Wild-type host / expression vector combinations can be used for the expression of the DNA sequences of the present invention. For example, useful expression vectors may consist of segments of chromosomal, non-chromosomal, and synthetic DNA sequences. Suitable vectors include SV40 and derivatives of known bacterial plasmids, e.g., E. coli plasmids col E1, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNA, e.g., a great many derivatives of phage X, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single-stranded phage DNA; yeast plasmids, e.g., 2u plasmid or its derivatives; vectors useful in eukaryotic cells, e.g., vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNA, e.g., plasmids modified to utilize phage DNA or other expression regulatory sequences; and similar vectors.

[0268] The DNA sequences of the present invention can be expressed in these vectors by using any of the various expression regulatory sequences—sequences that control the expression of DNA sequences linked to them. Such useful expression regulatory sequences include, for example, SV40, CMV, vaccinia, polyoma or adenovirus early or late promoters, lac systems, trp systems, TAC systems, TRC systems, LTR systems, major operator and promoter regions of phage λ, regulatory regions of fd coat proteins, promoters for 3-phosphoglycerate kinase or other glycoseptic enzymes, promoters for acid phosphatases (e.g., Pho5), promoters for yeast junction factors, and other sequences known to control the expression of genes in prokaryotic or eukaryotic cells or these viruses, as well as various combinations thereof.

[0269] Various single-cell host cells are also useful for the expression of the DNA sequence of the present invention. These hosts include well-known eukaryotic and prokaryotic hosts, e.g., E. coli, Pseudomonas, Bacillus, Streptomyces, fungi, e.g., yeast, and animal cells, e.g., CHO, YB / 20, NSO, SP2 / 0, R1.1, BW, and LM cells, African green monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g., Sf9), and human and plant cells (in tissue culture).

[0270] It will be understood that not all vectors, regulatory sequences, and hosts will function equally well to express the DNA sequence of the present invention. Nor will all hosts function equally well using the same expression system. However, those skilled in the art will be able to select appropriate vectors, regulatory sequences, and hosts to achieve desired expression without departing the scope of the present invention and without excessive experimentation. For example, when selecting a vector, the host must be considered, since the vector must function within it. The copy number of the vector, the ability to control this copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, will also be considered.

[0271] When selecting an expression regulatory sequence, various factors are usually considered. These include, for example, the relative strength of the system, its regulatory capacity, and its compatibility with the specific DNA sequence or gene to be expressed, particularly with respect to potential secondary structures. A suitable single-cell host would be selected by considering, for example, its compatibility with the chosen vector, its selectivity, its ability to correctly fold the protein, its fermentation requirements, as well as the toxicity of the product encoded by the expressed DNA sequence to the host and the ease of purifying the expression product.

[0272] Taking these and other factors into consideration, those skilled in the art will be able to construct various vector / expression control sequence / host combinations that will express the DNA sequence of the present invention using fermentation or in large-scale animal culture.

[0273] It is further intended that specific binding member analogs can be prepared from the nucleotide sequences of protein complexes / subunits induced within the scope of the present invention. Analogs such as fragments can be produced, for example, by pepsin digestion of the specific binding member material. Other analogs such as mutaines can be produced by standard site-directed mutagenesis of the specific binding member coding sequence. Analogs exhibiting "specific binding member activity," such as small molecules, whether functioning as promoters or inhibitors, can be identified by known in vivo and / or in vitro assays.

[0274] As mentioned above, DNA sequences encoding specific binding members can be prepared synthetically rather than by cloning. DNA sequences with appropriate codons for specific binding member amino acid sequences can be designed. Generally, when this sequence is to be used for expression, preferred codons are selected for the intended host. Complete sequences are assembled by overlapping oligonucleotides prepared by standard methods to create a complete coding sequence. See, for example, Edge, Nature, 292:756 (1981); Nambair et al, Science, 223:1299 (1984); Jay et al., J. Biol. Chem., 259:6311 (1984).

[0275] Synthetic DNA sequences enable the convenient construction of genes that will express specific binding member analogs or "mutein." Alternatively, DNA encoding mutein can be produced by site-directed mutagenesis of natural specific binding member genes or cDNA, and mutein can be produced directly using conventional polypeptide synthesis.

[0276] A general method for site-specific incorporation of non-natural amino acids into proteins is described in Christopher J. Noren, Spencer J. Anthony-Cahill, Michael C. Griffith, and Peter G. Schultz, Science, 244:182-188 (April 1989). This method can be used to create analogs of non-natural amino acids.

[0277] This invention relates to the preparation of antisense oligonucleotides and ribozymes that can be used to interfere with EGFR expression at the translational level. This approach utilizes antisense nucleic acids and ribozymes to block the translation of a specific mRNA by either masking this mRNA with an antisense nucleic acid or cleaving it with a ribozyme.

[0278] 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). Within the cell, they hybridize to this mRNA to form a double-stranded molecule. The cell does not translate this double-stranded mRNA. Therefore, antisense nucleic acids do not interfere with the expression of mRNA into protein. Oligomers of approximately 15 nucleotides and molecules that hybridize to the AUG start codon are particularly effective because they are easy to synthesize and are likely to pose fewer problems than larger molecules when introduced into production cells. Antisense methods have been used to inhibit the expression of many genes in vitro (Marcus-Sekura, 1988; Hambor et al., 1988).

[0279] Ribozymes are RNA molecules that have the ability to specifically cleave other single-stranded RNA molecules in a manner somewhat similar to DNA restriction endonucleases. Ribozymes were discovered through the observation that certain mRNAs have the ability to excise specific introns. By modifying the nucleotide sequences of these RNAs, researchers were able to genetically engineer molecules that recognize and cleave specific nucleotide sequences within RNA molecules (Cech, 1988). Because these are sequence-specific, only mRNAs with specific sequences are inactivated.

[0280] Researchers identified two types of ribozymes: the Tetrahymena type and the "hammerhead" type (Hasselhoff and Gerlach, 1988). The Tetrahymena type ribozyme recognizes a 4-nucleotide sequence, while the "hammerhead" type recognizes an 11- to 18-nucleotide sequence. The longer the recognition sequence, the more likely it is to occur exclusively in the target mRNA species. Therefore, the hammerhead type ribozyme is preferable to the Tetrahymena type ribozyme for inactivating specific mRNA sequences, and the 18-nucleotide recognition sequence is preferable to shorter recognition sequences.

[0281] Thus, using the DNA sequences described herein, antisense molecules against mRNA for EGFR and its ligands, and ribozymes for cleaving said mRNA can be prepared.

[0282] The present invention can be better understood by referring to the following non-limiting embodiments provided as illustrative examples. The following embodiments are presented to better illustrate preferred embodiments of the present invention, but should not be considered to limit the broad scope of the present invention in any way. [Examples]

[0283] Antibody production and isolation cell line For immunological and specificity analysis, several cell lines were used, which were either natural, normal, wild-type, or transfected with the "wtEGFR" gene or the ΔEGFR gene with the Δ2-7 deletion mutation: mouse fibroblast cell line NR6, NR6 ΔEGFR (Transfected with ΔEGFR) and NR6 wtEGFR (transfected with wtEGFR), human glioblastoma cell lineage U87MG (expressing low levels of endogenous wtEGFR), U87MG wtEGFR (Transfected with wtEGFR), U87MG ΔEGFR (Transfected with ΔEGFR), as well as human squamous cell carcinoma cell line A431 (expressing high levels of wtEGFR).

[0284] For immunological and specificity analysis, several cell lines were used, which were either natural, normal, wild-type, or transfected with the "wtEGFR" gene or with the ΔEGFR gene having the de207 or Δ2-7 deletion mutation: mouse fibroblast cell line NR6, NR6 ΔEGFR (Transfected with ΔEGFR) and NR6 wtEGFR (transfected with wtEGFR), human glioblastoma cell lineage U87MG (expressing low levels of endogenous wtEGFR), U87MG wtEGFR Alternatively, "U87MG.wtEGFR" (transfected with wtEGFR), U87MG ΔEGFR Alternatively, "U87MG.Δ2-7" (transfected with ΔEGFR), and human squamous cell carcinoma cell line A431 (expressing high levels of wtEGFR). NR6, NR6 ΔEGFR , and NR6 wtEGFRCell lines have been described previously (Batra et al. (1995) Epidermal Growth Factor Ligand-independent, Unregulated, Cell-Tranforming Potential of a Naturally Occurring Human Mutant EGFRvIII Gene. Cell Growth Differ. 6(10):1251-1259). The NR6 cell line does not contain normal endogenous EGFR (Batra et al., 1995). The U87MG cell line and transfection have been described previously (Nishikawa et al. (1994) A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc. Natl. Acad. Sci. U.S.A. 91, 7727-7731).

[0285] The U87MG astrocytoma cell line that endogenously expresses low levels of wtEGFR (Ponten, J. and Macintyre, E.H. (1968) Long term culture of normal and neoplastic human glia. Acta. Pathol. Microbiol. Scand. 74, 465-86) was infected with a retrovirus containing de2-7 EGFR to produce the U87MG.Δ2-7 cell line (Nishikawa et al., 1994). The transfected cell line U87MG.wtEGFR was produced as described in Nagane et al. (1996) Cancer Res. 56, 5079-5086. U87MG cells have approximately 1×10 5 EGFR expression, while U87MG.wtEGFR cells have approximately 1×10 6The cells express EGFR, and therefore closely resemble the situation observed in gene amplification. The mouse pro-B cell line BaF / 3, which does not express any known EGFR-related molecules, was also transfected with de2-7 EGFR, resulting in the BaF / 3.Δ2-7 cell line (Luwor et al. (2004) The tumor-specirid de2-7 epidermal growth factor receptor (EGFR) promotes cells survival and heterodimerizes with the wild-type EGFR, Oncogene 23:6095-6104). Human squamous cell carcinoma A431 cells were obtained from ATCC (Rockville, Maryland). The epidermal carcinoma cell line A431 has been previously described (Sato et al. (1987) Derivation and assay of biological effects of monoclonal antibodies to epidermal growth factor receptors. Methods Enzymol. 146, 63-81).

[0286] All cell lines were cultured in DMEM / F-12 with GlutaMAX™ (Life Technologies, Inc., Melbourne, Australia and Grand Island, New York) supplemented with 10% FCS (CSL, Melbourne, Australia); 2 mM glutamine (Sigma Chemical Co., St. Louis, Missouri); and penicillin / streptomycin (Life Technologies, Inc., Grand Island, New York). In addition, U87MG.Δ2-7 and U87MG.wtEGFR cell lines were maintained in 400 mg / mL genethicin (Life Technologies, Inc., Melbourne, Victoria, Australia). Cell lines were grown at 37°C in an undenatured atmosphere of 5% CO2.

[0287] reagent The de2-7 EGFR unique junction peptide has the amino sequence: LEEKKGNYVVTDH (SEQ ID NO: 13). Specific biotinylated junction peptides (biotin-LEEKKGNYVVTDH (SEQ ID NO: 5) and LEEKKGNYVVTDH-biotin (SEQ ID NO: 6)) were synthesized from de2-7 EGFR using standard Fmoc chemistry, and their purity (>96%) was determined by reverse-phase HPLC and mass spectrometry (Auspep, Melbourne, Australia).

[0288] Antibodies used in the study To compare with our findings using other reagents, we included additional mAbs in our study. These reagents were mAb528 against wtEGFR (Sato et al. (1983) Mol. Biol. Med. 1(5), 511-529) and DH8.3 produced against a synthetic peptide spanning the junctional sequence of the Δ2-7 EGFR deletion mutation. The DH8.3 antibody (IgG1) specific to de2-7 EGFR was previously described (Hilles et al. (1995) Specific targeting of a mutant, activated EGF receptor found in glioblastoma using a monoclonal antibody. Int. J. Cancer. 63, 537-43, 1995), and the antibody was obtained after immunization of mice with a specific junctional peptide found in de2-7 EGFR (Hills et al., 1995).

[0289] The 528 antibody recognizing both de2-7 EGFR and wild-type EGFR has been described previously (Masui et al. (1984) Growth inhibition of human tumor cells in athymic mice by anti-epidermal growth factor receptor monoclonal antibodies. Cancer Res. 44, 1002-7), and the antibody was produced at the Biological Production Facility, Ludwig Institute for Cancer Research (Melbourne, Australia) using hybridoma (ATCC HB-8509) obtained from the American Type Culture Collection (Rockville, Maryland). Polyclonal antibody SC-03 is an affinity-purified rabbit polyclonal antibody raised against the carboxy-terminal peptide of EGFR (Santa Cruz Biotechnology Inc.).

[0290] Antibody Production Mouse fibroblast cell line NR6 ΔEGFR was used as the immunogen. BALB / c mice received 5×10 5 -2×10 6 cells for immunization five times subcutaneously at intervals of 2 to 3 weeks to produce mouse hybridomas. Complete Freund's adjuvant was used for the first injection. Thereafter, incomplete Freund's adjuvant (Difco™, Voigt Global Distribution, Lawrence, Kansas) was used. Splenocytes from immunized mice were fused with the mouse myeloma cell line SP2 / 0 (Shulman et al. (1978) Nature 276:269-270). Supernatants from newly generated clones were screened for reactivity with NR6, NR6 wtEGFR and NR6 ΔEGFR in a hemadsorption assay, and further screened against human glioblastoma cell lines U87MG, U87MG wtEGFR and U87MGΔEGFR The samples were analyzed using a hematopoietic adsorption assay. Subsequently, the selected hybridoma supernatant was tested by Western blotting and further analyzed by immunohistochemistry. Newly produced mAbs that showed the expected reactivity were purified.

[0291] Five hybridomas were established, and NR6 was detected in the rosette hemagglutination assay. ΔEGFR High titer (1:2500) and NR6 and NR6 wtEGFR For further characterization based on the low background of the cells, three clones, 124(IgG2a), 806(IgG2b), and 1133(IgG2a), were first selected. A fourth clone, 175(IgG2a), was then further characterized. This will be discussed separately in Example 23 below. In subsequent hemagglutination analysis, these antibodies were found to be native human glioblastoma cell lines U87MG and U87MG wtEGFR It did not show reactivity with U87MG (undiluted supernatant ≤10%), but ΔEGFR It showed strong reactivity with [unspecified substance]; however, it showed little reactivity with A431. In contrast, FACS analysis showed that 806 was unreactive with natural U87MG, and U87MG ΔEGFR Stain strongly and use U87MG to a lower degree wtEGFR The sample was stained. This indicates that 806 binds to both ΔEGFR and wtEGFR (see below).

[0292] Next, mAb124, mAb806, and mAb1133 were analyzed for their reactivity with wtEGFR and ΔEGFR using a Western blot assay. The detergent lysates were then NR6. ΔEGFR U87MG ΔEGFRExtracted from A431. All three mAbs showed a reactivity pattern similar to cell lysates staining both wtEGFR protein (170kDa) and ΔEGFR protein (140kDa). As a reference reagent, mAbR.I., known to be reactive with wtEGFR (Waterfield et al. (1982) J. Cell Biochem. 20(2), 149-161), was used instead of mAb528, which is known to be unreactive in Western blot analysis. mAbR.I. showed reactivity with wild-type and ΔEGFR. All three newly acidified clones showed reactivity with ΔEGFR and less strong reactivity with wtEGFR. U87MG ΔEGFR and NR6 ΔEGFR Of the lysates, only DH8.3 was positive.

[0293] Xenograft tumor U87MG, U87MG ΔEGFR Table 1 shows the immunohistochemical analyses of clones 124, 806, and 1133 related to A431, as well as mAb528 and mAbDH8.3. All mAbs were obtained from xenograft U87MG. ΔEGFR These showed strong staining. Only mAb528 showed weak reactivity in natural U87MG xenografts. In A431 xenografts, mAb528 showed strong, homogeneous reactivity. mAb124, mAb806, and mAb1133 showed reactivity with cells mainly located in the basal region of A431 squamous cell carcinoma, but did not reactivity with the upper cell layer and keratinized components. DH8.3 was negative in A431 xenografts.

[0294] [Table 2] Micro-interstitial staining for detection of endogenous mouse antibodies

[0295] Sequencing The variable heavy chain (VH) and variable light chain (VL) of mAb806, mAb124, and mAb1133 were sequenced, and their complementarity-determining regions (CDRs) were identified as follows: mAb806 The mAb806 VH chain: nucleic acid sequence (SEQ ID NO: 1) and amino acid sequence with signal peptide (SEQ ID NO: 2) are shown in Figures 14A and 14B, respectively (the signal peptide is underlined in Figure 14B). In Figure 16, the complementarity-determining regions CDR1, CDR2, and CDR3 (SEQ ID NOs: 15, 16, and 17, respectively) are highlighted by underlining. The mAb806 VH chain amino acid sequence without this signal peptide (SEQ ID NO: 11) is shown in Figure 16.

[0296] The mAb806 VL chain: nucleic acid sequence (SEQ ID NO: 3) and amino acid sequence with signal peptide (SEQ ID NO: 4) are shown in Figures 15A and 15B, respectively (the signal peptide is underlined in Figure 15B). In Figure 17, the complementarity-determining regions CDR1, CDR2, and CDR3 (SEQ ID NOs: 18, 19, and 20, respectively) are highlighted by underlining. The mAb806 VL chain amino acid sequence without this signal peptide (SEQ ID NO: 12) is shown in Figure 17.

[0297] mAb124 The mAb124 VH chain: nucleic acid (SEQ ID NO: 21) and amino acid (SEQ ID NO: 22) sequences are shown in Figures 51A and 51B, respectively. The complementarity-determining regions CDR1, CDR2, and CDR3 (SEQ ID NOs: 23, 24, and 25, respectively) are indicated by underlining.

[0298] The mAb124 VL chain: nucleic acid (SEQ ID NO: 26) and amino acid (SEQ ID NO: 27) sequences are shown in Figures 51C and 51D, respectively. The complementarity-determining regions CDR1, CDR2, and CDR3 (SEQ ID NOs: 28, 29, and 30, respectively) are indicated by underlining.

[0299] mAb1133 The mAb1133 VH chain: nucleic acid (SEQ ID NO: 31) and amino acid (SEQ ID NO: 32) sequences are shown in Figures 52A and 52B, respectively. The complementarity-determining regions CDR1, CDR2, and CDR3 (SEQ ID NOs: 33, 34, and 35, respectively) are indicated by underlining.

[0300] The mAb1133 VL chain: nucleic acid (SEQ ID NO: 36) and amino acid (SEQ ID NO: 37) sequences are shown in Figures 52C and 52D, respectively. The complementarity-determining regions CDR1, CDR2, and CDR3 (SEQ ID NOs: 38, 39, and 40, respectively) are indicated by underlining. [Examples]

[0301] Antibody binding to cell lines by FACS As shown in this specification and the following examples, mAb806 was initially selected for further characterization. As discussed in Example 26 below, mAb124 and mAb1133 were also selected for further characterization and were found to have properties corresponding to the unique properties of mAb806 discussed herein.

[0302] To determine the specificity of mAb806, its binding to U87MG, U87MG.Δ2-7, and U87MG.wtEGFR cells was analyzed by flow-activated cell sorting (FACS). Briefly, as previously described (Nishikawa et al., 1994), cells were labeled with appropriate antibodies (10 μg / mL) and then labeled with fluorescein-conjugated goat anti-mouse IgG (1:100 dilution; Calbiomechem, San Diego, California, USA; Becton-Dickinson PharMingen, San Diego, California, USA). FACS data were obtained by observing a minimum of 5,000 events using Coulter Epics Elite ESP and analyzed using EXPO (version 2) for Windows®. Unrelated IgG2b was included as an isotype control for mAb806, and 528 antibody was included as it recognizes both de2-7 and wtEGFR.

[0303] Only antibody 528 was able to stain the parental U87 cell line (Figure 1). This is consistent with previous reports demonstrating that these cells express wtEGFR (Nishikawa et al, 1994). mAb806 and DH8.3 had binding levels similar to the control antibody. This clearly indicates that they cannot bind to the wild-type receptor (Figure 1). Binding of isotype control antibodies to U87MG.Δ2-7 and U87MG.wtEGFR cells was similar to that observed for U87MG cells.

[0304] mAb806 stained U87MG.Δ2-7 and U87MG.wtEGFR cells. This indicates that mAb806 specifically recognizes de2-7 EGFR and amplified EGFR (Figure 1). The DH8.3 antibody stained U87MG.Δ2-7 cells. This confirms that the DH8.3 antibody specifically recognizes de2-7 EGFR (Figure 1). As expected, the 528 antibody stained both U87MG.Δ2-7 and U87MG.wtEGFR cell lines (Figure 1). As expected, the 528 antibody stained U87MG.Δ2-7 with higher intensity than the parent cells. This is because it binds to both de2-7 and wild-type receptors co-expressed in these cells (Figure 1). Similar results were obtained using protein A mixed hematopoietic adsorption, which detects surface-bound IgG by the appearance of human erythrocyte (type O) coated protein A on target cells. Monoclonal antibody 806 was reactive with U87MG.Δ2-7, but did not show significant reactivity with U87MG expressing wild-type EGFR (less than 10% undiluted supernatant). Importantly, mAb806 also bound to the BaF / 3.Δ2-7 cell line. This clearly indicates that co-expression of wtEGFR is not a necessary condition for mAb806 reactivity (Figure 1). [Examples]

[0305] Antibody binding in assays To further characterize the specificity of the mAb806 and DH8.3 antibodies, 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 hours and then blocked with 2% human serum albumin (HSA) in PBS. sEGFR is the recombinant extracellular domain (amino acids 1-621) of wild-type EGFR, which was produced as previously described (Domagala 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, 11-29). Antibodies were added to the wells in triple repeats at increasing concentrations in 2% HSA in phosphate-buffered saline (PBS). Using ABTS (Sigma, Sydney, Australia) as a substrate and absorbance measured at 405 nm, we detected conjugated antibodies using horseradish peroxidase conjugate sheep anti-mouse IgG (Silenus, Melbourne, Australia).

[0306] Both the mAb806 antibody and the 528 antibody presented dose-dependent saturation binding curves to immobilized wild-type sEGFR (Figure 2A). Since the specific junction peptide found in de2-7 EGFR is not present in sEGFR, mAb806 must bind to an epitope located within the wild-type EGFR sequence. Binding of the 528 antibody was lower than that observed for the mAb806 antibody, presumably because it recognizes higher-order structure determinants. As expected, the DH8.3 antibody did not bind to wild-type sEGFR even at concentrations up to 10 μg / mL (Figure 2A). Lysized sEGFR inhibited the binding of the 528 antibody to immobilized sEGFR in a dose-dependent manner, but could not inhibit binding to mAb806 (Figure 2B). This suggests that mAb806, when immobilized on an ELISA plate, can only bind to wild-type EGFR and can induce higher-order structure changes in a specific way. Using BIAcore, a similar result was observed: mAb806 binds to immobilized sEGFR, but immobilized mAb806 cannot bind to soluble sEGFR (Figure 2C).

[0307] After denaturation by heating at 95°C for 10 minutes, soluble sEGFR could not inhibit the binding of mAb806 to immobilized sEGFR (Figure 2C). This confirms that mAb806 can bind to wild-type EGFR under certain conditions. Interestingly, denatured sEGFR could not inhibit the binding of the 528 antibody (Figure 2C). This clearly indicates that this antibody recognizes a higher-order epitope. The DH8.3 antibody showed dose-dependent saturable binding to the specific de2-7 EGFR peptide (Figure 2D). Neither the mAb806 antibody nor the 528 antibody bound to the peptide, even at concentrations higher than those used to achieve saturation binding of DH8.3. This further indicates that mAb806 does not recognize the epitope determinant within this peptide.

[0308] In the second assay, the biotinylated de2-7 specific peptide (biotinLEEKKGNYVVTDH (SEQ ID NO: 5)) did not bind to a streptavidin-precoated ELISA plate (Pierce, Rockford, Illinois). Antibodies were conjugated and detected as in the first assay. Neither the mAb806 antibody nor the 528 antibody bound to the peptide, even at higher concentrations than those used to achieve saturated binding of DH8.3. This further indicates that mAb806 does not recognize the epitope determinant within this peptide.

[0309] Additional experiments were conducted to further demonstrate that mAb806 recognizes an epitope different from the aforementioned junction peptide. The C-terminally biotinylated de2-7 peptide (LEEKKGNYVVTDH-biotin (SEQ ID NO: 6)) was used in the study along with mAb806 and mAbL8A4 produced in response to this de2-7 peptide (Reist et al. (1995) Cancer Res. 55(19), 4375-4382; Foulon et al. (2000) Cancer Res. 60(16), 4453-4460).

[0310] Reagents used in peptide research Junction peptide: LEEKKGNYVVTDH-OH (Biosource, Camarillo, California); Peptide C:LEEKKGNYVVTDH(K-Biot)-OH (Biosource, Camarillo, California); sEGFR: Recombinant soluble extracellular domain of wild-type EGFR derived from CHO cells (amino acids 1-621) (LICR Melbourne); mAb806: Mouse monoclonal antibody, IgG 2b (LICR NYB); mAbL8A4: Mouse monoclonal antibody, IgG1 (Duke University); IgG1 isotype control mAb; IgG 2b Isotype reference mAb.

[0311] Peptide C was immobilized on a streptavidin microsensor chip at a surface density of 350 RU (+ / - 30 RU). A series of mAb dilutions were tested for reaction with this peptide. Specificity was assessed by performing blockage experiments using non-biotinized peptides.

[0312] mAbL8A4 showed strong reactivity with peptide C even at low antibody concentrations (6.25 M) (Figure 2E). mAb806 did not show detectable specific reactivity with peptide C up to an antibody concentration of 100 nM (the highest concentration tested) (Figures 2E and 2F). The reaction of mAbL8A4 with peptide C was expected, as this peptide was used as an immunogen in the production of mAbL8A4. The addition of the junction peptide (non-biotinized, 50 μg / mL) completely blocked the reactivity between mAbL8A4 and peptide C. This confirms the specificity of this antibody against the junction peptide epitope.

[0313] In the second set of BIAcore experiments, sEGFR was immobilized on a CM microsensor chip at a surface density of ~4000 RU. A series of mAb dilutions were tested for reactivity with sEGFR.

[0314] mAb806 was strongly reactive with denatured sEGFR, but mAbL8A4 did not react with denatured sEGFR. The reactivity of mAb806 with denatured sEGFR decreased with decreasing antibody concentration. It was expected that mAbL8A4 would not react with sEGFR, because mAbL8A4 was produced using a junction peptide as an immunogen, while sEGFR does not contain a junction peptide.

[0315] Dot blot immunohistochemistry was also performed. Serial dilutions of the peptide were spotted at 0.5 μL on PVDF or nitrocellulose membranes. The membranes were blocked with 2% BSA in PBS and then probed with antibodies 806, L8A4, DH8.3, and a control antibody. Antibodies L8A4 and DH8.3 bound to the peptides on the membrane (data not shown). mAb806 did not bind to the peptides at the concentration at which L8A4 clearly showed binding (data not shown). The control antibody was also negative for peptide binding.

[0316] mAb806 bound to wtEGFR in the cell lysate according to immunoblotting (results not shown). This differs from the results obtained with the DH8.3 antibody, which reacts with de2-7 EGFR but not with wtEGFR. Therefore, mAb806 can recognize wtEGFR after denaturation, but cannot recognize this receptor when it is present on the cell surface in its native state. [Examples]

[0317] Scattered Analysis To determine the relative affinity of each antibody, a scatchard analysis was performed using U87MG.Δ2-7 cells after correction for the immune response. Antibodies were analyzed using the Chloramine T method. 125 The antibodies were labeled with I (Amrad, Melbourne, Australia), and their immunoreactivity was determined by the Lindmo assay (Lindmo et al. (1984) Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. J.Immunol.Methods. 72,77-89).

[0318] In 1% HSA / PBS, 1-2 × 10 6All binding assays were performed using live U87MG.Δ2-7 or A431 cells, with gentle rotation for 90 minutes at 4°C. The set concentration was 10 ng / mL. 125 I-labeled antibody was used in the presence of appropriately increasing concentrations of unlabeled antibody. Nonspecific binding was assessed in the presence of a 10,000-fold excess of unlabeled antibody. 125 Neither the radiolabeled mAb806 nor the DH8.3H antibody bound to the parent U87MG cells. After incubation was complete, the cells were washed and bound using a COBRA II gamma counter (Packard Instrument Company, Meriden, Connecticut, USA). 125 I-labeled antibodies were counted.

[0319] Both mAb806 and DH8.3 antibodies retained high immunoreactivity when iodized, typically greater than 90% for mAb806 and 45-50% for DH8.3 antibodies. mAb806 showed a reactivity of 1.1 × 10⁶. 9 M -1 While it had affinity for the de2-7 EGFR receptor, the affinity for DH8.3 was 1.0 × 10⁻⁶. 8 M -1 The rate was several tens of times lower. Iodized antibodies also did not bind to U87MG parent cells. mAb806 averaged 2.4 × 10⁶ per cell. 5 Recognizing the binding site, the DH8.3 antibody produced an average of 5.2 × 10⁶ 5 It bound to the site. Thus, not only is there good agreement in receptor numbers between antibodies, but a previous report (Reist et al. (1997) 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) showed that when measured using different de2-7 EGFR-specific antibodies on the same cell line, it yielded 2.5 × 10⁶ per cell. 5This shows the de2-7 receptor. [Examples]

[0320] Antibody internalization by U87MG.Δ2-7 cells The rate of antibody internalization after binding to target cells affects both the tumor targeting characteristics and the treatment options. Therefore, we examined the internalization of mAb806 and DH8.3 antibodies after binding to U87MG.Δ2-7 cells by FACS. U87MG.Δ2-7 cells were incubated in DMEM at 4°C for 1 hour with either mAb806 antibody or DH8.3 antibody (10 μg / mL). After washing, the cells were transferred to DMEM preheated to 37°C, and aliquots were taken at various time points after incubation at 37°C. Internalization was stopped by immediately washing the aliquots with ice-cold washing buffer (1% HSA / PBS). At the completion of this time course, the cells were stained by FACS as described above. Formula: Internalized antibody = (time) x The internalization percentage was calculated by comparing surface antibody staining at various time points to the zero point using the formula (average fluorescence - background fluorescence at time 0) / (average fluorescence - background fluorescence at time 0) × 100. This method was validated in one assay that uses an iodized antibody (mAb806) to measure internalization, as previously described (Huang et al. (1997) 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). Differences in internalization rates at different time points were compared using Student's t-test. Throughout this study, data were analyzed for significance using Student's t-test, except for the in vivo survival assay analyzed by Wilcoxone analysis.

[0321] Both antibodies showed relatively rapid internalization, reaching steady-state levels at 10 minutes for mAb806 and 30 minutes for DH8.3 (Figure 3). DH8.3 internalization was significantly higher in both rate (80.5% of DH8.3 internalized at 10 minutes compared to 36.8% for mAb806, p<0.001) and total internalized amount at 60 minutes (93.5% vs. 30.4%, p<0.001). mAb806 showed significantly lower internalization levels at 30 and 60 minutes compared to 20 minutes in all four assays performed (Figure 3). This was also confirmed using an internalization assay based on iodized mAb806 (data not shown). [Examples]

[0322] Electron microscopy analysis of antibody internalization In light of the differences in internalization rates between antibodies mentioned above, we performed a detailed analysis of intracellular antibody transport using an electron microscope.

[0323] U87MG.Δ2-7 cells were grown to a concentration of 80% on gelatin-coated chamber slides (Nunc, Naperville, Illinois) and then washed with ice-cold DMEM. The cells were then incubated in DMEM with mAb806 or DH8.3 antibody at 4°C for 45 minutes. After washing, the cells were further incubated at 4°C for 30 minutes with gold-conjugate (20 nm particle) anti-mouse IgG (BB International, Cardiff, UK). After further washing, preheated DMEM / 10%PCS was added to the cells and incubated at 37°C for varying times from 1 to 60 minutes. Antibody internalization was stopped with ice-cold medium, and the cells were fixed with 2.5% glutaraldehyde in PBS / 0.1% HSA, followed by post-fixation with 2.5% osmium tetroxide. After dehydration with a stepwise series of acetone, the samples were embedded in Epon / Araldite resin, cut into ultrathin sections using a Reichert Ultracut-S microtome (Leica), and recovered on nickel grid. These sections were stained with uranyl acetate and lead citrate, and then visually inspected at 80kV using a Philips CM12 transmission electron microscope. Statistical analysis of gold particles contained within the covering pits was performed using the chi-square test.

[0324] The DH8.3 antibody appeared to be internalized primarily by covering pits, while mAb806 appeared to be internalized by macropinocytosis (Figure 19). Indeed, detailed analysis of 32 covering pits formed in cells incubated with mAb806 showed that neither contained the antibody. In contrast, approximately 20% of all covering pits from cells incubated with DH8.3 were positive for the antibody and contained numerous gold particles. Statistical analysis of the total number of gold particles contained within the covering pits revealed this difference to be highly significant (p<0.01). After 20–30 minutes, both antibodies could be observed in structures that morphologically resembled ribosomes (Figure 19C). The presence of cell disruption fragments within these structures was also consistent with their ribosomal properties. [Examples]

[0325] In vivo distribution of antibodies in tumor-carrying nude mice In nude mice carrying a U87MG xenograft on one side of the body and a U87MG.Δ2-7 xenograft on the other side, the in vivo distribution of mAb806 and DH8.3 antibodies was compared. Since previous reports (Hills et al. (1995) Specific targeting of a mutant, activated EGF receptor found in glioblastoma using a monoclonal antibody. Int.J.Cancer.63,537-43) have demonstrated that DH8.3 antibodies exhibit peak tumor targeting levels between 4 and 24 hours, a relatively short timeframe was chosen for this study.

[0326] 3 × 10 in nude BALB / c mice 6 Tumor xenografts were established by sc injection of U87MG, U87MG.Δ2-7, or A431 cells. Immunohistochemistry at various time points showed that de2-7 EGFR expression in U87MG.Δ2-7 xenografts remained stable throughout the entire lifecycle (data not shown). A431 cells retained their mAb806 reactivity when grown as tumor xenografts, as determined by immunohistochemistry. U87MG or A431 cells were injected into one side of the body, and 7-10 days later, U87MG.Δ2-7 cells were injected into the other side of the body because de2-7 EGFR-expressing xenografts grow faster. Antibodies were radiolabeled as described above and assessed for immunoreactivity. When the tumors reached a weight of 100-200 mg, they were injected into mice via the posterior orbital route. Each mouse received two different antibodies (2 μg per antibody): 2 μCi 125 I-labeled mAb806 and 2μCi 131 I-labeled DH8.3 or 528. Unless otherwise indicated, groups of five were sacrificed at various time points after injection, and blood was obtained by cardiac puncture. Tumors, liver, spleen, kidneys, and lungs were obtained by dissection. All tissues were weighed using a dual-channel counting window. 125 I and 131I activity was assayed. For each antibody, the data was expressed as %ID / g tumor, determined by comparison with the injection dose standard, or converted to tumor-to-blood / liver ratio (i.e., %ID / g tumor divided by %ID / g blood or liver). Differences between groups were analyzed by Student's t-test. After injection of radiolabeled mAb806, some tumors were fixed with formalin, embedded in paraffin, cut into 5 μm sections, and then exposed to X-ray film (AGFA, Belgium, Moltzel) to determine antibody localization by autoradiography.

[0327] In terms of %ID / g tumor, mAb806 reached this peak level of 18.6% m / g tumor at 8 hours in the U87MG.Δ2-7 xenograft (Figure 4), which was considerably higher than any other tissue except blood. DH8.3 also showed a peak tumor level at 8 hours, but this level was statistically lower (p<0.001) compared to mAb806 at 8.8% m / g tumor (Figure 4B). Levels of both antibodies decreased slowly at 24 and 48 hours. 125 Autoradiography of U87MG.Δ2-7 xenograft tissue sections collected 8 hours after injection of I-labeled mAb806 alone clearly showed antibody localization to viable tumors (Figure 20). Neither antibody showed specific targeting of U87MG parental xenografts (Figures 4A and 4B). Regarding tumor-to-blood / liver ratio, mAb806 showed the highest ratio for both blood (ratio of 1.3) and liver (ratio of 6.1) at 24 hours (Figures 5A and 5B). The DH8.3 antibody showed this highest ratio for blood at 8 hours (ratio of 0.38) and for liver at 24 hours (ratio of 1.5) (Figures 5A and 5B). These uses were considerably lower than the values ​​obtained for mAb806.

[0328] As explained above, mAb806 levels in tumors peaked at 8 hours. This peak is relatively early compared to many tumor-targeting antibodies, but it is in complete agreement with other studies using de2-7 EGFR-specific antibodies (Hills et al., 1995; Reist et al., 1997; Reist et al. (1996) Radioiodination of internalizing monoclonal antibodies using N-succinimidyl 5-iodo-3-pyridinecarboxylate. Cancer Res. 56, 4970-7) where all peaks occurred at 4–24 hours post-injection using similar volumes of antibody. In fact, contrary to previous reports, the 8-hour time point was included under the assumption that antibody targeting would peak rapidly. The %ID / g tumor observed with mAb806 was similar to those reported for other de2-7 EGFR-specific antibodies using standard iodization techniques (Hills et al., 1995; Huang et al., 1997; Reist et al. (1995) 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).

[0329] There are likely two reasons for this early peak. Firstly, tumors expressing de2-7 EGFR, including transfected U87MG cells, grow extremely rapidly as tumor xenografts. Therefore, even during the relatively short periods used in these in vivo distribution studies, tumor size increases to such an extent that the %ID / g tumor is reduced compared to slow-growing tumors (a 5-10-fold increase in mass in 4 days). Secondly, internalization of mAb806 was relatively slow compared to DH8.3, but still rapid for many other tumor antibody / antigen systems. Internalized antibodies undergo rapid proteolysis with degradation products expelled from the cell (Press et al. (1990) Inhibition of catabolism of radiolabeled antibodies by tumor cells using lysosomotropic amines and carboxylic ionophores. Cancer Res. 50, 1243-50). This process of internalization, degradation, and efflux reduces the amount of iodized antibodies retained within the cell. As a result, internalized antibodies exhibit lower targeting levels than their non-internalized counterparts. Electron microscopy data reported herein clearly demonstrate that internalized mAb806 is rapidly transported to lysosomes, where rapid degradation likely occurs. This observation is consistent with the rapid elimination of iodine from the cell.

[0330] Previously described L8A4 monoclonal antibodies against specific junction peptides found in de2-7 EGFR operate in a manner similar to mAb806 (Reist et al. (1997) In vitro and in vivo behavior of radiolabeled chimeric anti-EGFRvIII monoclonal antibody: comparison with its murine parent. Nucl. Med. Biol. 24, 639-47). When using U87MG cells transfected with de2-7 EGFR, this antibody exhibited similar internalization rates (35% at 1 hour compared to 30% at 1 hour for mAb806) and comparable in vivo targeting when using 3T3 fibroblasts transfected with de2-7 EGFR (peak of 24% ID / g tumors at 24 hours compared to 18% ID / g tumors at 8 hours for mAb806) (Reist et al. (1997) 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).

[0331] Interestingly, in vivo retention of this antibody in tumor xenografts was enhanced when labeled with N-succinimidyl 5-iodo-3-pyridinecarboxylate (Reist et al., 1997). This labeled junction had a positive charge at lysosomal pH and therefore enhanced cellular retention (Reist et al. (1996) Radioiodination of internalizing monoclonal antibodies using N-succinimidyl 5-iodo-3-pyridinecarboxylate. Cancer Res. 56, 4970-7). The enhanced retention holds potential usefulness when considering antibodies for radioimmunotherapy, and this method could be used to improve the retention of iodized mAb806 or this fragment. [Examples]

[0332] Binding of mAb806 to cells containing amplified EGFR To examine whether mAb806 can recognize EGFR expressed in cells containing the amplified receptor gene, we analyzed its binding to A431 cells. As previously described, A431 cells are human squamous cell carcinoma cells that express high levels of wtEGFR. Low but highly reproducible binding of mAb806 to A431 cells was observed by FACS analysis (Figure 6). The DH8.3 antibody did not bind to A431 cells. This indicates that the binding of mAb806 was not a result of low levels of de2-7 EGFR expression (Figure 6). As expected, the anti-EGFR 528 antibody showed strong staining of A431 cells (Figure 6). In light of these results, we characterized the binding of mAb806 to A431 cells by scatchard analysis. Binding of iodized mAb806 was relatively low, but consistent data for scatchard analysis were obtained. The average of three such experiments is 2.5 × 10⁶ per cell. 5 The receptor is 9.5 × 10 7 M -1Affinity values ​​were obtained. Therefore, the affinity for this receptor was several tens of times lower than the affinity for de2-7 EGFR. Furthermore, mAb806 appears to recognize only a small portion of EGFR found on the surface of A431 cells. The 528 antibody yielded approximately 2 × 10⁶ values ​​per cell. 6 The receptor was identified. This is consistent with a great many other studies (Santon et al. (1986) Effects of epidermal growth factor receptor concentration on tumorigenicity of A431 cells in nude mice. Cancer Res. 46, 4701-5).

[0333] To ensure these results are not solely limited to the A431 cell line, mAb806 reactivity was examined in two other cell lines exhibiting EGFR gene amplification. Both the HN5 head and neck cell line (Kwok TT and Sutherland RM (1991) Differences in EGF-related radiosensitisation of human squamous carcinoma cells with high and low numbers of EGF receptors. Br.J.Cancer.64,251-4) and the MDA-468 breast cancer cell line (Filmus et al. (1985) 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.128,898-905) have been reported to contain multiple copies of the EGFR gene. Consistent with these reports, the 528 antibody presented strong staining of both cell lines (Figure 21). Similar to the A431 cell line, mAb806 clearly stained both cell lines, albeit at lower levels than observed with the 528 antibody (Figure 21). Therefore, mAb806 binding appears to be generally observed for cells containing EGFR gene amplification, not simply limited to A431.

[0334] Recognition of wild-type sEGFR by mAb806 clearly requires some degree of denaturation of this receptor to expose the epitope. The degree of denaturation required is very slight, as even the adsorption of wild-type sEGFR to a plastic surface induces robust binding of mAb806 in ELISA assays. Since mAb806 binds to only about 10% of the EGFR on the surface of A431 cells, it is inferred that this subset of the receptor would have a modified higher-order structure similar to that induced by de2-7 EGFR truncation. Indeed, the extremely high expression of EGFR mediated by gene amplification in A431 cells causes some receptors to process incorrectly, which leads to modified higher-order structures. Interestingly, semi-quantitative immunoblotting of A431 cell lysates with mAb806 showed that it can recognize a large portion of the A431 EGF receptor by SDS-PAGE and Western transfer. These results further support the claim that mAb806 binds to a subset of receptors on the surface of A431 cells that have a modified higher-order structure. These observations in A431 cells are consistent with immunohistochemical data demonstrating that mAb806 binds to gliomas with EGFR gene amplification. Since mAb806 binding is completely negative for parental U87MG cells, it would seem that this phenomenon could be limited to cells containing amplified EGFR, although the level of "denatured" receptors on the surface of U87MG cells may be below detection level. However, iodized mAb806 has a detection level of 1 × 10⁶ 7 This possibility seems unlikely, as the cells did not bind to the U87MG cell pellet containing the following cells. [Examples]

[0335] In vivo targeting of A431 cells using mAb806 A second in vivo distribution study was conducted with mAb806 to determine whether it could target A431 tumor xenografts. This study was performed over a longer time period to obtain more information regarding the targeting of U87MG.Δ2-7 xenografts by mAb806, which was included as a positive control in all mice. In addition, an anti-EGFR 528 antibody was included as a positive control for A431 xenografts. This was because previous studies had demonstrated low but significant targeting of A431 cells grown in nude mice with this antibody (Masui et al. (1984) Growth inhibition of human tumor cells in athymic mice by anti-epidermal growth factor receptor monoclonal antibodies. Cancer Res. 44, 1002-7).

[0336] During the first 48 hours, mAb806 exhibited nearly identical targeting characteristics to those observed in the initial experiment (Figure 7A compared to Figure 4A). In terms of %ID / g tumor, the level of mAb806 in the U87MG.Δ2-7 xenograft slowly decreased after 24 hours, but consistently remained higher than the levels detected in normal tissue. Uptake in the A431 xenograft was relatively low, but there was a small increase in %ID / g tumor during the first 24 hours that was not observed in normal tissues such as the liver, spleen, kidney, and lung (Figure 7A). Uptake of the 528 antibody was very low in both xenografts, expressed as %ID / g tumor, partly due to the faster clearance of this antibody from the blood (Figure 7B). 125Autoradiography of A431 xenograft tissue sections collected 24 hours after injection of radioactive mAb806 alone clearly showed antibody localization to viable tumor near the tumor periphery, but not to the central necrotic area (Figure 23). In terms of tumor-to-blood ratio, mAb806 peaked at 72 hours for U87MG.Δ2-7 xenografts and at 100 hours for A431 xenografts (Figures 8A, B). The tumor-to-blood ratio for mAb806 never exceeded 1.0 for A431 tumors, but this increased throughout the entire time course (Figure 8B) and was higher than all other tissues examined (data not shown). This indicates a low targeting level.

[0337] The tumor-to-blood ratio for 528 antibody showed a similar profile to mAb806, but the higher levels in A431 xenografts were noteworthy (Figure 8A, B). mAb806 had a peak tumor-to-liver ratio of 7.6 in U87MG.Δ2-7 at 72 hours. This clearly indicates preferential uptake in these tumors compared to normal tissues (Figure 8C). Other tumor-to-organ ratios for mAb806 were similar to those observed in the liver (data not shown). The peak tumor-to-liver ratio for mAb806 in A431 xenografts was 2.0 at 100 hours. This also indicates slightly preferential uptake in tumors compared to normal tissues (Figure 8D). [Examples]

[0338] treatment research The effects of mAb806 were evaluated in two xenograft models of the disease: a prophylactic model and a stochastic tumor model.

[0339] Xenograft model 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 wtEGFR. Therefore, it was not possible to grow both cell types in the same mouse.

[0340] Tumor cells (3 × 10) in 100 mL of PBS 6 mAb806 was subcutaneously inoculated into both flanks of 4-6 week old female nude mice (Animal Research Center, Western Australia, Australia). The therapeutic efficacy of mAb806 was investigated in both prophylactic and stochastic tumor models. In the prophylactic model, five mice, each carrying two xenografts, were intraperitoneally treated with either 1 or 0.1 mg of mAb806 or vehicle (PBS) starting the day before tumor cell inoculation. Treatment was continued for two weeks, three times a week, for a total of six sessions. In the stochastic model, the tumor size was 65 ± 6.42 mm. 3 (U87MG.Δ2-7), 84±9.07mm 3 (U87MG), 73±7.5mm 3 (U87MG.wtEGFR) or 201±19.09mm 3 Treatment was initiated when the average volume of the (A431 tumor) was reached. Formula (length × width) 2 Using ) / 2 (wherein the formula, length is the longest axis and width was measured perpendicular to the length), mm 3Tumor volume was determined using the following method (Clark et al. (2000) Therapeutic efficacy of anti-Lewis(y) humanized 3 S 193 radioimmunotherapy in a breast cancer model: enhanced activity when combined with taxol chemotherapy. Clin. Cancer Res. 6, 3621-3628). Data for each treatment group were expressed as mean tumor volume ± SE. Statistical analysis was performed at a given time point using Student's t-test. Xenograft size was 1.5 cm. 3 When the tumor reached an approximate volume, the animals were euthanized and the tumor was removed for histological examination. This research project was approved by the Animal Ethic Committee of Austin and the Repatriation Medical Centre.

[0341] Tissue examination of tumor xenografts The xenograft was excised and bisected. One half was fixed in 10% formalin / PBS and then embedded in paraffin. Subsequently, a 4-micrometer section was cut and stained with hematoxylin and eosin (H&E) for routine histological examination. The other half was embedded in Tissue Tek® OCT compound (Sakura Finetek, Trans, California), frozen in liquid nitrogen, and stored at -80°C. Thin (5-micrometer) cryostat sections were cut, fixed in ice-cold acetone for 10 minutes, and then air-dried for another 10 minutes. The sections were blocked with protein blocking reagent (Lipshaw Immunon, Pittsburgh, USA) for 10 minutes, and then incubated at room temperature (RT) for 30 minutes with biotinylated primary antibody (1 mg / mL). All antibodies were biotinylated using an ECL protein biotinylation module (Amersham, Australia, Baulkham Hills) as instructed by the manufacturer. After rinsing with PBS, sections were incubated for a further 30 minutes with streptavidin-horseradish peroxidase complex (Silenus, Australia, Melbourne). After the final PBS wash, these sections were exposed to 3-amino-9-ethylcarbozole (AEC) substrate (0.1M acetic acid, 0.1M sodium acetate, 0.02M AEC (Sigma Chemical Co., St. Louis, Missouri)) in the presence of hydrogen peroxide for 30 minutes. Sections were rinsed with water, counterstained with hematoxylin for 5 minutes, and mounted.

[0342] The efficacy of mAb806 in preventative models The efficacy of mAb806 against U87MG and U87MG.Δ2-7 was investigated in a prophylactic xenograft model. The antibody or vehicle was administered intravenously (ip) the day before tumor inoculation, three times a week for two weeks. mAb806, at a dose of 1 mg per injection, had no effect on the growth of wtEGFR-expressing parental U87MG xenografts (Figure 9A). In contrast, mAb806 significantly inhibited U87MG.Δ2-7 xenografts in a dose-dependent manner (Figure 9B). At day 20, when control animals were sacrificed, the mean tumor volume was 1637 ± 178.98 mm² in the control group. 3 For the group receiving 0.1 mg per injection, the result was statistically smaller at 526 ± 94.74 mm. 3 (p<0.0001), and for the 1 mg injection group, the result was 197±42.06 mm. 3 (p<0.0001). The treatment group was sacrificed at day 24, and the mean tumor volume at this point was 1287±243.03 mm for the 0.1 mg treatment group. 3 , and for the 1 mg group, 492 ± 100.8 mm 3 That was the case.

[0343] The efficacy of mAb806 in a probabilistic xenograft model. Considering the efficacy of mAb806 in a prophylactic xenograft model, we then examined its ability to probabilistically inhibit tumor xenograft growth. Antibody treatment reduced tumor growth to 65±6.42 mm for U87MG.Δ207 xenografts. 3 And for the parent U87MG xenograft, 84±9.07mm 3 Except for initiating this when the mean tumor volume reached the specified level, it was as described in the prophylactic model. Again, mAb806 was ineffective in inhibiting the growth of parental U87MG xenografts at a dose of 1 mg per injection (Figure 10A). In contrast, mAb806 significantly inhibited the growth of U87MG.Δ2-7 xenografts in a dose-dependent manner (Figure 10B). At day 17, the day before sacrificing the control animals, the mean tumor volume was 935 ± 215.04 mm for the control group. 3 For the group receiving 0.1 mg per injection, the result was 386 ± 57.51 mm. 3(p<0.01), and for the 1 mg injection group, the result was 217±58.17 mm. 3 (p<0.002).

[0344] To investigate whether the growth inhibition observed with mAb806 is limited to cells expressing de2-7 EGFR, this efficacy against U87MG.wtEGFR tumor xenografts was examined in a stochastic model. These cells serve as a model for tumors with EGFR gene amplification but without de2-7 EGFR expression. The tumor size was 73±7.5 mm. 3 mAb806 treatment was initiated when the mean tumor volume reached [value missing]. mAb806 significantly inhibited the growth of the U87MG.wtEGFR xenograft compared to control tumors treated with the vehicle (Figure 10C). On the day of sacrifice of the control animals, this mean tumor volume was 960 ± 268.9 mm for the control group. 3 For the group treated with 1 mg injection, the result was 468 ± 78.38 mm. 3 (p<0.04).

[0345] Histological and immunohistochemical analysis of stochastic tumors To assess potential histological differences between mAb806-treated and control U87MG.Δ2-7 and U87MG.wtEGFR xenografts (collected on days 24 and 42, respectively), formalin-fixed, paraffin-embedded sections were stained with H&E. Necrotic areas were observed in sections from both mAb806-treated U87MG.Δ2-7 (collected 3 days after the end of treatment) and U87MG.wtEGFR xenografts (collected 9 days after the end of treatment). This result was consistently observed in a large number of tumor xenografts (n=4). However, analysis of sections from control-treated xenografts did not show the same necrotic areas as observed in mAb806-treated xenografts. Sections from either mAb806-treated or control-treated U87MG xenografts were also stained with H&E, and these showed no difference in cell viability between the two groups. This further supports the hypothesis that mAb806 binding induces reduced cell viability / necrosis within tumor xenografts.

[0346] Immunohistochemical analysis of U87MG, U87MG.Δ2-7, and U87MG.wtEGFR xenograft sections was performed to determine de2-7 and wtEGFR expression levels after mAb806 treatment. Sections were collected at days 24 and 42 as shown above and immunostained with 528 and 806 antibodies. As expected, the 528 antibody stained all xenograft sections, with no significant decrease in intensity between treated and control tumors. Staining of U87MG sections was undetectable with mAb806, but positive staining was observed in U87MG.Δ2-7 and U87MG.wtEGFR xenograft sections. There was no difference in mAb806 staining density between control and treated U87MG.Δ2-7 and U87MG.wtEGFR xenografts. This suggests that antibody treatment does not reduce de2-7 or wtEGFR expression.

[0347] Treatment of A431 xenografts with mAb806 To demonstrate that the antitumor effect of mAb806 is not limited to U87MG cells, mice carrying A431 xenografts were administered this antibody. These cells contained amplified EGFR genes and approximately 2 × 10⁶ cells per cell. 6 It expresses the receptor for EGFR. As described above, mAb806 binds to approximately 10% of these EGFRs and targets A431 xenografts. When examined in the previously described prophylactic xenograft model, mAb806 significantly inhibited the growth of A431 xenografts (Figure 11A). At day 13, when control animals were sacrificed, the mean tumor volume was 135 ± 147.54 mm in the control group. 3 The results were as follows: and for the 1 mg injection treatment group, the average length was 260 ± 60.33 mm. 3 (p<0.0001).

[0348] In another experiment, a 0.1 mg mAb dose also significantly inhibited the growth of A431 xenografts in a prophylactic model.

[0349] In light of the efficacy of a prophylactic A431 xenograft model, we examined this ability to probabilistically inhibit tumor xenograft growth. Antibody therapy was used when the tumor was 201 ± 19.09 mm. 3 Except for not initiating the treatment until the mean tumor volume reached a certain level, the treatment proceeded as described in the prophylactic model. mAb806 significantly inhibited the growth of the probabilistic tumor xenograft (Figure 11B). At day 13, when the control animals were sacrificed, the mean tumor volume was 1142 ± 120.06 mm for the control group. 3 The results were as follows: and for the 1 mg injection group, the average was 451 ± 65.58 mm. 3 (p<0.0001).

[0350] In summary, the therapeutic studies with mAb806 described here clearly demonstrated dose-dependent inhibition of U87MG.Δ2-7 xenograft growth. In contrast, inhibition of parental U87MG xenografts was not observed, despite the fact that they continued to express wtEGFR in vivo. mAb806 not only significantly reduced xenograft volume but also induced significant necrosis within the tumor. This is the first report demonstrating the success of the in vivo therapeutic use of such an antibody against human de2-7 EGFR expressing glioma xenografts.

[0351] EGFR gene amplification has been reported in numerous different tumors and is observed in approximately 50% of gliomas (Voldberg et al., 1997). It has been suggested that subsequent EGFR overexpression mediated by receptor gene amplification can provide a growth advantage by increasing intracellular signaling and cell growth (Filmus et al., 1987). Glioma cells mimicking EGFR gene amplification were produced by transfecting U87MG cell lines with wtEGFR. Treatment of U87MG.wtEGFR xenografts with mAb806 resulted in significant growth inhibition. Therefore, mAb806 also mediates in vivo antitumor activity against cells with EGFR gene amplification. Interestingly, mAb806 inhibition of U87MG.wtEGFR xenografts appears to be less effective than that observed in U87MG.Δ2-7 tumors. This likely reflects the fact that mAb806 has a lower affinity for amplified EGFR and binds to only a small percentage of the receptor expressed on the cell surface. However, despite the small effect on U87MG.wtEGFR xenograft volume, mAb806 treatment resulted in large necrotic areas within these xenografts.

[0352] To rule out the possibility that mAb806 mediates inhibition solely of U87MG-derived cell lines, we tested its efficacy against A431 xenografts. These squamous cell carcinoma-derived cell lines possess significant EGFR gene amplification, which is retained both in vitro and in vivo. Treatment of A431 xenografts with mAb806 resulted in significant growth inhibition in both prophylactic and stochastic models. This indicates that the antitumor effect of mAb806 is not limited to the transfected U87MG cell line. [Examples]

[0353] Combination therapy with A431 xenografts using mAb806 and AG1478 The antitumor effect of mAb806 in combination with AG1478 was tested in mice carrying A431 xenografts. AG1478 (4-(3-chloroanilino)-6,7-dimethoxyquinazoline) is a potent and selective inhibitor of EGFR kinase against HER2-neu and is a platelet-derived growth factor receptor kinase (Calbiochem.Cat. No. 658552). Three controls were included: treatment with vehicle alone, vehicle + mAb806 alone, and vehicle + AG1478 alone. The results are shown in Figure 12. 0.1 mg of mAb806 was administered 1 day before xenografting and at 1, 3, 6, 8, and 10 days post-xenografting. 400 μg of AG1478 was administered at 0, 2, 4, 7, 9, and 11 days post-xenografting.

[0354] Both AG1478 and mAb806, when administered individually, resulted in a significant reduction in tumor volume. However, when used in combination, the reduction in tumor volume was greatly enhanced.

[0355] In addition, the binding of mAb806 to EGFR in A431 cells was evaluated in the absence and presence of AG1478. Cells were left overnight in serum-free medium, then treated with AG1478 for 10 minutes at 37°C, washed twice with PBS, and then lysed with 1% Triton. The lysates were prepared by centrifugation at 12,000 g for 10 minutes. Subsequently, the lysates were evaluated for 806 reactivity by ELISA using a modified version of the assay described by Schooler and Wiley, Analytical Biochemistry 277, 135-142 (2000). Plates were coated overnight at room temperature with 10 μg / mL of mAb806 in PBS / EDTA, and then washed twice. Subsequently, plates were blocked with 10% serum albumin / PBS for 2 hours at 37°C and washed twice. The cell lysate at a 1:20 ratio was added to 10% serum albumin / PBS at 37°C for 1 hour, followed by four washes. Anti-EGFR (SC-03; Santa Cruz Biotechnology Inc.) in 10% serum albumin / PBS was reacted at room temperature for 90 minutes, and the plate was washed four times. Anti-rabbit-HRP (1:2000 from Silenus) in 10% serum albumin / PBS was added at room temperature for 90 minutes, followed by four washes, and color development was performed using ABTS as a substrate. mAb806 binding was found to be significantly increased in the presence of gradually increasing doses of AG1478 (Figure 13). [Examples]

[0356] Immunoreactivity in human glioblastoma pre-classified by EGFR status Given the high incidence of EGFR expression, amplification, and mutation in glioblastoma, detailed immunohistochemical studies were performed to assess the specificity of 806 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. Of these tumors, six expressed only wtEGFR transcripts, 10 had wtEGFR gene amplification, of which five showed only wild-type EGFR transcripts, and five showed both wild-type EGFR and de2-7 gene transcripts.

[0357] Immunohistochemical analysis was performed using 5 mm sections of fresh frozen tissue, fixed in cold acetone for 10 minutes after being applied to histological slides. The bound primary antibody was detected by biotinylated equine anti-mouse antibody, followed by an avidin-biotin complex reaction. Diaminobenzidine tetrahydrochloride (DAB) was used as a chromogenic agent. The degree of immunohistochemical reaction in the tissue was predicted by light microscopy and graded according to the number of immunoreactive cells in 25% increments as follows: Localized = less than 5% +=5-25% ++=25-50% +++=50-75% ++++=>75%

[0358] 528 cells showed strong reactivity in all tumors, but DH8.3 immunostaining was limited to tumors expressing de2-7 EGFR (Table 2). Consistent with previous observations in FACS and rosette formation assays, mAb806 did not resonate with glioblastoma expressing wtEGFR transcripts from non-amplified EGFR genes (Table 2). This reactivity pattern of mAb806 is similar to that observed in xenograft studies and, conversely, suggests that this antibody recognizes de2-7 and amplified EGFR but not wtEGFR when expressed on the cell surface.

[0359] [Table 3] * localized staining [Examples]

[0360] EGFR immunoreactivity in normal tissues To determine whether de2-7 EGFR is expressed in normal tissues, immunohistochemical studies with mAb806 and DH8.3 were performed on a panel of 25 tissues. None of the tissues tested showed strong immunoreactivity with either mAb806 or DH8.3. This suggests that de2-7 EGFR is absent in normal tissues (Table 3). Some variable staining with mAb806 was observed in the tonsils, limited to the basal cell layer of the epidermis and mucosal squamous cells of the epithelium. In the placenta, incidental immunostaining was observed in the trophoblast epithelium. Interestingly, two tissues expressing high endogenous levels of wtEGFR, liver and skin, showed no significant mAb806 reactivity. No reactivity was observed in liver samples, and only weak, inconsistent, localized reactivity was incidentally detected (less than 10% of all samples studied) in basal keratinocytes in skin samples and tonsil mucosal squamous epithelium. This further demonstrates that this antibody does not bind to any significant amount of wtEGFR expressed on the cell surface (Table 3). All complement tissues were positive for wtEGFR, as evidenced by universal staining with the 528 antibody (Table 3).

[0361] [Table 4] * Some interstitial staining in various tissues [Examples]

[0362] EGFR immunoreactivity in various tumors A panel of 12 different malignant lesions was used to examine the extent of de2-7 EGFR in other tumor types. The 528 antibody showed homogeneous staining in most of the tumors analyzed, excluding melanoma and seminoma. Where present, DH8.3 immunoreactivity was limited to incidental lesion tumor cells. This suggests that, using this detection system, de2-7 EGFR expression in extrabrain tumors is minimal, if any (Table 4). There was also localized staining in blood vessels and various diffuse staining of connective tissue with the DH8.3 antibody in some tumors (Table 4). This staining was strongly dependent on the antibody concentration used and was considered nonspecific background reactivity. mAb806 showed positive staining in 64% of head and neck tumors and 50% of lung carcinomas (Table 4). mAb806 reactivity was minimal in other locations, except for urological tumors, where 30% of cases were positive.

[0363] Since head and neck cancers and lung cancers were negative for DH8.3 antibody, the reactivity observed with this mAb in these tumors can be associated with EGFR gene amplification.

[0364] [Table 5] * localized staining [Examples]

[0365] Immunoreactivity in human glioblastoma without EGFR status selection To confirm the specificity of mAb806 and evaluate its reactivity, it was compared to 528 and DH8.3 antibodies in a panel of 46 glioblastomas that were not pre-selected for their EGFR status. The 528 antibody was strongly and homogeneously positive in all but two samples (numbers 27 and 29) (44 / 46, 95.7%). These two cases were also negative for mAb806 and mAbDH8.3. mAb806 was positive in 27 / 46 (58.7%) cases, of which 22 cases exhibited homogeneous immunoreactivity in more than 50% of these tumors. The DH8.3 antibody was positive in 15 / 46 (32.6%) glioblastomas, of which 9 cases showed homogeneous immunoreactivity. Immunochemical staining of these unselected tumors is presented in Table 5.

[0366] In all cases except for one (case 35), there was a concordance between mAb806 and DH8.3. Molecular analysis for the presence of EGFR amplification was performed in 44 cases (Table 5). Of these, 30 cases were cotyped and possessed the previously established mAb806 immunoreactivity pattern: for example, 16 mAb806-negative cases did not show EGFR amplification, and 14 EGFR-amplified cases were also mAb806 immunopositive. However, 13 cases showing 806 immunoreactivity were negative for EGFR amplification, while one EGFR-amplified case was mAb806-negative. Further analysis of the mutation status of these amplification-negative and 806-positive cases is described below. This analysis explains the majority of the 13 cases that were negative for EGFR amplification and recognized by 806.

[0367] Subsequently, molecular analysis of deletion mutations was performed on 41 / 46 cases by RT-PCR (Table 5). Of these, 34 cases were cotyped and possessed DH8.3 specific to the deletion mutation: 12 cases were positive in both RT-PCR and immunohistochemistry, and 22 cases were negative / negative. Three cases (numbers 2, 34, and 40) were DH8.3 positive / RT-PCR negative for the deletion mutation, and three cases (numbers 12, 18, and 39) were DH8.3 negative / RT-PCR positive. As predicted by our previous specificity analysis, mAb806 immunoreactivity was observed in all DH8.3-positive tissues except for one case (number 35).

[0368] Case number 3 also showed a mutation containing a de2-7 mutation sequence (referred to as A2 in Table 5), but this mutation did not appear to be a classical de2-7 deletion with a missing base at 801 (data not shown). This case was negative for DH8.3 reactivity but showed reactivity with 806. This suggests that 806 can recognize additional, possibly specific, EGFR mutations.

[0369] [Table 6] TIFF2026143589000007.tif46170 * N = unamplified, A = amplified + WT = Wild type, 5' mutation nd = not done

[0370] 806 antibody reactivity was observed in 19 / 27 cases, or in over 70% of the aforementioned cases, for amplified or de2-7 mutant EGFR cotypes. It is noteworthy that two of these eight cases were also DH8.3 reactive. [Examples]

[0371] Systemic treatment and analysis of intracranial glioma tumors To test the efficacy of the anti-ΔEGFR monoclonal antibody, mAb806, the inventors treated nude mice with intracranial ΔEGFR overexpressing glioma xenografts with injections of mAb806, this isotype control IgG, or PBS.

[0372] Human glioblastoma primary explants rapidly lose amplified and rearranged receptor expression in culture, and no existing glioblastoma cell lines exhibit such expression. To maintain expression levels comparable to those seen in human tumors, U87MG, LN-Z308, and A1207 cells (donated by Dr. S. Aaronson of Amount Sinai Medial Center, New York, NY) were infected with ΔEGFR, kinase-deficient ΔEGFR (DK), or wild-type EGFR (wtEGFR) viruses. As previously described (Nishikawa et al. (1994) A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc. Natl. Acad. Sci. USA, 91, 7727-7731), these viruses also conferred resistance to G418.

[0373] Populations expressing various EGFR alleles at similar levels (these expression levels roughly correspond to amplification levels of 25 gene copies; human glioblastoma generally has amplification levels of 10 to 50 gene copies of the truncated receptor) were selected by FACS as previously described (Nishikawa et al., 1994) and named 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 a medium containing G418 (U87MG cell line, 400 μg / mL; LN-Z308 and A1207 cell lines, 800 μg / mL).

[0374] U87MG.ΔEGFR cells (1 × 10) in 5 μL of PBS 5 ) or 5 × 10 5 LN-Z308.ΔEGFR, A1207.ΔEGFR, U87MG, U87MG.DK, and U87MG.wtEGFR cells were implanted in the right striatum of nude mouse brains as previously described (Mishima et al. (2000) 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). From day 0 to day 14 after implantation, systemic therapy with mAb806 or IgG2b isotype control was performed by intravenous injection of 1 μg of mAb in 100 μL volume every other day. For direct therapy of intracerebral U87MG.ΔEGFR tumors, 10 μg of mAb806 or IgG2b isotype control was injected into the tumor injection site every other day for 5 days, starting on day 1.

[0375] Animals treated with PBS or isotype control IgG had a median survival time of 13 days, while mice treated with mAb806 had a median survival time of 21 days, a 61.5% increase (p<0.001; Figure 24A).

[0376] Treatment of mice 3 days after tumor probability and implantation extended the median survival time of mAb806-treated animals by 46.1% (from 13 to 19 days; p<0.01) compared to the control group (data not shown).

[0377] To determine whether these antitumor effects of mAb806 extend beyond the U87MG.ΔEGFR xenograft, animals with other glioma cell xenografts, LN-Z380.ΔEGFR and A1207.ΔEGFR, were treated similarly. The median survival time of mAb806-treated mice with the LN-Z308.ΔEGFR xenograft was extended from 19 days to 58 days compared to controls (P<0.001; Figure 24B). Surprisingly, four of the eight mAb806-treated animals survived for more than 60 days (Figure 24B). The median survival time of animals with the A1207.ΔEGFR xenograft was also extended from 24 days to 29 days compared to controls (P<0.01; data not shown).

[0378] mAb806 therapy inhibits the growth of ΔEGFR-overexpressing brain tumors. Mice carrying U87MG.ΔEGFR and LN-Z308.ΔEGFR xenografts were euthanized on days 9 and 15, respectively. Tumor sections were histopathologically analyzed to determine tumor volume. Consistent with observations regarding animal survival, mAb806 treatment reduced the volume of the xenografts, U87MG.ΔEGFR by approximately 90% (P<0.001; Figure 24C) and LN-Z308.ΔEGFR by more than 95% (P<0.001; Figure 24D), compared to the control group. Similar results were obtained for animals with A1207.ΔEGFR tumors (65% volume reduction, P<0.01; data not shown).

[0379] Intratumor therapy with mAb806 extends survival in mice with U87MG.ΔEGFR brain tumors. The efficacy of direct intratumoral injection of mAb806 for the treatment of U87MG.ΔEGFR xenografts was also evaluated. Animals were injected intratumorally with either mAb806 or isotype control IgG the day after tumor implantation. Control animals survived for 15 days, while mAb806-treated mice remained alive for 18 days (P<0.01; Figure 24E). Intratumoral treatment with mAb806 was somewhat effective, but it inevitably involved the difficulties of multiple intracranial injections and increased injection risks. Therefore, the inventors focused on systemic treatment for further research.

[0380] mAb806 treatment extends the survival time of mice with U87MG.wtEGFR, but does not extend the survival time of mice with U87MG and U87MG.DK intracranial xenografts. To determine whether growth inhibition by mAb806 is selective for mice expressing ΔEGFR, we treated animals with U87MG, U87MG.DK (kinase-deficient ΔEGFR), and U87MG.wtEGFR brain xenografts. mAb806 treatment did not extend the survival time of mice implanted with a U87MG tumor expressing low levels of endogenous wild-type EGFR (wtEGFR) (Huang et al. (1997) 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), nor did it extend the survival time of animals with U87MG.DK xenografts overexpressing kinase-deficient ΔEGFR in addition to low levels of endogenous wtEGFR (Figure 25). mAb806 treatment slightly extended the survival time of mice with U87MG.wtEGFR tumors that overexpress wtEGFR (P<0.05, median survival time of 23 days compared to 26 days for the control group) (Figure 25C).

[0381] mAb806 responsiveness correlates with in vivo antitumor efficacy. To understand the different effects of mAb806 on tumors expressing various levels or types of EGFR, we determined the reactivity of mAb806 with various tumor cells by FACS. Stained cells were analyzed using Cell Quest software (Becto-Dickinson PharMingen) with FACS Calibur. For the first antibody, we used the following mAbs: mAb806, anti-EGFR mAb clone 528, and clone EGFR.1. Mouse IgG2a or IgG2b was used as an isotype control.

[0382] Consistent with previous reports (Nishikawa et al. (1994) A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc. Natl. Acad. Sci. USA, 91, 7727-7731), anti-EGFR mAb 528 recognized both ΔEGFR and wtEGFR, and clearly showed strong staining for U87MG.ΔEGFR cells compared to U87MG cells (Figure 25A, 528).

[0383] In contrast, the antibody EGFR.1 reacted with wtEGFR but not with ΔEGFR (Nishikawara et al., 1994). This is because U87MG.ΔEGFR cells were weakly reactive, similar to U87MG cells (Figure 26A, panel EGFR.1).

[0384] This EGFR.1 antibody reacted more potently with U87MG.wtEGFR than with U87MG cells. This is because U87MG.wtEGFR cells overexpress wtEGFR (Figure 26A, panel EGFR.1). mAb806 reacted strongly with U87MG.ΔEGFR and U87MG.DK cells, but not with U87MG cells, although it reacted weakly with U87MG.wtEGFR. This indicates that mAb806 is selective for ΔEGFR and has weak cross-activity to overexpressed wtEGFR (Figure 26A, panel mAb806).

[0385] This level of reactivity with U87MG.wtEGFR was quantitatively and qualitatively similar to antibody-mediated survival extension (Figure 25C).

[0386] The inventors further determined the specificity of mAb806 by immunoprecipitation. EGFR in various cell lines was immunoprecipitated with the antibody mAb806, anti-EGFR mAb clone 528 (Oncogene Research Products, Boston, Massachusetts), or clone EGFR.1 (Oncogene Research Products).

[0387] In short, cells were lysed with a 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 phenylmethyl fluoride (phenylmethlsulfonyl fluoride), 2 mM Na3VO4, 5 μg / mL leupeptin, and 5 μg / mL aprotinin. The antibody was incubated with the cell lysate at 4°C for 1 hour, after which protein A and G Sepharose were added. The immunoprecipitation was washed twice with the lysis buffer and once with HNTG buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol], subjected to electrophoresis, and transferred to a nitrocellulose membrane.

[0388] Protein blots separated by electrophoresis were probed with the anti-EGFR antibody C13 (provided by Dr. GNGill of the University of California, San Diego, California), which is used to detect both wild-type EGFR and ΔEGFR in immunoblotting (Huang et al., 1997), and the proteins were visualized using an ECL chemiluminescence detection system (Amercham Pharmacia Biotech). Antibodies against Bcl-X (rabbit polyclonal antibody; Transduction Laboratories, Lexington, Kentucky) and phosphotyrosine (4G10, Upstate Biotechnology, Lake Placid, New York) were used for Western blot analysis as previously described (Nagane et al. (1998) 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).

[0389] Consistent with FACS analysis, antibody 528 recognized wtEGFR and the mutant receptor (Figure 26B - Panel IP: 528), while antibody EGFR.1 reacted with wtEGFR but not with the mutant (Figure 26B, Panel IP: EGFR.1). Furthermore, the levels of the mutant receptor in U87MG.ΔEGFR and U87MG.DK cells were comparable to those of wtEGFR in U87MG.wtEGFR cells (Figure 26B, Panel IP: 528).

[0390] However, the antibody mAb806 was only able to precipitate small amounts of wtEGFR from U87MG.wtEGFR cell lysates compared to the larger amounts of mutant receptors precipitated from U87MG.ΔEGFR and U87MG.DK cells, and was undetectable in amount from U87MG cells (Figure 26B, panel IP: mAb806). In summary, these data suggest that mAb806 recognizes an epitope within ΔEGFR that is present even in small portions of wtEGFR when overexpressed on the cell surface (see further discussion and references on the mAb806 epitope below).

[0391] mAb806 treatment reduces ΔEGFR autophosphorylation and Bcl.X in U87MG.ΔEGFR brain tumors. L Reduce expression Next, we investigated the underlying mechanism of growth inhibition by mAb806. The constitutively active kinase activity and carboxy-terminal autophosphorylation of ΔEGFR are indispensable for this biological function (Nishikawa et al. (1994) A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc. Natl. Acad. Sci. USA 91, 7727-7731; Huang et al., 1997; Nagane et al. (1996) A common mutant epidermal growth factor receptor confers enhanced tumorigenicity on human glioblastoma cells by increasing proliferation and redu...

Claims

1. An isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain multiple copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, wherein the antibody does not bind to the de2-7 junction peptide consisting of the amino acid sequence of SEQ ID NO: 13; binds to an epitope in the sequence of residues 287-302 of human wild-type EGFR (SEQ ID NO: 14); and does not contain a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 2, nor does it contain a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO:

4.

2. The isolated antibody according to claim 1, comprising a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence shown in SEQ ID NO: 42 and the light chain has the amino acid sequence shown in SEQ ID NO:

47.

3. The isolated antibody according to claim 1, comprising a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence shown in SEQ ID NO: 129 and the light chain has the amino acid sequence shown in SEQ ID NO:

134.

4. The isolated antibody according to claim 1, comprising a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence shown in SEQ ID NO: 22 and the light chain has the amino acid sequence shown in SEQ ID NO:

27.

5. The isolated antibody according to claim 1, comprising a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence shown in SEQ ID NO: 32 and the light chain has the amino acid sequence shown in SEQ ID NO:

37.

6. The isolated antibody according to claim 1, comprising a heavy chain and a light chain, wherein the variable region of the heavy chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequences shown in SEQ ID NOs: 44, 45, and 46.

7. The isolated antibody according to claim 1, comprising a heavy chain and a light chain, wherein the variable region of the light chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequences shown in SEQ ID NOs: 49, 50, and 51.

8. The isolated antibody according to claim 1, comprising a heavy chain and a light chain, wherein the variable region of the heavy chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequences shown in SEQ ID NOs: 130, 131, and 132.

9. The isolated antibody according to claim 1, comprising a heavy chain and a light chain, wherein the variable region of the light chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequences shown in SEQ ID NOs: 135, 136, and 137.

10. The isolated antibody according to claim 1, comprising a heavy chain and a light chain, wherein the variable region of the heavy chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequences shown in SEQ ID NOs: 23, 24, and 25.

11. The isolated antibody according to claim 1, comprising a heavy chain and a light chain, wherein the variable region of the light chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequences shown in SEQ ID NOs: 28, 29, and 30.

12. The isolated antibody according to claim 1, comprising a heavy chain and a light chain, wherein the variable region of the heavy chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequences shown in SEQ ID NOs: 33, 34, and 35.

13. The isolated antibody according to claim 1, comprising a heavy chain and a light chain, wherein the variable region of the light chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequences shown in SEQ ID NOs: 38, 39, and 40.

14. The isolated antibody according to claim 1, which is in the form of antibody F(ab')2, scFv fragment, diabody, triabody or tetrabody.

15. The isolated antibody according to claim 1, further comprising a detectable or functionally labeled substance.

16. The isolated antibody according to claim 15, wherein the detectable or functional label is a drug to which it is covalently attached.

17. The isolated antibody according to claim 15, wherein the label is a radiolabel.

18. The isolated antibody according to claim 1, which is PEG-modified.

19. An isolated nucleic acid comprising a sequence encoding the isolated antibody described in claim 1.

20. A method for preparing an isolated antibody according to claim 1, comprising expressing a nucleic acid under conditions that induce the expression of the antibody, and recovering the antibody.

21. A method for treating a tumor in a human patient, comprising administering an effective amount of the isolated antibody described in claim 1 to the patient.

22. A kit for the diagnosis of tumors in which EGFR is abnormally expressed or EGFR is expressed in a truncated protein form, comprising the isolated antibody of claim 1.

23. A kit for the diagnosis of major EGFR abnormal expression or expression of EGFR in a truncated protein form, according to claim 22, further comprising reagents and / or instructions for use.

24. A pharmaceutical composition comprising the isolated antibody described in claim 1.

25. The pharmaceutical composition according to claim 24, further comprising a pharmaceutically acceptable vehicle, carrier, or diluent.

26. The pharmaceutical composition according to claim 24, further comprising an anticancer agent selected from the group consisting of a chemotherapy agent, an anti-EGFR antibody, a radioimmunotherapy agent, and a combination thereof.

27. The pharmaceutical composition according to claim 26, wherein the chemotherapeutic agent is selected from the group consisting of tyrosine kinase inhibitors, phosphorylation cascade inhibitors, posttranslational regulators, cell growth or division inhibitors (e.g., antimitotic agents), signal transduction inhibitors, and combinations thereof.

28. The pharmaceutical composition according to claim 27, wherein the tyrosine kinase inhibitor is selected from the group consisting of AG1478, ZD1839, STI571, OSI-774, SU-6668, and combinations thereof.

29. The pharmaceutical composition according to claim 26, wherein the anti-EGFR antibody is selected from the group consisting of anti-EGFR antibodies 528, 225, SC-03, DR8.3, L8A4, Y10, ICR62, ABX-EGF, and combinations thereof.

30. A method for preventing and / or treating cancer in a mammal, comprising administering a therapeutically effective amount of the pharmaceutical composition described in claim 24 to a mammal.

31. A method for treating endogenous brain cancer that produces abnormally expressed EGFRs in a mammal, comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 24 to a mammal.

32. A method for treating an endogenous brain cancer in a mammal that produces an abnormally expressed EGFR as described in claim 30, wherein the endogenous brain cancer is selected from the group consisting of glioblastoma, medulloblastoma, meningioma, neoplastic astrocytoma, and neoplastic arteriovenous malformation.

33. A single-celled host transformed with a recombinant DNA molecule encoding the isolated antibody of claim 1.

34. A single-celled host transformed with a recombinant DNA molecule encoding the isolated antibody of claim 1, selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NSO, SP2 / 0, R1.1, B-W, L-M, COS 1, COS 7, BSC1, BSC40, and BMT10 cells, plant cells, insect cells, and human cells (in tissue culture).

35. A method for detecting the presence of amplified EGFR, de2-7EGFR, or EGFR having high mannose glycosylation, wherein the EGFR is measured by (a) contacting a biological sample from a mammal suspected to contain amplified EGFR, de2-7EGFR, or EGFR having high mannose glycosylation with an isolated antibody of claim 1 under conditions that allow the EGFR to bind to the isolated antibody; and (b) detecting whether binding has occurred between the EGFR from the sample and the isolated antibody, wherein the detection of binding indicates the presence or activity of the EGFR in the sample.

36. A method for detecting cancer in a mammal, comprising the presence or detection of EGFR according to the method of claim 35, wherein the detection of the presence of EGFR indicates the presence of a tumor or cancer in the mammal.

37. An isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain multiple copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, comprising a heavy chain and a light chain, wherein the heavy chain has an amino acid sequence substantially homologous to the amino acid sequence shown in SEQ ID NO: 42, and the light chain has an amino acid sequence substantially homologous to the amino acid sequence shown in SEQ ID NO:

47.

38. An isolated antibody according to claim 37, wherein the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 42, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:

47.

39. An isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain multiple copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, comprising a heavy chain and a light chain, wherein the variable region of the heavy chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NOs: 44, 45, and 46, and the variable region of the light chain comprises a polypeptide-binding domain having an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NOs: 49, 50, and 51.

40. An isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain numerous copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, comprising a heavy chain and a light chain, wherein the heavy chain has an amino acid sequence substantially homologous to the amino acid sequence shown in SEQ ID NO: 129, and the light chain has an amino acid sequence substantially homologous to the amino acid sequence shown in SEQ ID NO:

134.

41. An isolated antibody according to claim 40, wherein the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 129, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:

134.

42. An isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain multiple copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, comprising a heavy chain and a light chain, wherein the variable region of the heavy chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NOs: 130, 131 and 132, and the variable region of the light chain comprises a polypeptide-binding domain having an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NOs: 135, 136 and 138.

43. An isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain numerous copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, comprising a heavy chain and a light chain, wherein the heavy chain has an amino acid sequence substantially homologous to the amino acid sequence shown in SEQ ID NO: 22, and the light chain has an amino acid sequence substantially homologous to the amino acid sequence shown in SEQ ID NO:

27.

44. An isolated antibody according to claim 43, wherein the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 22, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:

27.

45. An isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain multiple copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, comprising a heavy chain and a light chain, wherein the variable region of the heavy chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NOs: 23, 24, and 25, and the variable region of the light chain comprises a polypeptide-binding domain having an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NOs: 28, 29, and 30.

46. An isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain numerous copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, comprising a heavy chain and a light chain, wherein the heavy chain has an amino acid sequence substantially homologous to the amino acid sequence shown in SEQ ID NO: 32, and the light chain has an amino acid sequence substantially homologous to the amino acid sequence shown in SEQ ID NO:

37.

47. An isolated antibody according to claim 46, wherein the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 32, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:

37.

48. An isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain multiple copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, comprising a heavy chain and a light chain, wherein the variable region of the heavy chain comprises a polypeptide-binding domain region having an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NOs: 33, 34, and 35, and the variable region of the light chain comprises a polypeptide-binding domain having an amino acid sequence highly homologous to the amino acid sequence shown in SEQ ID NOs: 38, 39, and 40.

49. An isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain multiple copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, Sequence ID: It does not bind to the de2-7 junction peptide, which consists of 13 amino acid sequences; It binds to the epitope within the sequence of residues 287-302 (SEQ ID NO: 14) of human wild-type EGFR; Including a light chain and a heavy chain, the variable region of the light chain is The amino acid sequence shown in formula I: HSSQDIX ａａ１ SNIG (I) (In the formula, X ａａ１ (This refers to an amino acid residue having a non-charged R group.) A first polypeptide-binding domain region having the corresponding amino acid sequence, The amino acid sequence shown in formula II: HGTNLX ａａ２ D (II) (In the formula, X ａａ２ (This refers to an amino acid residue that has a charged R group.) A second polypeptide-binding domain region having the corresponding amino acid sequence, and The amino acid sequence shown in formula III: VQYX ａａ３ QFPWT (III) (In the formula, X ａａ３ (This is selected from the group consisting of amino acid residues A, G, and those in which A or G is conservatively substituted.) It comprises a third polypeptide-binding domain region having the corresponding amino acid sequence, and the variable region of the heavy chain is The amino acid sequence shown in formula VI: SADX ａａ４ AWN (IV) (In the formula, X ａａ４ (This is selected from the group consisting of amino acid residues F, Y, and F or Y in which they are conservatively substituted.) A first polypeptide-binding domain region having the corresponding amino acid sequence, The amino acid sequence shown in formula V, formula VI, or formula VII: YISYSGNTRYX ａａ５ PSLKS (V) (In the formula, X ａａ５ This is an amino acid residue having a non-charged R group. YISYSX ａａ６ NTRYNPSLKS (VI) (In the formula, X ａａ６ (This is selected from the group consisting of amino acid residues G, A, and G or A in which they are conservatively substituted.) YISYSGNTRYNPSLX ａａ７ S (VII) (In the formula, X ａａ７ (This is a basic amino acid residue.) A second polypeptide binding region having the corresponding amino acid sequence, and The amino acid sequence shown in formula VIII: X ａａ８ TAGRGFPY (VIII) (In the formula, X ａａ８ (This is selected from the group consisting of amino acid residues V, A, and V or A in which they are conservatively substituted.) A third polypeptide-binding domain region having the corresponding amino acid sequence. It includes; Furthermore, an antibody that does not contain a heavy chain variable region sequence having the amino acid sequence shown in SEQ ID NO: 2, and does not contain a light chain variable region sequence having the amino acid sequence shown in SEQ ID NO:

4.

50. X ａａ１ However, N is X ａａ２ However, D is X ａａ３ However, A is; X ａａ４ However, F is X ａａ５ However, it is an amino acid residue having a non-charged R group; X ａａ６ However, it is G; X ａａ７ However, K is; and X ａａ８ The isolated antibody according to claim 49, wherein V.

51. X ａａ５ The isolated antibody according to claim 50, wherein the antibody is N or Q.

52. X ａａ１ The antibody according to claim 49, wherein the antibody is N or S.

53. X ａａ２ The antibody according to claim 49, wherein the antibody is D or E.

54. X ａａ３ The antibody according to claim 49, wherein the antibody is A or G.

55. X ａａ４ The antibody according to claim 49, wherein the antibody is F or Y.

56. X ａａ５ The antibody according to claim 49, wherein the antibody is N or Q.

57. X ａａ６ However, G or A, and X ａａ７ The antibody according to claim 49, wherein the antibody is independently K or R.

58. X ａａ８ The antibody according to claim 49, wherein the antibody is V or A.

59. The isolated antibody according to claim 49, which is in the form of antibody F(ab')2, scFv fragment, diabody, triabody or tetrabody.

60. The isolated antibody according to claim 49, further comprising a detectable or functional label.

61. The isolated antibody according to claim 60, wherein the detectable or functional label is a drug to which it is covalently attached.

62. The isolated antibody according to claim 60, wherein the label is a radiolabel.

63. The isolated antibody according to claim 49, which is PEG-modified.

64. An isolated nucleic acid comprising a sequence encoding the isolated antibody of claim 49.

65. A method for preparing an isolated antibody according to claim 49, comprising expressing a nucleic acid under conditions that induce the expression of the antibody, and recovering the antibody.

66. A method for treating a tumor in a human patient, comprising administering an effective amount of the isolated antibody described in claim 49 to the patient.

67. A kit for the diagnosis of tumors in which EGFR is abnormally expressed or EGFR is expressed in a truncated protein form, comprising the isolated antibody of claim 49.

68. A kit for the diagnosis of tumors in which EGFR is abnormally expressed or expressed in a truncated protein form, according to claim 67, further comprising reagents and / or instructions for use.

69. A pharmaceutical composition comprising the isolated antibody described in claim 49.

70. The pharmaceutical composition according to claim 69, further comprising a pharmaceutically acceptable vehicle, carrier, or diluent.

71. The pharmaceutical composition according to claim 69, further comprising an anticancer agent selected from the group consisting of a chemotherapy agent, an anti-EGFR antibody, a radioimmunotherapy agent, and a combination thereof.

72. The pharmaceutical composition according to claim 71, wherein the chemotherapeutic agent is selected from the group consisting of tyrosine kinase inhibitors, phosphorylation cascade inhibitors, posttranslational regulators, cell growth or division inhibitors (e.g., antimitotic agents), signal transduction inhibitors, and combinations thereof.

73. The pharmaceutical composition according to claim 72, wherein the tyrosine kinase inhibitor is selected from the group consisting of AG1478, ZD1839, STI571, OSI-774, SU-6668, and combinations thereof.

74. The pharmaceutical composition according to claim 71, wherein the anti-EGFR antibody is selected from the group consisting of anti-EGFR antibodies 528, 225, SC-03, DR8.3, L8A4, Y10, ICR62, ABX-EGF, and combinations thereof.

75. A method for preventing and / or treating cancer in a mammal, comprising administering a therapeutically effective amount of the pharmaceutical composition described in claim 69 to a mammal.

76. A method for treating endogenous brain cancers that produce abnormally expressed EGFRs in mammals, comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 69 to a mammal.

77. A method for treating an endogenous brain cancer in a mammal that produces an abnormally expressed EGFR according to claim 76, wherein the endogenous brain cancer is selected from the group consisting of glioblastoma, medulloblastoma, meningioma, neoplastic astrocytoma, and neoplastic arteriovenous malformation.

78. A single-celled host transformed with a recombinant DNA molecule encoding the isolated antibody of claim 49.

79. A single-celled host transformed with a recombinant DNA molecule encoding the isolated antibody of claim 49, selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, YB / 20, NSO, SP2 / 0, R1.1, B-W, L-M, COS 1, COS 7, BSC1, BSC40, and BMT10 cells, plant cells, insect cells, and human cells (in tissue culture).

80. A method for detecting the presence of amplified EGFR, de2-7EGFR, or EGFR having high mannose glycosylation, wherein the EGFR is measured by (a) contacting a biological sample from a mammal suspected to contain amplified EGFR, de2-7EGFR, or EGFR having high mannose glycosylation with an isolated antibody according to claim 49 under conditions that allow the EGFR to bind to the isolated antibody; and (b) detecting whether binding has occurred between the EGFR from the sample and the isolated antibody, wherein the detection of binding indicates the presence or activity of the EGFR in the sample.

81. A method for detecting cancer in a mammal, comprising the presence or detection of EGFR according to the method of claim 80, wherein the presence or detection of EGFR indicates the presence of a tumor or cancer in the mammal.

82. An isolated antibody capable of binding to EGFR in tumors having amplification of the EGFR gene (the cells of the tumor contain multiple copies of the EGFR gene) and in tumors expressing a truncated version of the EGFR receptor de2-7, wherein it does not bind to the de2-7 junction peptide consisting of the amino acid sequence of SEQ ID NO: 13; it binds to an epitope in the sequence of residues 273-501 of human wild-type EGFR; it comprises a light chain and a heavy chain, wherein the variable region of the light chain comprises a first polypeptide-binding domain region having the amino acid sequence HSSQDINSNIG (SEQ ID NO: 18), a second polypeptide-binding domain region having the amino acid sequence HGTNLDD (SEQ ID NO: 19), and a third polypeptide-binding domain region having the amino acid sequence VQYAQFPWT (SEQ ID NO: 20); and the variable region of the heavy chain comprises a first polypeptide-binding domain region having the amino acid sequence SDFAWN (SEQ ID NO: 15), the amino acid sequence shown in formula IX: YISYSGNTRYX ａａ９ PSLKS (IX) (In the formula, X ａａ９ This is an amino acid residue having a non-charged R group. An antibody comprising a second polypeptide-binding region having the corresponding amino acid sequence, and a third polypeptide-binding domain region having the amino acid sequence VTAGRGFPY (SEQ ID NO: 17).

83. The isolated antibody according to claim 82, which binds to an epitope in the sequence of residues 287-302 of human wild-type EGFR (SEQ ID NO: 14).

84. X ａａ９ The isolated antibody according to claim 82, wherein the antibody is N or Q.

85. The isolated antibody according to claim 82, wherein the binding domain region is held by a human antibody framework.

86. The isolated antibody according to claim 85, wherein the human antibody framework is a human IgG1 antibody framework.

Images