Anti-EGFR antibodies and antibody drug conjugates

Abstract

To provide anti-epidermal growth factor (EGFR) antibodies and antibody drug conjugates (ADCs), including compositions and methods of using the antibodies and ADCs.SOLUTION: There is provided an anti-hEGFR antibody or an antigen binding portion thereof, a) that binds to an epitope within a specific amino acid sequence or competes, for binding to epidermal growth factor receptor variant III (EGFRvIII), with a second anti-hEGFR antibody comprising a heavy chain variable domain containing a specific amino acid sequence and a light chain variable domain containing a specific amino acid sequence; b) that binds to EGFR with a dissociation constant (Kd) of about 1×10-6M or less as determined by surface plasmon resonance; and c) that inhibits tumor growth in an in vivo human non-small-cell lung carcinoma xenograft assay with a tumor growth inhibition percentage (%) of at least about 50% relative to a human IgG antibody which is not specific for EGFR.SELECTED DRAWING: Figure 4

Description

[Background technology]

[0001] Human epidermal growth factor receptor (also known as HER-1 or Erb-B1, referred to herein as "EGFR") is a 170 kDa transmembrane receptor encoded by the c-erbB proto-oncogene and exhibits intrinsic tyrosine kinase activity (Modjtahedi et al., Br. J. Cancer 73:228-235 (1996); Herbst and Shin, Cancer 94:1593-1611 (2002)). SwissProt database entry P00533 provides the sequence of human EGFR. EGFR regulates numerous intracellular processes through tyrosine kinase-mediated signaling pathways, including but not limited to, activation of signaling pathways that control cell proliferation, differentiation, cell survival, apoptosis, angiogenesis, mitosis, and metastasis (Atalay et al., Ann. Oncology 14:1346-1363 (2003); Tsao and Herbst, Signal 4:4-9 (2003); Herbst and Shin, Cancer 94:1593-1611 (2002); Modjtahedi et al., Br. J. Cancer 73:228-235 (1996)).

[0002] Known ligands for EGFR include EGF, TGFA / TGF-α, amphiregulin, epigen / EPGN, BTC / betacellulin, epiregulin / EREG, and HBEGF / heparin-binding EGF. Ligand binding to EGFR induces receptor homo- and / or heterodimerization and autophosphorylation of key cytoplasmic residues. Phosphorylated EGFR recruits adaptor proteins such as GRB2, resulting in activation of a complex downstream signaling cascade that includes at least the following major downstream signaling cascades: RAS-RAF-MEK-ERK, PI3 kinase-AKT, PLCγ-PKC, and STAT modules. This autophosphorylation further triggers downstream activation and signaling by several other proteins that associate with the phosphotyrosine-binding SH2 domain of EGFR. These downstream signaling proteins initiate multiple signaling cascades, including the MAPK, Akt, and JNK pathways, leading to cell proliferation. Ligand binding to EGFR can also activate the NF-κB signaling cascade. Ligand binding also directly phosphorylates other proteins, such as RGS16, activating its GTPase activity, potentially coupling EGF receptor signaling to G protein-coupled receptor signaling. Ligand binding also phosphorylates MUC1, enhancing its interaction with SRC and CTNNB1 / β-catenin.

[0003] Overexpression of EGFR has been reported in numerous human malignancies, including bladder, brain, head and neck, pancreatic, lung, breast, ovarian, colon, prostate, and kidney cancers (Atalay et al., Ann. Oncology 14:1346-1363 (2003); Herbst and Shin, Cancer 94:1593-1611 (2002); and Modjtahedi et al., Br. J. Cancer 73:228-235 (1996)). In many of these conditions, overexpression of EGFR correlates with or is associated with poor patient prognosis (Herbst and Shin, Cancer 94:1593-1611 (2002); and Modjtahedi et al., Br. J. Cancer 73:228-235 (1996)). EGFR is also expressed in cells of normal tissues, particularly epithelial tissues of the skin, lung, and gastrointestinal tract, although generally at lower levels than in malignant cells (Herbst and Shin, Cancer 94:1593-1611 (2002)).

[0004] A significant proportion of tumors containing EGFR gene amplification (i.e., multiple copies of the EGFR gene) co-express a truncated form of the receptor (Wikstrand et al. (1998) J. Neurovirol. 4, 148-158) called de2-7EGFR, ΔEGFR, EGFRvIII, or Δ2-7 (these terms are used interchangeably herein) (Olapade-Olaopa et al. (2000) Br. J. Cancer. 82, 186-94). The rearrangement seen in de2-7EGFR results in the generation of an in-frame mature mRNA lacking 801 nucleotides spanning exons 2 to 7 (Wong et al. (1992) Proc. Natl. Acad. Sci. USA 89, 2965-9; Yamazaki et al. (1990) Jpn. J. Cancer Res. 81, 773-9; Yamazaki et al. (1988) Mol. Cell. Biol. 8, 1816-20; and Sugawa et al. (1990) Proc. Natl. Acad. Sci. USA 87, 8602-6). The corresponding EGFR protein lacks 267 amino acids, consisting of residues 6 to 273 of the extracellular domain, and contains a novel glycine residue at the fusion junction (Sugawa et al., 1990). This deletion, combined with the insertion of a glycine residue, generates a unique junction peptide at the deletion interface ( Sugawa et al., 1990 ).

[0005] EGFRvIII has been reported in numerous tumor types, including glioma, breast cancer, lung cancer, ovarian cancer, and prostate cancer (Wikstrand et al. (1997) Cancer Res. 57, 4130-40; Olapade-Olaopa et al. (2000) Br. J. Cancer. 82, 186-94; Wikstrand, et al. (1995) Cancer Res. 55, 3140-8; Garcia de Palazzo et al. (1993) Cancer Res. 53, 3217-20). This truncated receptor does not bind ligand but has low constitutive activity and confers significant growth effects on glioma cells grown as tumor xenografts in nude mice (Nishikawa et al. (1994) Proc. Natl. Acad. Sci. USA 91, 7727-31), and can transform NIH3T3 cells (Batra et al. (1995) Cell Growth Differ. 6, 1251-9) and MCF-7 cells. The intracellular mechanisms utilized by de2-7EGFR in glioma cells are not fully understood, but they have been reported to be associated with reduced apoptosis (Nagane et al. (1996) Cancer Res. 56, 5079-86) and a slight increase in proliferation (Nagane et al., 1996). Because expression of this truncated receptor is restricted to tumor cells, it provides a highly specific target for antibody therapy.

[0006] Antibody-drug conjugates (ADCs) are a novel class of therapeutic agents in which antibodies are linked to cytotoxic drugs via a chemical linker. The therapeutic concept of ADCs is to combine the binding capacity of antibodies with drugs and use antibodies to deliver drugs to tumor cells by binding to target surface antigens. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Modjtahedi et al.,Br.J.Cancer 73:228-235(1996) [Non-patent document 2] Herbst and Shin, Cancer 94:1593-1611(2002) [Non-licensed document 3] Atalay et al., Ann. Oncology 14:1346-1363(2003)

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Non-licensed literature 9

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[0008] Thus, there remains a need in the art for anti-EGFR antibodies and ADCs that can be used therapeutically in cancer treatment. [Means for solving the problem]

[0009] In certain embodiments, the present invention provides anti-EGFR antibodies and antibody drug conjugates (ADCs) that specifically bind to EGFRvIII.

[0010] In one embodiment, the invention provides an anti-human epidermal growth factor receptor (anti-hEGFR) antibody, or antigen-binding portion thereof, that binds to an epitope within the amino acid sequence CGADSYEMEEDGVRKC (SEQ ID NO: 45) or competes with a second anti-hEGFR antibody for binding to epidermal growth factor receptor variant III (EGFRvIII) (SEQ ID NO: 33) in a competitive binding assay, wherein the second anti-EGFR antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5; and has an antigen-binding activity of about 1 x 10 as measured by surface plasmon resonance. -6 The dissociation constant (K dand inhibits tumor growth in an in vivo human non-small cell lung cancer (NSCLC) xenograft assay by at least about 50% tumor growth inhibition % (TGI%) compared to a human IgG antibody that is not specific for EGFR, wherein the human IgG antibody is administered at the same dose and frequency as the anti-hEGFR antibody or antigen-binding portion thereof.

[0011] In certain embodiments of the invention, the antibody, or antigen-binding portion thereof, has a denaturing activity of about 1 x 10 as measured by surface plasmon resonance. -6 M ~ approx. 1×10 -10 K of M d It binds to EGFR(1-525) (SEQ ID NO: 47).

[0012] In another embodiment of the invention, the antibody, or antigen-binding portion thereof, has a denaturing activity of about 1 x 10 as measured by surface plasmon resonance. -6 M ~ approx. 1×10 -7 K of M d It binds to EGFR(1-525) (SEQ ID NO: 47).

[0013] In certain embodiments, the antibody, or antigen-binding portion thereof, has a molecular weight of about 8.2 x 10 as measured by surface plasmon resonance. -9 K below M d In other embodiments, the antibody, or antigen-binding portion thereof, binds to EGFRvIII (SEQ ID NO: 33) at a binding affinity of about 8.2 x 10 as measured by surface plasmon resonance. -9 M ~ approx. 6.3×10 -10 K of M d In some embodiments, the antibody, or antigen-binding portion thereof, binds to EGFRvIII (SEQ ID NO: 33) at a binding affinity of about 8.2 x 10 as measured by surface plasmon resonance. -9 M ~ approx. 2.0×10 -9 K of M d It binds to EGFRvIII (SEQ ID NO: 33) at

[0014] In yet other embodiments of the invention, the antibody, or antigen-binding portion thereof, inhibits tumor growth by at least about 60% in an in vivo human non-small cell lung cancer (NSCLC) xenograft assay compared to a human IgG antibody non-specific for EGFR.

[0015] In certain embodiments, the invention relates to an antibody or antigen-binding portion thereof that inhibits tumor growth by at least about 70% in an in vivo human non-small cell lung cancer (NSCLC) xenograft assay compared to a non-EGFR-specific human IgG antibody. In certain embodiments, the antibody or antigen-binding portion thereof inhibits tumor growth by at least about 80% in an in vivo human non-small cell lung cancer (NSCLC) xenograft assay compared to a non-EGFR-specific human IgG antibody.

[0016] In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 12, a heavy chain CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 11, and a heavy chain CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 10; and a light chain CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 8, a light chain CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 6. In yet other embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5. In other embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 41 and / or a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 43. In another embodiment, the antibody, or antigen-binding portion thereof, comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 15 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 13. In another embodiment, the antibody, or antigen-binding portion thereof, is conjugated to an auristatin.

[0017] The present invention further provides, in certain embodiments, isolated nucleic acids encoding antibodies, or antigen-binding portions thereof, such as those described herein.

[0018] The present invention further encompasses, in certain embodiments, an anti-hEGFR antibody or antigen-binding portion thereof, comprising: a light chain CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40; a light chain CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 39; and a light chain CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 38; and a heavy chain CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 37; a heavy chain CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 36; and a heavy chain CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0019] In certain embodiments, the invention relates to an anti-hEGFR antibody, or antigen-binding portion thereof, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78; and a light chain variable region comprising an amino acid sequence selected from the group consisting of 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, and 79.

[0020] In other embodiments, the invention relates to a nucleic acid sequence encoding a nucleotide sequence encoding a nucleotide sequence encoding a heavy chain CDR set (CDR1, CDR2, and CDR3) selected from the group consisting of SEQ ID NOs: 10, 11, and 12; SEQ ID NOs: 16, 17, and 18; SEQ ID NOs: 10, 11, and 19; SEQ ID NOs: 20, 11, and 12; SEQ ID NOs: 21, 3, and 22; SEQ ID NOs: 16, 17, and 19; SEQ ID NOs: 2, 3, and 4; SEQ ID NOs: 10, 3, and 12; SEQ ID NOs: 80, 11, and 18; SEQ ID NOs: 80, 3, and 18; SEQ ID NOs: 20, 3, and 12; SEQ ID NOs: 80, 11, and 12; and SEQ ID NOs: 81, 11, and 22; and SEQ ID NOs: 82, 83, and 31; and SEQ ID NOs: 82, 27, and 85, provided that the antibody or antigen-binding portion thereof does not simultaneously comprise the heavy chain CDR set of SEQ ID NOs: 2, 3, and 4 and the light chain CDR set of SEQ ID NOs: 6, 7, and 8. In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 41 and / or a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 43.

[0021] In some embodiments of the invention, the antibody, or antigen-binding portion thereof, comprises a heavy chain immunoglobulin constant region selected from the group consisting of a human IgG constant region, a human IgM constant region, a human IgE constant region, and a human IgA constant region. In some embodiments, the IgG constant region is selected from the group consisting of an IgG1 constant region, an IgG2 constant region, an IgG3 constant region, and an IgG4 constant region. In other embodiments, the antibody is a multispecific antibody.

[0022] In other embodiments of the invention, the antibody or antigen-binding portion thereof comprises a Fab, a Fab', a F(ab')2, an Fv, a disulfide-linked Fv, an scFv, a single domain antibody, and a diabody.

[0023] In yet other embodiments of the invention, the antibody or antigen-binding portion thereof is conjugated to an imaging agent. In certain embodiments of the invention, the imaging agent is selected from the group consisting of a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. In other embodiments of the invention, the radiolabel is indium. In yet other embodiments, the invention encompasses a pharmaceutical composition comprising the antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier.

[0024] The present invention further encompasses, in certain embodiments, antibody-drug conjugates (ADCs) comprising an antibody or antigen-binding portion thereof described herein conjugated to at least one drug. In certain embodiments, the antibody is an anti-human epidermal growth factor receptor (anti-hEGFR) antibody or antigen-binding portion thereof, which binds to an epitope within the amino acid sequence CGADSYEMEEDGVRKC (SEQ ID NO: 45) or competes with a second anti-hEGFR antibody for binding to epidermal growth factor receptor variant III (EGFRvIII) (SEQ ID NO: 33) in a competitive binding assay, wherein the second anti-EGFR antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5; and has a binding affinity of about 1 x 10 as measured by surface plasmon resonance. -6 The dissociation constant (K d) to EGFR(1-525) (SEQ ID NO: 47); and inhibits tumor growth in an in vivo human non-small cell lung cancer (NSCLC) xenograft assay by at least about 50% tumor growth inhibition % (TGI%) compared to a human IgG antibody that is not specific for EGFR in the NSCLC xenograft assay, wherein the human IgG antibody that is not specific for EGFR is administered at the same dose and frequency as the anti-hEGFR antibody or antigen-binding portion thereof. In one embodiment of the present invention, the at least one drug is selected from the group consisting of an anti-apoptotic agent, an antimitotic agent, an antitumor antibiotic, an immunomodulatory agent, a nucleic acid for gene therapy, an alkylating agent, an anti-angiogenic agent, an antimetabolite, a boron-containing agent, a chemotherapeutic agent, a hormonal agent, an antihormonal agent, a corticosteroid, a photosensitive therapeutic agent, an oligonucleotide, a radionuclide agent, a radiosensitizer, a topoisomerase inhibitor, and a tyrosine kinase inhibitor. In certain embodiments, the antimitotic drug is a dolastatin, auristatin, maytansinoid, or plant alkaloid. In certain embodiments, the drug is a dolastatin, auristatin, maytansinoid, or plant alkaloid. An example of an auristatin is monomethyl auristatin F (MMAF) or monomethyl auristatin E (MMAE). Examples of maytansinoids include, but are not limited to, DM1, DM2, DM3, and DM4. In certain embodiments, the antitumor antibiotic is selected from the group consisting of actinomycin, anthracycline, calicheamicin, and duocarmycin. In certain embodiments, the actinomycin is a pyrrolobenzodiazepine (PBD).

[0025] The present invention further encompasses, in some embodiments, an ADC comprising an anti-EGFR antibody conjugated to an auristatin, wherein the antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10; and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 6. In one embodiment, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5. In yet another embodiment, the invention encompasses an antibody, or antigen-binding portion thereof, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 15, and a light chain comprising the amino acid sequence of SEQ ID NO: 13.

[0026] The present invention further encompasses, in certain embodiments, ADCs in which an anti-EGFR antibody is conjugated to at least one drug (such as, but not limited to, MMAE), with 1 to 8 drug molecules conjugated to the antibody. In one embodiment, 1 to 4 drug molecules are conjugated to the antibody of the ADC. In one embodiment, 2 to 4 drug molecules are conjugated to the antibody of the ADC.

[0027] The invention further encompasses ADCs comprising an anti-EGFR antibody conjugated to at least one drug, in some embodiments, the drug being conjugated via a maleimidocaproyl, valine-citrulline linker, and in other embodiments, the drug being conjugated via a maleimidocaproyl, valine-citrulline, p-aminobenzyloxycarbamoyl (PABA) linker.

[0028] The present invention further encompasses, in certain embodiments, an ADC in which an anti-EGFR IgG1 antibody is covalently linked to monomethyl auristatin E (MMAE) via a linker (e.g., maleimidocaproyl, valine-citrulline). In certain embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, 1 to 4 MMAE molecules are linked to the antibody.

[0029] The present invention further encompasses, in certain embodiments, ADCs comprising an anti-EGFR IgG1 antibody covalently linked to maleimidocaproyl, valine-citrulline, p-aminobenzyloxycarbamoyl-monomethyl auristatin E (mc-vc-PABA-MMAE), the antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:5, and one to four MMAE molecules are linked to the antibody. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:15 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:13. In certain embodiments, two to four MMAE molecules are linked to the antibody. In certain embodiments, the EGFR antibody is linked to mc-vc-PABA-MMAE, as shown in FIG. 11.

[0030] The present invention further encompasses, in some embodiments, an EGFR-specific ADC comprising an IgG1 antibody specific for human EGFR, MMAE, and a linker covalently linking MMAE to the antibody. In certain embodiments, the antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10; and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 6. In one embodiment, the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5. In yet another embodiment, the invention encompasses an antibody, or antigen-binding portion thereof, comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 15, and a light chain comprising the amino acid sequence of SEQ ID NO: 13.

[0031] In yet another embodiment, the invention encompasses a pharmaceutical composition comprising an ADC mixture comprising a plurality of ADCs described herein and a pharmaceutically acceptable carrier. In certain embodiments, the ADC mixture has an average drug-to-antibody ratio (DAR) of 2 to 4. In other embodiments, the ADC mixture comprises ADCs, each having a DAR of 2 to 8. In certain embodiments, the ADC mixture has an average drug-to-antibody ratio (DAR) of about 2.4 to about 3.6.

[0032] In certain embodiments, the present invention encompasses a method of treating a subject with cancer, comprising administering to the subject a pharmaceutical composition described herein to treat the subject. In one embodiment, the cancer is selected from the group consisting of breast cancer, lung cancer, glioblastoma, prostate cancer, pancreatic cancer, colon cancer, head and neck cancer, and kidney cancer. In one embodiment, the cancer is selected from the group consisting of breast cancer, lung cancer, glioblastoma, prostate cancer, pancreatic cancer, colon cancer, colorectal cancer, head and neck cancer, mesothelioma, kidney cancer, squamous cell carcinoma, triple-negative breast cancer, and non-small cell lung cancer. In one embodiment, the cancer is breast cancer. In one embodiment, the cancer is lung cancer. In one embodiment, the cancer is prostate cancer. In one embodiment, the cancer is pancreatic cancer. In one embodiment, the cancer is colon cancer. In one embodiment, the cancer is head and neck cancer. In one embodiment, the cancer is kidney cancer. In one embodiment, the cancer is mesothelioma. In one embodiment, the cancer is squamous cell carcinoma. In one embodiment, the cancer is triple-negative breast cancer. In one embodiment, the cancer is non-small cell lung cancer. In certain embodiments, the squamous cell carcinoma is lung squamous cell carcinoma or head and neck squamous cell carcinoma.

[0033] In yet another embodiment, the cancer comprises EGFR amplification or overexpresses EGFR. In certain embodiments, the cancer is characterized by EGFR overexpression. In certain embodiments, the cancer is characterized by EGFR amplification.

[0034] The present invention further encompasses, in certain embodiments, a method of inhibiting or suppressing solid tumor growth in a subject having a solid tumor, comprising administering to the subject a pharmaceutical composition described herein to inhibit or suppress growth of the solid tumor. In certain embodiments, the solid tumor is characterized by EGFR overexpression. In certain embodiments, the solid tumor is characterized by EGFR amplification.

[0035] In one embodiment of the invention, the invention encompasses a method for inhibiting or suppressing solid tumor growth in a subject having a solid tumor, comprising administering to the subject having the solid tumor an effective amount of an antibody or ADC described herein, such that growth of the solid tumor is inhibited or suppressed.

[0036] In certain embodiments, the solid tumor is an EGFR-expressing solid tumor or an EGFRvIII-positive solid tumor. In other embodiments, the solid tumor is non-small cell lung cancer or glioblastoma. In other embodiments, the solid tumor is squamous cell carcinoma.

[0037] In one embodiment, the invention provides a method for treating a subject with cancer, comprising administering an effective amount of an ADC comprising an anti-EGFR antibody, or antigen-binding portion thereof, conjugated to at least one auristatin, wherein the anti-EGFR antibody or antigen-binding portion thereof is of the IgG isotype and comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 10, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 8, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 7, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the antibody or antigen-binding portion thereof is linked to mc-vc-PABA-MMAE.

[0038] In certain embodiments, the present invention encompasses methods of treating a subject with cancer, comprising administering a pharmaceutical composition described herein in combination with another agent or therapy. In certain embodiments, the additional agent is selected from the group consisting of an anti-PD1 antibody (e.g., pembrolizumab (Keytruda®) or nivolumab), an anti-CTLA-4 antibody (e.g., ipilimumab), ibrutinib, duvelisib, idelalisib, venetoclax, and temozolomide. In certain embodiments, the additional therapy is radiation. In certain embodiments, the additional agent is an anti-PD1 antibody (e.g., pembrolizumab (Keytruda®) or nivolumab). In certain embodiments, the additional agent is an anti-CTLA-4 antibody (e.g., ipilimumab). In certain embodiments, the additional agent is ibrutinib. In certain embodiments, the additional agent is duvelisib. In certain embodiments, the additional agent is idelalisib. In certain embodiments, the additional agent is venetoclax. In certain embodiments, the additional agent is temozolomide.

[0039] The present invention further provides, in certain embodiments, isolated nucleic acids encoding antibodies or antigen-binding portions thereof, such as those described herein. The invention also encompasses vectors containing the nucleic acids and host cells, such as prokaryotic or eukaryotic cells (e.g., animal cells, protozoan cells, plant cells, and fungal cells), containing the vectors. In embodiments of the invention, the animal cells are selected from the group consisting of mammalian cells, insect cells, and avian cells. In one embodiment, the mammalian cells are selected from the group consisting of CHO cells, COS cells, and Sp2 / 0 cells.

[0040] In certain embodiments, the present invention relates to an anti-hEGFR antibody-drug conjugate (ADC) comprising an anti-hEGFR antibody conjugated with an auristatin, wherein the antibody comprises a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10; and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 8, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 6. In one embodiment, the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5. In yet another embodiment, the antibody comprises an IgG heavy chain immunoglobulin constant region. In yet another embodiment, the IgG is an IgG1 or IgG4 heavy chain immunoglobulin constant region.

[0041] In one embodiment, the invention encompasses an ADC in which the auristatin is monomethyl auristatin F (MMAF) or monomethyl auristatin E (MMAE). In one embodiment, the invention encompasses an ADC in which the auristatin is monomethyl auristatin F (MMAF). In one embodiment, the invention encompasses an ADC in which the auristatin is monomethyl auristatin E (MMAE).

[0042] In another embodiment, the invention comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:15 and a light chain comprising the amino acid sequence of SEQ ID NO:13.

[0043] In yet another embodiment of the invention, the anti-EGFR antibody is covalently attached to the auristatin via a linker comprising maleimidocaproyl, valine-citrulline, p-aminobenzyl alcohol (mc-vc-PABA).

[0044] In one embodiment, the invention encompasses an ADC comprising an anti-EGFR antibody and a radiolabel (e.g., indium).

[0045] In one embodiment, the anti-EGFR antibodies described herein are covalently linked to at least one pyrrolobenzodiazepine (PBD). In certain embodiments, the anti-EGFR antibodies disclosed herein are linked to a PBD as shown in Figure 21 (i.e., SGD-1882).

[0046] In certain embodiments, the invention relates to pharmaceutical compositions containing an ADC described herein and a pharmaceutically acceptable carrier. In certain embodiments, the invention relates to pharmaceutical compositions containing an ADC mixture comprising the ADCs described herein, wherein the ADC mixture has an average drug-to-antibody ratio (DAR) ranging from 2 to 4. In certain embodiments, the ADC mixture has an average drug-to-antibody ratio (DAR) ranging from 2.4 to 3.6.

[0047] In one embodiment, the present invention relates to a pharmaceutical composition comprising an ADC mixture containing anti-hEGFR antibody-drug conjugates (ADCs) and a pharmaceutically acceptable carrier, wherein the ADC mixture has an average drug-to-antibody ratio (DAR) of 2 to 4, and the ADC comprises a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10, and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 8, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 6, conjugated to monomethyl auristatin E (MMAE).

[0048] In one embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO:9, and the light chain variable region of the anti-EGFR antibody comprises the amino acid sequence set forth in SEQ ID NO:5.

[0049] In other embodiments of the invention, the antibody comprises an IgG heavy chain immunoglobulin constant region. In other embodiments, the invention encompasses antibodies comprising an IgG1 or IgG4 heavy chain immunoglobulin constant region. In one embodiment, the invention encompasses antibodies that are of the IgG1 isotype.

[0050] In yet another embodiment, the invention encompasses an antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 15 and a light chain comprising the amino acid sequence of SEQ ID NO: 13. In one embodiment, the invention conjugates MMAE to the antibody through a maleimidocaproyl, val-cit, PABA linker.

[0051] In one embodiment of the present invention, the present invention provides a method for treating a subject with cancer, comprising administering to the subject a pharmaceutical composition containing an antibody or ADC described herein to treat the subject. In one embodiment, the cancer is selected from the group consisting of breast cancer, lung cancer, glioblastoma, prostate cancer, pancreatic cancer, colon cancer, head and neck cancer, and kidney cancer. In one embodiment, the cancer is selected from the group consisting of breast cancer, lung cancer, glioblastoma, prostate cancer, pancreatic cancer, colon cancer, colorectal cancer, head and neck cancer, mesothelioma, kidney cancer, squamous cell carcinoma, triple-negative breast cancer, and non-small cell lung cancer. In yet another embodiment, the cancer comprises EGFR amplification or overexpresses EGFR. In one embodiment, the squamous cell carcinoma is lung squamous cell carcinoma or head and neck squamous cell carcinoma. In one embodiment, the cancer is an EGFR-overexpressing cancer. In one embodiment, the cancer is characterized by EGFR amplification. In one embodiment, the cancer is breast cancer. In one embodiment, the cancer is lung cancer. In one embodiment, the cancer is prostate cancer. In one embodiment, the cancer is pancreatic cancer. In one embodiment, the cancer is colon cancer. In one embodiment, the cancer is head and neck cancer. In one embodiment, the cancer is kidney cancer. In one embodiment, the cancer is colorectal cancer. In one embodiment, the cancer is mesothelioma. In one embodiment, the cancer is squamous cell carcinoma. In one embodiment, the cancer is triple-negative breast cancer. In one embodiment, the cancer is non-small cell lung cancer. In certain embodiments, the squamous cell carcinoma is lung squamous cell carcinoma or head and neck squamous cell carcinoma.

[0052] In certain embodiments, the present invention also provides a method for inhibiting or suppressing solid tumor growth in a subject having a solid tumor, the method comprising administering a pharmaceutical composition described herein to the subject having the solid tumor to inhibit or suppress solid tumor growth. In one embodiment, the solid tumor is non-small cell lung cancer or glioblastoma. In yet another embodiment, the solid tumor is an EGFRvIII-positive tumor or an EGFR-expressing solid tumor. In yet another embodiment, the solid tumor is an EGFR-overexpressing solid tumor. In yet another embodiment, the solid tumor is an EGFR-amplified tumor. In one embodiment, the solid tumor is EGFR-amplified non-small cell lung cancer. In one embodiment, the solid tumor is EGFR-overexpressing non-small cell lung cancer. In one embodiment, the solid tumor is EGFR-amplified glioblastoma. In one embodiment, the solid tumor is EGFR-overexpressing glioblastoma.

[0053] In certain embodiments, the present invention provides combination therapies comprising administering to a subject in need thereof (e.g., a subject with cancer or a solid tumor) a pharmaceutical composition described herein. The pharmaceutical composition described herein can be administered simultaneously with, before, or after administration of another agent or therapy. In certain embodiments, the additional agent is selected from the group consisting of an anti-PD1 antibody, an anti-CTLA-4 antibody, temozolomide, a bcl-xl inhibitor, and a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor. In still other embodiments, the additional agent is a chemotherapeutic agent. In certain embodiments, the additional therapy is radiation. In other embodiments, the additional agent is ibrutinib (Imbruvica®, Pharmacyclics). In other embodiments, the additional agent is duvelisib. In other embodiments, the additional agent is idelalisib (Zydelig®, Gilead Sciences, Inc.). In other embodiments, the additional agent is venetoclax (ABT-199 / GDC-0199, AbbVie, Inc.). In certain embodiments, the additional agent is an anti-PD1 antibody (e.g., pembrolizumab (Keytruda®) or nivolumab). In certain embodiments, the additional agent is an anti-CTLA-4 antibody (e.g., ipilimumab). In certain embodiments, the additional agent is temozolomide.

[0054] In certain embodiments, the invention relates to a chimeric antigen receptor (CAR) comprising an antigen-binding region (e.g., CDR) of an antibody described herein or an scFv described herein. In certain embodiments, the invention relates to a CAR comprising a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 39, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 38; and a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 37, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 36, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0055] In certain embodiments, the invention relates to a CAR comprising a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 10; and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 8, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 7, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 6. [Brief explanation of the drawings]

[0056] [Figure 1] The heavy chain variable region (VH) and light chain variable region (VL) amino acid sequences of Ab1 (SEQ ID NOs: 1 and 5) and AbA (SEQ ID NOs: 9 and 5) are shown. The CDR sequences within the VH and VL regions are boxed, and mismatches between the Ab1 VH sequence and the AbA VH sequence are shaded. [Figure 2] The full-length light and heavy chains of Ab1 (SEQ ID NOs: 13 and 14) and AbA (SEQ ID NOs: 13 and 15) are shown. Differences between the Ab1 and AbA sequences in the heavy chain are shaded. [Figure 3] This table summarizes the affinity measurements of several Ab1 variant antibodies compared with Ab1 and Ab2 using Biacore binding assays. EGFR(1-525) and EGFRvIII were used for binding analysis. In Figure 3, ka (M-1s-1) represents the rate constant for the antibody to associate with the antigen to form an antibody / antigen complex, kd (s-1) represents the dissociation rate constant for the antibody to dissociate from the antibody / antigen complex, and Kd (M) represents the equilibrium dissociation constant. [Figure 4] Graph summarizing the results of FACS analysis, which clearly shows that AbA has improved binding to A431 tumor cells (human squamous cell carcinoma cells) compared to Ab1, but has lower binding affinity compared to Ab2. [Figure 5] The results of a FACS competition assay are shown, demonstrating that the Ab1 variant antibodies recognize the same EGFR epitope as Ab1. [Figure 6]The results of Ab1 and Ab1 variant antibody binding to EGFR(1-525) are summarized. Black circles represent Ab1 or Ab2 (control), and black circles represent Ab1 variant antibodies. Circles represent Group 1 and Group 2. The data are summarized in Figure 7. [Figure 7] These figures show the results of Western blot analysis in vitro testing the activity of Ab1 and Ab1 variant antibodies in various cell lines. Cells from SCC-15 (FIG. 7A) and H292 cells were exposed to the conditions described in Example 4 and analyzed by Western blot analysis using anti-phosphotyrosine EGFR (pY EGFR), anti-EGFR (tot EGFR (full-length EGFR)), and anti-actin (actin) antibodies. Figure 7A shows results demonstrating the ability of Ab1, Ab2, and Ab1 variant antibodies to inhibit EGF-mediated tyrosine phosphorylation of EGFR in SCC-15 cells. Figure 7B shows results demonstrating the ability of Ab1, Ab2, and Ab1 variant antibodies to inhibit EGF-mediated tyrosine phosphorylation of EGFR in H292 cells. [Figure 8] The results of the pEGFR ELISA assay using Ab1, Ab2, and Ab1 variants (Figure 8A) and the inhibition levels of Ab1 compared to Ab2 and AbP in the A431 inhibition assay (Figure 8B) are shown graphically. The Y-axis in Figure 8A is optical density (OD) at 450 nm. [Figure 9] 10 graphically depicts the results of testing Ab1, Ab2, and Ab1 variant antibodies for binding to normal human epidermal keratinocytes expressing wild-type EGFR using a FACS binding assay. [Figure 10] 1 graphically depicts the results of a mouse xenograft inhibition assay comparing the ability of AbA, AbG, AbK, AbM, and AbP to inhibit tumor growth in human NSCLC cancer xenografts with Ab1, Ab2, and a human IgG (huIgG) control. Arrows indicate the time points of administration of the various antibodies. [Figure 11] 1 shows the structure of AbA-maleimidocaproyl-vc-PABA-MMAE ADC (referred to herein as "AbA-vcMMAE"). [Figure 12] 1 shows the results of hydrophobic interaction chromatography (HIC) analysis of the purification of AbA-vcMMAE. [Figure 13] 1 shows the results of size exclusion chromatography (SEC) analysis of AbA-vcMMAE. [Figure 14] The results of two mouse xenograft inhibition assays using anti-EGFR ADCs are graphically depicted. Figure 14A shows the results of a mouse xenograft inhibition assay comparing inhibition of tumor growth in NCI-H1703 cells derived from a human NSCLC cancer xenograft, demonstrating increased inhibition with AbA-vcMMAE compared to Ab1 and Ab1-mcMMAF ADCs. Figure 14B shows the results of a mouse xenograft inhibition assay comparing inhibition of tumor growth in EBC-1 cells derived from a human NSCLC cancer xenograft, demonstrating increased inhibition with AbA-vcMMAE compared to Ab1 and Ab1-mcMMAF ADCs. Arrows indicate the time points of antibody administration. [Figure 15]

[00149] Figure 15A graphically depicts the results of a mouse xenograft inhibition assay comparing inhibition of tumor growth in NCI-H292 cells, demonstrating increased inhibition by purified AbA-vcMMAE (AbA-vcMMAEp) and AbA-vcMMAE compared to purified Ab1-vcMMAE (Ab1-vcMMAEp), Ab1-vcMMAE, purified Ab1-mcMMAF ADC (Ab1-mcMMAFp), and Ab1-mcMMAF (compared to three controls). Figure 15B shows the results of a mouse xenograft inhibition assay comparing tumor growth inhibition in NCI-H292 cells, demonstrating the increased inhibitory activity of AbA-vcMMAE compared to purified AbA-vcMMAE (AbA-vcMMAEp), and compared to purified Ab1-vcMMAE (Ab1-vcMMAEp), Ab1-vcMMAE, Ab1-mcMMAF, and Ab1-mcMMAFp. The dosages of the molecules in Figures 15A and B are indicated in parentheses (i.e., 3 mg / kg or 6 mg / kg). Arrows indicate the time points of antibody or ADC administration. Control 2 in Figure 15 represents a negative control, an anti-tetanus toxoid antibody that does not bind to EGFR. [Figure 16]The amino acid sequences of the Ab1 variant heavy chain variable region (VH) library design (Figure 16A) and Ab1 variant light chain variable region (VL) library design (Figure 16B) are shown. [Figure 17] Schematic diagram of EGFR showing the regions to which Ab1 and Ab2 bind. [Figure 18] Figure 1 shows graphically the results of a mouse xenograft inhibition assay (with NCI-H292 (NSCLC) cells) using anti-EGFR ADCs. The dose of the molecule is indicated in parentheses (i.e., 3 mg / kg or 6 mg / kg). Arrows indicate the time of antibody or ADC administration. [Figure 19] The graphs show the results of a mouse glioblastoma xenograft inhibition assay using anti-EGFR MMAE and MMAF ADCs. The dose of the molecules in Figure 19 is shown in parentheses (i.e., 1 mg / kg). The arrows indicate the time of antibody or ADC administration. Control 2 in Figure 19 represents a negative control, an anti-tetanus toxoid antibody that does not bind to EGFR. [Figure 20] Figures 20A and B graphically show the results of a single-photon emission computed tomography (SPECT) imaging assay comparing the efficiency of antibody uptake by EGFR-expressing tumors in two tumor models (SW48 (Figure 20A) and EBC1 (Figure 20B) tumor models, respectively) using In-labeled AbA, Ab1, or a control antibody. [Figure 21] 1 shows the structure of a PBD dimer (SGD-1882) (collectively referred to as SGD-1910) conjugated to an antibody (Ab) via a maleimidocaproyl-valine-alanine linker. DETAILED DESCRIPTION OF THE INVENTION

[0057] Various aspects of the present invention relate to anti-EGFR antibodies and antibody fragments, anti-EGFR ADCs, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for producing such antibodies and fragments. Also covered by the present invention are methods of using the antibodies or ADCs described herein to detect human EGFR, inhibit human EGFR activity (in vitro or in vivo), and treat cancers such as epithelial cancer, breast cancer, colon cancer, head and neck cancer (e.g., glioblastoma), lung cancer, kidney cancer, pancreatic cancer, mesothelioma, squamous cell carcinoma (e.g., lung squamous cell carcinoma or head and neck squamous cell carcinoma), triple-negative breast cancer, non-small cell lung cancer, and prostate cancer.

[0058] I. Definition To facilitate understanding of the present invention, certain terms are first defined. Furthermore, it should be understood that when a numerical value or range of values ​​for a parameter is stated, all intermediate values ​​and ranges within the stated numerical values ​​are also encompassed within the present invention.

[0059] The terms "anti-epidermal growth factor (EGF) receptor antibody" or "anti-EGFR antibody" are used interchangeably herein and refer to an antibody that specifically binds to EGFR. An antibody that "binds" to an antigen of interest (i.e., EGFR) is an antibody that can bind to the antigen with sufficient affinity to be useful for targeting cells expressing the antigen. In a preferred embodiment, the antibody specifically binds to human EGFR (hEGFR). Examples of anti-EGFR antibodies are disclosed in Example 1 below. Unless otherwise specified, the term "anti-EGFR antibody" shall refer to an antibody that binds to wild-type EGFR or any mutant of EGFR (e.g., EGFRvIII).

[0060] The amino acid sequence of wild-type human EGFR is shown below as SEQ ID NO: 32, with the signal peptide (amino acid residues 1-24) underlined and the extracellular domain (ECD, amino acid residues 25-645) highlighted in bold. The truncated wild-type ECD of EGFR (also referred to herein as EGFR(1-525)) corresponds to SEQ ID NO: 47, which is equivalent to the amino acids in SEQ ID NO: 32. The mature form of wild-type EGFR is this protein minus the signal peptide, i.e., corresponds to amino acid residues 25-1210 of SEQ ID NO: 32.

[0061] [ka]

[0062] The amino acid sequence of the ECD of human EGFR, including the signal sequence (underlined), is shown below as SEQ ID NO: 34.

[0063] [ka]

[0064] The overall structure of EGFR is shown in Figure 17. The ECD of EGFR consists of four regions (Cochran et al. (2004) J. Immunol. Methods, 287, 147-158). Regions I and III are suggested to contribute to the formation of high-affinity binding sites for ligands. Regions II and IV are cysteine-rich laminin-like regions that stabilize the protein fold and contain the predicted EGFR dimerization interface.

[0065] EGFR mutants appear to arise as a result of gene rearrangements involving EGFR gene amplification.

[0066] The most common EGFR variant present in human cancers is EGFRvIII (Kuan et al. Endocr Relat Cancer. 8(2):83-96(2001)). Gene amplification results in a deletion of 267 amino acids in the extracellular domain of EGFR, with a glycine residue inserted at the fusion junction. Thus, EGFRvIII lacks amino acids 6-273 of the extracellular domain of wild-type EGFR, with a glycine residue inserted at the fusion junction. The EGFRvIII mutant of EGFR lacks 267 amino acid residues in the extracellular domain and a glycine residue inserted at the deletion junction. The EGFRvIII amino acid sequence is shown below as SEQ ID NO: 33 (the ECD is highlighted in bold and corresponds to SEQ ID NO: 46; the signal sequence is underlined).

[0067] [ka]

[0068] EGFRvIII contributes to tumor progression in a ligand-independent manner through constitutive signaling. EGFRvIII is not known to be expressed in normal tissues (Wikstrand et al. Cancer Research 55(14):3140-3148(1995); Olapade-Olaopa et al. Br J Cancer. 82(1):186-94(2000)), but shows significant expression in tumor cells, including breast cancer, glioma, NSCL cancer, ovarian cancer, and prostate cancer (Wikstrand et al. Cancer Research 55(14):3140-3148(1995); Ge et al. Int J Cancer. 98(3):357-61(2002); Wikstrand et al. Cancer Research 55(14):3140-3148(1995); Moscatello et al. Cancer Res. 55(23):5536-9(1995); Garcia de Palazzo et al. Cancer Res.53(14):3217-20(1993); Moscatello et al. Cancer Res.55(23):5536-9(1995); and Olapade-Olaopa et al.2(1):186-94(2000)).

[0069] As used herein, "biological activity of EGFR" refers to all inherent biological properties of EGFR, including, but not limited to, binding to epidermal growth factor (EGF), binding to transforming growth factor alpha (TGFα), homodimerization, activation of JAK2 kinase activity, activation of MAPK kinase activity, and activation of transmembrane receptor protein tyrosine kinase activity.

[0070] As used herein, the terms "specific binding" or "specifically binds" with respect to the interaction of an antibody or ADC with a second chemical species means that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than recognizing proteins in general. If an antibody or ADC is specific for epitope "A," the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction mixture containing labeled "A" and the antibody will reduce the amount of labeled A that binds to the antibody or ADC.

[0071] As used herein, the phrase "specifically binds to hEGFR" or "specific binding to hEGFR" means that an anti-EGFR antibody or ADC can interact with hEGFR with equal or greater affinity than Ab1 or Ab1 ADC.

[0072] As used herein, the term "specific binding to EGFR(1-525)" or "specifically binds to EGFR(1-525)" refers to binding to EGFR(1-525) and the dissociation constant (K) as measured by surface plasmon resonance. D ) is 2.3 × 10 -6 It means an antibody or ADC having a β-amyloid ratio of 0.01 to 0.01 M or less.

[0073] The term "antibody" refers broadly to an immunoglobulin (Ig) molecule, generally composed of four polypeptide chains: two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof that retains the essential target-binding characteristics of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art, and non-limiting embodiments thereof are described below.

[0074] In a full-length antibody, each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further divided into hypervariable regions called complementarity-determining regions (CDRs) and highly conserved regions called framework regions (FRs) located between them. Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) and class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass.

[0075] As used herein, the term "antigen-binding portion" (or simply "antibody portion") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., hIL-13). It has been shown that fragments of a full-length antibody can perform the antigen-binding function of an antibody. Embodiments of such antibodies may be in a bispecific, dual-specific, or multispecific format that specifically binds to two or more different antigens. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single antibody arm; (v) a dAb fragment consisting of a single variable domain (Ward et al., (1989) Nature 341:544-546, Winter et al., PCT Publication No. WO 90 / 05144 A1, which is incorporated herein by reference); and (vi) an isolated complementarity-determining region (CDR). Furthermore, the two domains of an Fv fragment, the VL and VH, are encoded by separate genes but can be recombinantly linked by a synthetic linker to form a single protein chain in which the VL and VH domains pair to form a monovalent molecule (referred to as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also encompassed within the term "antigen-binding portion" of an antibody. In certain embodiments of the present invention, scFv molecules may be incorporated into fusion proteins. Other forms of single-chain antibodies, such as diabodies, are also included.Diabodies are bivalent, bispecific antibodies in which a VH domain and a VL domain are expressed on a single polypeptide chain; however, due to the use of a linker that is too short, pairing between the two domains on the same chain is not possible. Instead, these domains pair with complementary domains on another chain to form two antigen-binding sites (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123). Such antibody-binding moieties are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag, New York, 790 pp. (ISBN 3-540-41354-5)).

[0076] The term "antibody construct" as used herein refers to a polypeptide comprising one or more antigen-binding moieties of the present invention linked to a linker polypeptide or immunoglobulin constant region. A linker polypeptide comprises two or more amino acid residues linked by peptide bonds and is used to link one or more antigen-binding moieties. Such linker polypeptides are well known in the art (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123). An immunoglobulin constant region refers to a heavy or light chain constant region. Examples of human IgG heavy and light chain constant region amino acid sequences are known in the art and are as follows:

[0077] [Table 1]

[0078] Furthermore, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule formed by covalent or noncovalent association of the antibody or antigen-binding portion thereof with one or more other proteins or peptides. Examples of such immunoadhesion molecules include tetrameric scFv molecules formed using a streptavidin core region (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101) and bivalent biotinylated scFv molecules formed using cysteine ​​residues, a marker peptide, and a C-terminal polyhistidine tag (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from full-length antibodies using conventional techniques, such as papain or pepsin digestion of full-length antibodies, respectively. Furthermore, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

[0079] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to EGFR is substantially free of antibodies that specifically bind to antigens other than EGFR). However, an isolated antibody that specifically binds to EGFR may be cross-reactive with other antigens (e.g., EGFR molecules from other species). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0080] The term "humanized antibody" refers to an antibody containing heavy and light chain variable region sequences derived from a non-human species (e.g., mouse), but in which at least a portion of the VH and / or VL sequences has been modified to be more "human-like," i.e., more similar to human germline variable sequences. In particular, the term "humanized antibody" refers to an antibody or variant, derivative, analog, or fragment thereof that immunospecifically binds to an antigen of interest and contains framework (FR) regions having substantially the amino acid sequence of a human antibody and complementarity-determining regions (CDRs) having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" refers to a CDR having an amino acid sequence that is at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of the non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv), in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. Preferably, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, the humanized antibody comprises at least the variable regions of a light chain and a heavy chain. The antibody may also comprise the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody comprises only a humanized light chain. In other embodiments, the humanized antibody comprises only a humanized heavy chain. In certain embodiments, the humanized antibody comprises only a humanized variable region of the light chain and / or a humanized heavy chain.

[0081] The humanized antibody can be selected from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4. The humanized antibody can comprise sequences from more than one class or isotype, and particular constant regions can be selected to optimize desired effector functions using techniques well known in the art.

[0082] The terms "Kabat numbering," "Kabat definition," and "Kabat labeling" are used interchangeably herein. These terms are art-recognized and refer to a numbering system for amino acid residues that are more variable than other amino acid residues (i.e., hypervariable) in the heavy and light chain variable regions of an antibody or antigen-binding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). In the heavy chain variable region, the hypervariable regions are located at amino acids 31-35 of CDR1, 50-65 of CDR2, and 95-102 of CDR3. In the light chain variable region, the hypervariable region is located at amino acid positions 24-34 of CDR1, amino acid positions 50-56 of CDR2, and amino acid positions 89-97 of CDR3.

[0083] As used herein, the term "CDR" refers to a complementarity-determining region within an antibody variable region sequence. There are three CDRs in each of the heavy chain (HC) and light chain (LC) variable regions, designated CDR1, CDR2, and CDR3 (or specifically HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3) in each variable region. As used herein, the term "CDR set" refers to a set of three CDRs present in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs have been defined differently by various systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only defines an unambiguous residue numbering system applicable to any antibody variable region, but also defines precise residue boundaries defining the three CDRs. These CDRs are sometimes referred to as Kabat CDRs. Chothia et al. (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) have used the Kabat CDRs. We have found that certain subportions within the CDRs, despite their diverse amino acid sequences, have nearly identical peptide backbone structures. These subportions are designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" represent the light chain and heavy chain regions, respectively. These regions are sometimes referred to as Chothia CDRs, and their boundaries overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)).Still other CDR boundary definitions exist that overlap with the Kabat CDRs, even if they do not strictly adhere to one of the above systems, and include shortening or lengthening based on predictions or experimental results that certain residues or groups of residues, or the entire CDR, do not significantly affect antigen binding. While the methods used herein can use CDRs defined according to any of these systems, preferred embodiments use CDRs defined by Kabat or Chothia.

[0084] As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of a variable region excluding CDRs. The precise definition of a CDR sequence can be determined using various systems, and the meaning of the "framework sequence" is interpreted accordingly. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 in the light chain and CDR-H1, CDR-H2, and CDR-H3 in the heavy chain) further divide the framework regions of the light and heavy chains into four subregions (FR1, FR2, FR3, and FR4) in each chain, with CDR1 located between FR1 and FR2, CDR2 located between FR2 and FR3, and CDR3 located between FR3 and FR4. The term "framework region" may be used without specifying the individual subregions as FR1, FR2, FR3, or FR4. In such cases, the term "framework region" refers to the entire FR in the variable region of a single native immunoglobulin chain. As used herein, FR refers to one of the four subregions, and FRs refers to two or more of the four subregions that make up a framework region.

[0085] The framework and CDR regions of a humanized antibody need not strictly correspond to the parental sequences; for example, the donor antibody CDR or consensus framework may be mutagenized by substitution, insertion, and / or deletion of at least one amino acid residue so that the CDR or framework residue at that position does not correspond to either the donor antibody or the consensus framework. However, in a preferred embodiment, such mutations will not be extensive. Typically, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of closely related immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). Within an immunoglobulin family, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in that family. If two amino acids occur equally frequently, either can be added to the consensus sequence.

[0086] "Percent amino acid sequence identity" with respect to a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that match those in the peptide or polypeptide sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignment for purposes of determining amino acid sequence identity can be performed by a variety of methods known to those of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. In one embodiment, the invention encompasses amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any one of SEQ ID NOS: 1-31, 35-40, or 50-85.

[0087] The term "multivalent antibody" is used herein to mean an antibody that contains two or more antigen-binding sites. In certain embodiments, multivalent antibodies can be engineered to have three or more antigen-binding sites and are generally other than naturally occurring antibodies.

[0088] The term "multispecific antibody" refers to an antibody capable of binding to two or more unrelated antigens. In one embodiment, a multispecific antibody is a bispecific antibody capable of binding to two unrelated antigens, for example, a bispecific antibody or antigen-binding portion thereof that binds to EGFR (e.g., EGFRvIII) and CD3.

[0089] The terms "dual variable domain" or "DVD" are used interchangeably herein and refer to an antigen-binding protein that contains two or more antigen-binding sites and is a tetravalent or greater binding protein. Such DVDs can be monospecific (i.e., capable of binding to one antigen) or multispecific (i.e., capable of binding to two or more antigens). A DVD-binding protein containing two heavy chain DVD polypeptides and two light chain DVD polypeptides is referred to as a DVDIg. Each half of a DVDIg contains a heavy chain DVD polypeptide and a light chain DVD polypeptide and two antigen-binding sites. Each binding site contains a heavy chain variable region and a light chain variable region, with a total of six CDRs per antigen-binding site involved in antigen binding. In one embodiment, the CDRs described herein are used in an anti-EGFR DVD.

[0090] The term "chimeric antigen receptor" or "CAR" refers to a recombinant protein that includes at least (1) an antigen-binding region (e.g., an antibody heavy or light chain variable region), (2) a transmembrane region for anchoring the CAR to a T cell, and (3) one or more intracellular signaling regions.

[0091] The term "activity" encompasses activities such as the binding specificity / affinity of an antibody (e.g., an anti-hEGFR antibody that binds to an hEGFR antigen) or ADC with an antigen and / or the neutralizing potency of an antibody (e.g., an anti-hEGFR antibody that binds to hEGFR and inhibits the biological activity of hEGFR), such as inhibition of EGFR phosphorylation in an EGFR-expressing cell line (e.g., human lung cancer cell line H292), or inhibition of proliferation of an EGFR-expressing cell line (e.g., human H292 lung cancer cells, human H1703 lung cancer cell line, or human EBC1 lung cancer cells).

[0092] As used herein, the term "non-small cell lung cancer (NSCLC) xenograft assay" refers to an in vivo assay used to determine whether an anti-EGFR antibody or ADC can inhibit tumor growth (e.g., further growth) and / or suppress tumor growth resulting from the implantation of NSCLC cells into immunodeficient mice. The NSCLC xenograft assay is performed after tumors have grown to a desired size (e.g., 200-250 mm). 3After transplanting NSCLC cells into immunodeficient mice so that they grow to a tumor size of 1000 μg / ml, the antibody or ADC is administered to the mice to determine whether the antibody or ADC can inhibit and / or suppress tumor growth. In certain embodiments, the activity of the antibody or ADC is measured by the percentage of tumor growth inhibition (%TGI) compared to a control antibody, e.g., a human IgG antibody (or collection thereof) that does not specifically bind to tumor cells, e.g., an antibody specific for an antigen not associated with cancer or an antibody obtained from non-cancerous material (e.g., normal human serum). In such embodiments, the antibody (or ADC) and the control antibody are administered to the mice at the same dose, frequency, and route. In one embodiment, the mice used in the NSCLC xenograft assay are severe combined immunodeficient (SCID) mice and / or athymic CD-1 nude mice. Examples of NSCLC cells that can be used in the NSCLC xenograft assay include, but are not limited to, H292 cells (e.g., NCIH292 [H292] (ATCC® CRL1848™)).

[0093] The term "epitope" refers to the region of an antigen to which an antibody or ADC binds. In certain embodiments, epitopic determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. In certain embodiments, antibodies of the invention bind to the epitope represented by the amino acid sequence CGADSYEMEEDGVRKC (SEQ ID NO: 45) (corresponding to amino acid residues 287-302 of the mature form of hEGFR).

[0094] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detecting changes in protein concentration within a biosensor matrix, e.g., using a BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For a detailed description, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnson, B., et al. (1991) Anal. Biochem. 198:268-277. In one embodiment, surface plasmon resonance is measured according to the method described in Example 2.

[0095] As used herein, "k on " or "k a The term "antibody / antigen complex rate" refers to the rate constant at which an antibody associates with an antigen to form an antibody / antigen complex.

[0096] As used herein, "k off " or "k d The term "dissociation rate constant" refers to the dissociation rate constant of an antibody from an antibody / antigen complex.

[0097] As used herein, "K D The term "K" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction (e.g., AbA antibody and EGFR). D is k a / k d It is calculated as follows.

[0098] As used herein, the term "competitive binding" refers to a situation in which a first antibody competes with a second antibody for a binding site on a third molecule (e.g., an antigen). In one embodiment, FACS analysis is used to measure competitive binding between two antibodies.

[0099] The term "competitive binding assay" refers to an assay used to determine whether two or more antibodies bind to the same epitope. In one embodiment, a competitive binding assay is a competitive fluorescence-activated cell sorting (FACS) assay used to determine whether two or more antibodies bind to the same epitope by examining whether the fluorescent signal of a labeled antibody is reduced by the introduction of an unlabeled antibody, resulting in a decrease in the level of fluorescence if they compete for the same epitope. An example of a competitive binding FACS assay is provided in Example 3, which describes a competitive FACS assay using U87MG cells (expressing EGFRvIII).

[0100] As used herein, the term "labeled antibody" refers to an antibody or antigen-binding portion thereof incorporating a label that allows for identification of the binding protein (e.g., antibody). Preferably, the label is a detectable marker, such as by incorporating a radiolabeled amino acid or by attaching a biotinyl moiety to the polypeptide that is detectable by labeled avidin (e.g., streptavidin conjugated with a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H 、 14 C 、 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho or 153 Sm), fluorescent labels (e.g., FITC, rhodamine, and lanthanide fluorophores), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), and magnetic materials (e.g., gadolinium chelators).

[0101] The term "antibody drug conjugate" or "ADC" refers to a binding protein, such as an antibody or antigen-binding fragment thereof, chemically linked to one or more chemical agents (also referred to herein as drugs), which may optionally be therapeutic or cytotoxic agents. In a preferred embodiment, an ADC comprises an antibody, a cytotoxic or therapeutic agent, and a linker that attaches or connects the agent to the antibody. ADCs typically have any number of drugs attached to the antibody, ranging from one to eight, including two-, four-, six-, or eight-drug loaded species. Non-limiting examples of drugs that can be included in ADCs include antimitotic agents, antitumor antibiotics, immunomodulatory agents, gene therapy vectors, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemotherapeutic agents, hormones, antihormones, corticosteroids, phototherapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, tyrosine kinase inhibitors, and radiosensitizers.

[0102] The terms "anti-epidermal growth factor antibody-drug conjugate," "anti-EGFR antibody-drug conjugate," and "anti-EGFR ADC" are used interchangeably herein to refer to an ADC in which an antibody that specifically binds to EGFR is conjugated to one or more chemical entities. In one embodiment, the anti-EGFR ADC comprises antibody AbA conjugated to an auristatin (e.g., MMAE or MMAF). The amino acid sequences corresponding to the light and heavy chains of antibody AbA are set forth in SEQ ID NO: 13 and SEQ ID NO: 15, respectively.

[0103] As used herein, the term "auristatin" refers to a family of antimitotic drugs. Auristatin derivatives are also included within the definition of the term "auristatin." Examples of auristatins include, but are not limited to, auristatin E (AE), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and synthetic analogs of dolastatins. In one embodiment, an anti-EGFR antibody described herein is conjugated to an auristatin to form an anti-EGFR ADC.

[0104] As used herein, the term "AbA-vcMMAE" refers to an ADC in which antibody AbA is linked to monomethyl auristatin E (MMAE) via a maleimidocaproyl, valine-citrulline, p-aminobenzyloxycarbamoyl (PABA) linker. AbA-vcMMAE is shown in Figure 11.

[0105] As used herein, the term "mcMMAF" refers to the linker / drug combination maleimidocaproyl-monomethylauristatin F (MMAF).

[0106] The term "drug-to-antibody ratio" or "DAR" refers to the number of drugs (e.g., auristatins) conjugated to antibodies in an ADC. The DAR of an ADC can range from 1 to 8, but can be higher (e.g., 10) depending on the number of linkage sites on the antibody. The term DAR can refer to the number of drugs loaded onto an individual antibody, or it can refer to the average DAR of a group of ADCs.

[0107] As used herein, the term "undesired ADC species" refers to any drug-loaded species that should be separated from ADC species with different drug loadings. In one embodiment, the term "undesired ADC species" can refer to ADCs with a DAR of 6 or more, such as drug-loaded species of 6 or more, i.e., ADCs with a DAR of 6 or more, including DAR6, DAR7, DAR8, and greater than DAR8 (i.e., drug-loaded species of 6, 7, 8, or greater than 8). In another embodiment, the term "undesired ADC species" can refer to any drug-loaded species of 8 or more, such as ADCs with a DAR of 8 or more, including DAR8 and greater than DAR8 (i.e., drug-loaded species of 8 or greater).

[0108] As used herein, the term "ADC mixture" refers to a composition containing ADCs with a heterogeneous DAR distribution. In one embodiment, the ADC mixture contains ADCs with a DAR distribution of 1 to 8, e.g., 2, 4, 6, and 8 (i.e., drug-loaded species of 2, 4, 6, and 8). In particular, degradation products containing DARs of 1, 3, 5, and 7 may also be included in the mixture. Furthermore, the ADCs in the mixture may have a DAR greater than 8. The ADC mixture is obtained by conjugation after interchain disulfide reduction. In one embodiment, the ADC mixture contains both ADCs with a DAR of 4 or less (i.e., drug-loaded species of 4 or less) and ADCs with a DAR of 6 or more (i.e., drug-loaded species of 6 or more).

[0109] The term "cancer" refers to or describes the physiological condition in mammals typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include glioblastoma, non-small cell lung cancer, lung cancer, colon cancer, colorectal cancer, head and neck cancer, breast cancer (e.g., triple-negative breast cancer), pancreatic cancer, squamous cell tumor, squamous cell carcinoma (e.g., lung squamous cell carcinoma or head and neck squamous cell carcinoma), anal cancer, skin cancer, and vulvar cancer. In one embodiment, an antibody or ADC of the invention is administered to a patient with a tumor that expresses EGFRvIII, a truncated form of EGFR, due to amplification of the EGFR gene. In one embodiment, an antibody or ADC of the invention is administered to a patient with a solid tumor that may overexpress EGFR. In one embodiment, an antibody or ADC of the invention is administered to a patient with squamous non-small cell lung cancer (NSCLC). In one embodiment, an antibody or ADC of the invention is administered to a patient with a solid tumor, including an advanced solid tumor.

[0110] As used herein, the term "EGFR-expressing tumor" refers to a tumor that expresses the EGFR protein. In one embodiment, EGFR expression in a tumor is determined using immunohistochemical staining of tumor cell membranes, and if immunohistochemical staining above background levels is detected in a tumor sample, the tumor is determined to be an EGFR-expressing tumor. Methods for detecting EGFR expression in tumors are known in the art, including, for example, the EGFR pharmDx™ Kit (Dako). On the other hand, an "EGFR-negative tumor" is defined as a tumor that does not have EGFR membrane staining above background in a tumor sample as measured by immunohistochemical techniques.

[0111] As used herein, the term "EGFRvIII-positive tumor" refers to a tumor that expresses EGFRvIII protein. In one embodiment, EGFRvIII expression in a tumor is determined using immunohistochemical staining of tumor cell membranes, and if immunohistochemical staining above background levels is detected in a tumor sample, the tumor is determined to be an EGFRvIII-expressing tumor. Methods for detecting EGFR expression in tumors are known in the art and include immunohistochemical assays. In contrast, an "EGFRvIII-negative tumor" is defined as a tumor that does not have EGFRvIII membrane staining above background levels in a tumor sample as measured by immunohistochemical techniques.

[0112] The terms "overexpress," "overexpression," or "overexpressing" are synonymous and generally refer to genes that are transcribed or translated at detectably higher levels in cancer cells compared to normal cells. Overexpression, therefore, refers to both protein and RNA overexpression (due to increased transcription, post-transcriptional processing, translation, post-translational processing, altered stability, and altered proteolysis) and localized overexpression due to altered protein trafficking patterns (increased nuclear translocation) and increased functional activity, such as in the case of increased enzymatic hydrolysis of a substrate. Overexpression therefore refers to protein or RNA levels. Overexpression can also be 50%, 60%, 70%, 80%, 90%, or more compared to normal or control cells. In certain embodiments, the anti-EGFR antibodies or ADCs of the invention are used to treat solid tumors that may overexpress EGFR.

[0113] As used herein, the term "administering" refers to delivering a substance (e.g., an anti-EGFR antibody or ADC) to achieve a therapeutic goal (e.g., treatment of an EGFR-related disorder). Administration can be parenteral, enteral, or topical. Parenteral administration is typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intra-articular, intraorbital, intracardiac, intradermal, intraperitoneal, transbronchial, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0114] As used herein, the term "combination therapy" refers to the administration of two or more therapeutic agents (e.g., an anti-EGFR antibody or ADC and another therapeutic agent), which can be administered simultaneously with, before, or after administration of the anti-EGFR antibody or ADC.

[0115] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a drug (e.g., an antibody or ADC) sufficient to reduce or ameliorate the severity and / or duration of a disorder (e.g., cancer) or one or more symptoms thereof, prevent progression of the disorder, induce regression of the disorder, prevent the recurrence, onset, onset, or progression of one or more symptoms associated with the disorder, detect the disorder, or enhance or improve the prophylactic or therapeutic effects of another therapy (e.g., a prophylactic or therapeutic agent). An effective amount of an antibody or ADC can, for example, inhibit tumor growth (e.g., inhibit an increase in tumor volume), suppress tumor growth (e.g., reduce tumor volume), reduce the number of cancer cells, and / or alleviate to some extent one or more symptoms associated with cancer. An effective amount can, for example, improve disease-free survival (DFS), improve overall survival (OS), or reduce the likelihood of recurrence.

[0116] The following subsections describe various aspects of the invention in further detail.

[0117] II. Anti-EGFR antibody One aspect of the present invention provides anti-EGFR antibodies or antigen-binding portions thereof that have improved characteristics (e.g., increased binding affinity to EGFR) over Ab1 and other antibodies known in the art. Another aspect of the present invention relates to antibody-drug conjugates (ADCs) comprising the anti-EGFR antibodies described herein and at least one drug (e.g., but not limited to, an auristatin). The antibodies or ADCs of the present invention have characteristics, including, but not limited to, binding to tumor cells expressing EGFRvIII, binding to wild-type EGFR on tumor cells expressing EGFR, recognizing the epitope CGADSYEMEEDGVRKC (SEQ ID NO: 45) on EGFR, binding to EGFR on normal human epithelial keratinocytes, and suppressing or inhibiting xenograft tumor growth in a mouse model.

[0118] Ab1 (Antibody 1) is a humanized anti-EGFR antibody. The light and heavy chain sequences of Ab1 are set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively (see also U.S. Patent Application Publication No. 20120183471, incorporated herein by reference). The light chain variable region of Ab1 is set forth in SEQ ID NO: 5 and contains the CDR1 amino acid sequence set forth in SEQ ID NO: 6, the CDR2 amino acid sequence set forth in SEQ ID NO: 7, and the CDR3 amino acid sequence set forth in SEQ ID NO: 8. The heavy chain variable region of Ab1 is set forth in SEQ ID NO: 1 and contains the CDR1 amino acid sequence set forth in SEQ ID NO: 2, the CDR2 amino acid sequence set forth in SEQ ID NO: 3, and the CDR3 amino acid sequence set forth in SEQ ID NO: 4.

[0119] Generally, Ab1 variant antibodies of the invention retain the epitope specificity of the parent antibody Ab1. Thus, in one embodiment, an anti-EGFR antibody of the invention can bind to the epitope on EGFR set forth in SEQ ID NO: 45 and / or compete with Ab1 for binding to EGFR. In various embodiments, the binding can be assayed according to the protocol described in Example 3 below. In a preferred embodiment of the invention, the anti-EGFR antibody competes with Ab1 and exhibits improved binding affinity to EGFR 1-525 (SEQ ID NO: 47), e.g., as measured by surface plasmon resonance, resulting in a lower dissociation constant (K d ) is about 1 × 10 -6 M ~ approx. 1×10 -10 It's M.

[0120] In one embodiment, the present invention relates to anti-EGFR antibodies that are variants of Ab1 and have improved characteristics (e.g., improved binding affinity and the ability to inhibit NSCLC tumor cell growth in vivo, as described in the Examples below). These novel antibodies are collectively referred to herein as "Ab1 variant antibodies." Generally, Ab1 variant antibodies retain the same epitope specificity as Ab1. Thus, in one embodiment, an anti-EGFR antibody or antigen-binding portion thereof of the present invention binds to an epitope within the amino acid sequence set forth in SEQ ID NO: 45 and competes with an anti-EGFR antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5 for binding to EGFRvIII in a competitive binding assay. In contrast to Ab1, the anti-EGFR antibodies of the present invention can inhibit or suppress tumor growth in vivo in an H292 human non-small cell lung carcinoma (NSCLC) xenograft assay in nude mice and / or can bind to wild-type EGFR on normal human epithelial keratinocytes. In various embodiments, the anti-EGFR antibody or antigen-binding fragment thereof of the invention is capable of modulating the biological function of EGFR. In other embodiments of the above aspects, the anti-EGFR antibody or antigen-binding fragment thereof binds to EGFRvIII, binds to EGFR on cells overexpressing EGFR, and recognizes the epitope CGADSYEMEEDGVRKC (SEQ ID NO: 45) on EGFR. In another embodiment, the anti-EGFR antibody or antigen-binding fragment thereof binds to EGFRvIII at an epitope that is different from the EGFRvIII junction peptide. In other embodiments of the above aspects, the anti-EGFR antibody or antigen-binding fragment thereof does not compete with cetuximab for binding to EGFR. The AbA antibody and Ab1 variant described in the Examples below have the following characteristics:

[0121] That is, the present invention encompasses anti-EGFR antibodies or antigen-binding portions thereof that can compete with Ab1 in competitive binding assays but are more effective at inhibiting or suppressing tumor growth. In one embodiment, the anti-EGFR antibodies or antigen-binding portions thereof of the present invention can bind to an epitope within the amino acid sequence CGADSYEMEEDGVRKC (SEQ ID NO: 45) and can compete with Ab1 (i.e., an anti-EGFR antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5) for binding to epidermal growth factor receptor variant III (EGFRvIII) (SEQ ID NO: 33) in competitive binding assays.

[0122] In one embodiment, the anti-EGFR antibody, or antigen-binding portion thereof, of the invention has a cytotoxicity of about 1 x 10 as measured by surface plasmon resonance. -6 The dissociation constant (K d ) to EGFR(1-525) (SEQ ID NO: 47). Alternatively, the antibody, or antigen-binding portion thereof, binds to EGFR(1-525) (SEQ ID NO: 47) at a concentration of about 1 x 10 as measured by surface plasmon resonance. -6 M ~ approx. 1×10 -10 K of M d Alternatively, the antibody or antigen-binding portion thereof binds to EGFR(1-525) (SEQ ID NO: 47) at a binding affinity of about 1 x 10 as measured by surface plasmon resonance. -6 M ~ approx. 1×10 -7 K of M d Alternatively, the antibody or antigen-binding portion thereof of the invention binds to EGFR(1-525) (SEQ ID NO: 47) at a binding affinity of about 1 x 10 as measured by surface plasmon resonance. -6 M ~ approx. 5×10 -10 K of M d , about 1×10 -6 M ~ approx. 1×10 -9 K of M d , about 1×10 -6 M ~ approx. 5×10 -9 K of M d , about 1×10 -6 M ~ approx. 1×10 -8 K of M d , about 1×10 -6 M ~ approx. 5×10 -8 K of Md , about 5.9×10 -7 M ~ approx. 1.7×10 -9 K of M d , about 5.9×10 -7 M ~ approx. 2.2×10 -7 K of M d In certain embodiments, the dissociation constant (K d ) is in one embodiment less than the dissociation constant of Ab1 and greater than the rate of the anti-EGFR antibody cetuximab.

[0123] One advantage of the anti-EGFR antibodies, and antigen-binding portions thereof, of the invention is that the antibodies can bind to tumor cells that express EGFRvIII. Although EGFRvIII is associated with certain types of cancer, many anti-EGFR antibodies known in the art (e.g., cetuximab) are not effective at inhibiting or suppressing tumor growth in tumors that express EGFRvIII. Thus, in one embodiment, the antibodies, or antigen-binding portions thereof, of the invention exhibit a cell proliferation rate of about 8.2 x 10 6 cells / mL, as measured by surface plasmon resonance. -9 K below M d Alternatively, the antibody, or antigen-binding portion thereof, of the invention binds to EGFRvIII (SEQ ID NO: 33) at a binding affinity of about 8.2 x 10 as measured by surface plasmon resonance. -9 M ~ approx. 6.3×10 -10 K of M d , about 8.2×10 -9 M ~ approx. 2.0×10 -9 K of M d , about 2.3×10 -9 M ~ approx. 1.5×10 -10 K of M d It binds to EGFRvIII (SEQ ID NO: 33) at

[0124] In one embodiment, the antibodies of the invention can inhibit or suppress tumor growth in an in vivo xenograft mouse model. For example, the antibodies of the invention, or antigen-binding portions thereof, can inhibit tumor growth by at least about 50% in an in vivo human non-small cell lung cancer (NSCLC) xenograft assay compared to a non-EGFR-specific human IgG antibody. In certain embodiments, the antibodies of the invention, or antigen-binding portions thereof, can inhibit or suppress tumor growth by at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% in an in vivo human non-small cell lung cancer (NSCLC) xenograft assay compared to a non-EGFR-specific human IgG antibody when administered at the same dose and dosing cycle. In certain embodiments, the antibodies of the invention, or antigen-binding portions thereof, are capable of inhibiting or suppressing tumor growth by about 80% to about 90%, or about 84% to about 90%, or about 88% to about 90%, in an in vivo human non-small cell lung cancer (NSCLC) xenograft assay when administered at the same dose and dosing cycle, compared to a human IgG antibody non-specific for EGFR.

[0125] As used herein, the term "xenograft assay" refers to a human tumor xenograft assay in which human tumor cells are implanted subcutaneously or into the organ of origin of the tumor in immunocompromised mice that do not reject the human cells.

[0126] Anti-EGFR antibodies or antigen-binding portions thereof having both of the above characteristics are also considered embodiments of the present invention. For example, the antibodies of the present invention have a cytotoxicity of about 1×10 when measured by surface plasmon resonance. -6 The dissociation constant (K d ) to EGFR(1-525) (SEQ ID NO: 47), binds to an epitope within the amino acid sequence CGADSYEMEEDGVRKC (SEQ ID NO: 45), and can compete with Ab1 (i.e., an anti-EGFR antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5) for binding to epidermal growth factor receptor variant III (EGFRvIII) (SEQ ID NO: 33) in a competitive binding assay.

[0127] In certain embodiments, the anti-EGFR antibody, or antigen-binding portion thereof, binds to an epitope within the amino acid sequence CGADSYEMEEDGVRKC (SEQ ID NO: 45), competes with Ab1 (i.e., an anti-EGFR antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5) for binding to epidermal growth factor receptor variant III (EGFRvIII) (SEQ ID NO: 33) in a competitive binding assay, and has a binding affinity of about 8.2 x 10 as measured by surface plasmon resonance. -9 K below M d It binds to EGFRvIII (SEQ ID NO: 33) at

[0128] In certain embodiments, the anti-EGFR antibody, or antigen-binding portion thereof, binds to an epitope within the amino acid sequence CGADSYEMEEDGVRKC (SEQ ID NO: 45), competes with Ab1 (i.e., an anti-EGFR antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5) for binding to epidermal growth factor receptor variant III (EGFRvIII) (SEQ ID NO: 33) in a competitive binding assay, and inhibits or suppresses tumor growth in an in vivo xenograft mouse model. More specifically, the antibody, or antigen-binding portion thereof, of the invention can inhibit tumor growth by at least about 50% in an in vivo human non-small cell lung cancer (NSCLC) xenograft assay compared to a human IgG antibody non-specific for EGFR when administered at the same dose and dosing cycle. Alternatively, the antibodies, or antigen-binding portions thereof, of the invention can inhibit or suppress tumor growth by at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% in an in vivo human non-small cell lung cancer (NSCLC) xenograft assay when administered at the same dose and dosing cycle, compared to a non-EGFR-specific human IgG antibody. In certain embodiments, the antibodies, or antigen-binding portions thereof, can inhibit or suppress tumor growth by about 80% to about 90%, or about 84% to about 90%, or about 88% to about 90% in an in vivo human non-small cell lung cancer (NSCLC) xenograft assay when administered at the same dose and dosing cycle, compared to a non-EGFR-specific human IgG antibody.

[0129] An embodiment of the present invention is an antibody that combines any of the above characteristics. The ADCs of the present invention, which will be described in detail below, can also have any of the above characteristics.

[0130] In one embodiment, the present invention encompasses an anti-hEGFR antibody, or an antigen-binding portion thereof, comprising an LC CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40, an LC CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 39, and an LC CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 38, and an HC CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 37, an HC CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 36, and an HC CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0131] In one embodiment, the invention encompasses an anti-hEGFR antibody, or antigen-binding portion thereof, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78; and a light chain variable region comprising an amino acid sequence selected from the group consisting of 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, and 79.

[0132] In one embodiment, the present invention relates to a HC selected from the group consisting of SEQ ID NOs: 10, 11 and 12; SEQ ID NOs: 16, 17 and 18; SEQ ID NOs: 10, 11 and 19; SEQ ID NOs: 20, 11 and 12; SEQ ID NOs: 21, 3 and 22; SEQ ID NOs: 16, 17 and 19; SEQ ID NOs: 2, 3 and 4; SEQ ID NOs: 10, 3 and 12; SEQ ID NOs: 80, 11 and 18; SEQ ID NOs: 80, 3 and 18; SEQ ID NOs: 20, 3 and 12; SEQ ID NOs: 80, 11 and 12; and SEQ ID NOs: 81, 11 and 22. and an LC light chain CDR set (CDR1, CDR2, and CDR3) selected from the group consisting of: SEQ ID NOs: 6, 7, and 8; SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 26, 27, and 28; SEQ ID NOs: 29, 30, and 31; SEQ ID NOs: 6, 7, and 84; SEQ ID NOs: 82, 83, and 31; and SEQ ID NOs: 82, 27, and 85 (provided that the antibody or antigen-binding portion thereof does not simultaneously comprise the HC CDR set of SEQ ID NOs: 2, 3, and 4 and the LC CDR set of SEQ ID NOs: 6, 7, and 8).

[0133] Preferably, the anti-EGFR antibodies of the invention exhibit a high ability to reduce or neutralize EGFR activity, e.g., as assessed by any one of several in vitro and in vivo assays known in the art. For example, inhibition of EGFR phosphorylation in an EGFR-expressing cell line (e.g., the h292 cell line) can be measured. In certain embodiments, the isolated antibody, or antigen-binding portion thereof, binds to human EGFR, and the antibody, or antigen-binding portion thereof, exhibits a potency of about 5.9 x 10 as measured by surface plasmon resonance. -7 K below M D Alternatively, the antibody, or antigen-binding portion thereof, dissociates from human EGFR (EGFR1-525) with a rate constant of about 4.2 x 10 as measured by surface plasmon resonance. -7 K of M D Alternatively, the antibody, or antigen-binding portion thereof, may dissociate from human EGFR(1-525) with a rate constant of about 2.5 x 10 as measured by surface plasmon resonance. -7 Estimate of M D In certain embodiments, the anti-EGFR antibody, or antigen-binding portion thereof, of the invention can dissociate from human EGFR(1-525) with a rate constant of 5.9 x 10 -7 M~5×10 -9 K of M D It has a rate constant.

[0134] Alternatively, the antibody, or antigen-binding portion thereof, has a molecular weight of about 6.1 x 10 as measured by surface plasmon resonance. -9 K below M D Alternatively, the antibody, or antigen-binding portion thereof, may dissociate from human EGFRvIII with a rate constant of about 3.9 x 10 as measured by surface plasmon resonance. -9 K below M D Alternatively, the antibody, or antigen-binding portion thereof, may dissociate from human EGFRvIII with a rate constant of about 2.3 x 10 as measured by surface plasmon resonance. -9 K below M D It can dissociate from human EGFRvIII with a rate constant.

[0135] In one embodiment, the present invention relates to an anti-EGFR antibody, or antigen-binding portion thereof, that is antibody AbA. AbA has improved binding affinity to EGFR relative to Ab1 and, further, exhibits unique in vitro and in vivo characteristics relative to Ab1. AbA binds to EGFR with significantly higher affinity than Ab1 in an in vitro keratinocyte binding assay. Furthermore, AbA can inhibit or suppress tumor growth in a xenograft H292 cell assay. Notably, AbA has improved in vitro and in vivo characteristics and is comparable to other Ab1 variant antibodies that have higher binding affinity than AbA. Despite AbA's lower binding affinity compared to other Ab1 variant antibodies (see, e.g., Figure 3 comparing AbP and AbQ to AbA), it exhibited comparable cell growth inhibition in an in vivo assay.

[0136] The term "AbA" encompasses IgG antibodies having at least six CDRs of AbA. AbA antibodies have the same light chain as Ab1, but the heavy chain contains six amino acid sequence variations relative to the parent antibody Ab1 (four amino acid variations in the variable region of the heavy chain and two variations in the constant region). AbA antibodies contain a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO:12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:10, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO:8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:7, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:6. The heavy chain variable region of AbA is represented by the amino acid sequence set forth in SEQ ID NO:9, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:5. The full-length heavy chain of antibody AbA is shown in the amino acid sequence set forth in SEQ ID NO:15, and the full-length light chain of antibody AbA is shown in the amino acid sequence set forth in SEQ ID NO:13 (see Figure 2). The nucleic acid sequence of the heavy chain of AbA is shown below.

[0137] [ka]

[0138] The nucleic acid sequence of the light chain of AbA is shown below.

[0139] [ka]

[0140] In one embodiment, the invention relates to an anti-EGFR antibody, or antigen-binding portion thereof, that is antibody AbB. The AbB antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 19, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 17, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 16, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 6. In another embodiment, the invention provides an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 64 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 65.

[0141] In one embodiment, the invention relates to an anti-EGFR antibody, or antigen-binding portion thereof, that is antibody AbC. The AbC antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 3, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 6. In another embodiment, the invention provides an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 66 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 67.

[0142] In one embodiment, the invention relates to an anti-EGFR antibody, or antigen-binding portion thereof, that is antibody AbD. The AbD antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 3, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 31, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 83, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 82. In another embodiment, the invention provides an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 68 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 69.

[0143] In one embodiment, the invention relates to an anti-EGFR antibody, or antigen-binding portion thereof, that is antibody AbE. The AbE antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 3, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 2, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 85, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 27, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 82. In another embodiment, the invention provides an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 50 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 51.

[0144] In one embodiment, the invention relates to an anti-EGFR antibody, or an antigen-binding portion thereof, that is antibody AbF. The AbF antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 3, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 6. In another embodiment, the invention provides an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 52 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 53.

[0145] In one embodiment, the invention relates to an anti-EGFR antibody, or antigen-binding portion thereof, that is antibody AbG. The AbG antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 17, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 16, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 25, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 24, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 23. In another embodiment, the invention provides an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 72 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 73.

[0146] In one embodiment, the invention relates to an anti-EGFR antibody, or antigen-binding portion thereof, that is antibody AbH. The AbH antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 80, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 25, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 24, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 23. In another embodiment, the invention provides an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 54 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 55.

[0147] In one embodiment, the invention relates to an anti-EGFR antibody, or antigen-binding portion thereof, that is antibody AbJ. The AbJ antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 3, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 80, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 25, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 24, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 23. In another embodiment, the invention provides an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 56 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 57.

[0148] In one embodiment, the invention relates to an anti-EGFR antibody, or antigen-binding portion thereof, that is antibody AbK. The AbK antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 19, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 28, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 27, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 26. In another embodiment, the invention provides an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 74 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 75.

[0149] In one embodiment, the invention relates to an anti-EGFR antibody, or antigen-binding portion thereof, that is antibody AbL. The AbL antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 80, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 28, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 27, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 26. In another embodiment, the invention provides an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 58 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59.

[0150] In one embodiment, the invention relates to an anti-EGFR antibody, or antigen-binding portion thereof, that is antibody AbM. The AbM antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 20, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 28, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 27, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 26. In another embodiment, the invention provides an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 76 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 77.

[0151] In one embodiment, the invention relates to an anti-EGFR antibody, or antigen-binding portion thereof, that is antibody AbN. The AbN antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 3, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 20, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 28, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 27, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 26. In another embodiment, the invention provides an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 60 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 61.

[0152] In one embodiment, the invention relates to an anti-EGFR antibody, or antigen-binding portion thereof, that is an AbO antibody. The AbO antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 80, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 28, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 27, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 26. In another embodiment, the invention provides an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 62 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 63.

[0153] In one embodiment, the invention relates to an anti-EGFR antibody, or antigen-binding portion thereof, that is antibody AbP. The AbP antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 22, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 3, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 21, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 31, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 30, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 29. In another embodiment, the invention provides an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 78 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 79.

[0154] In one embodiment, the invention relates to an anti-EGFR antibody, or antigen-binding portion thereof, that is antibody AbQ. The AbQ antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 22, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 81, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 31, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 30, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 29. In another embodiment, the invention provides an antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 70 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 71.

[0155] As described in Table 1 in the Examples below, the Ab1 variant antibody sequences provide consensus amino acid sequences corresponding to the CDR regions that improve Ab1 binding to the EGFR epitope. Accordingly, in one embodiment, the present invention relates to an anti-EGFR antibody, or antigen-binding portion thereof, comprising a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO:40, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO:39, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO:38, and a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO:37, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO:36, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO:35.

[0156] In one embodiment, the anti-epidermal growth factor receptor (anti-EGFR) antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of 50, 52, 53, 56, 58, 60, 62, 64, 66, and 68, and a light chain variable region comprising an amino acid sequence selected from the group consisting of 51, 53, 55, 57, 59, 61, 63, 65, 67, and 69.

[0157] In another embodiment, the anti-EGFR antibody, or antigen-binding portion thereof, of the invention comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 12, 18, 19, and 22, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 11 or 17, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 10, 16, 20, and 21; and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 8, 25, 28, and 31, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 7, 24, 27, and 30, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 6, 23, 26, and 29.

[0158] Phosphorylation and proliferation assays demonstrate that the antibodies described herein inhibit EGFR-mediated phosphorylation and tumor cell proliferation. For example, as described in Example 6, the EGFR antibodies of the invention (as tested) were found to inhibit tumor cell proliferation in vivo.

[0159] The above anti-EGFR antibody CDR sequences have been isolated in accordance with the present invention and establish a novel family of EGFR binding proteins comprising polypeptides comprising the CDR sequences shown in Tables 1-3 below.

[0160] To generate and select CDRs with favorable EGFR-binding and / or neutralizing activity for hEGFR, standard methods known in the art can be used to generate antibodies or antigen-binding portions thereof and evaluate the EGFR-binding and / or neutralizing properties of these antibodies or antigen-binding portions thereof, including, but not limited to, the methods specifically described herein.

[0161] In certain embodiments, the antibody comprises a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. In certain embodiments, the anti-EGFR antibody, or antigen-binding portion thereof, comprises a heavy chain immunoglobulin constant region selected from the group consisting of a human IgG constant region, a human IgM constant region, a human IgE constant region, and a human IgA constant region. In other embodiments, the antibody, or antigen-binding portion thereof, comprises an IgG1 heavy chain constant region, an IgG2 heavy chain constant region, an IgG3 heavy chain constant region, or an IgG4 heavy chain constant region. Preferably, the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. Additionally, the antibody can comprise a light chain constant region, either a κ light chain constant region or a λ light chain constant region. Preferably, the antibody comprises a κ light chain constant region. Alternatively, the antibody portion can be, for example, a Fab fragment or a single-chain Fv fragment.

[0162] In certain embodiments, the anti-EGFR antibody binding portion is a Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single domain antibody, or diabody.

[0163] In certain embodiments, the anti-EGFR antibody, or antigen-binding portion thereof, is a multispecific antibody (e.g., a bispecific antibody).

[0164] In certain embodiments, the anti-EGFR antibody, or antigen-binding portion thereof, comprises a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO:41 and / or a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO:43.

[0165] Substitution of amino acid residues in the Fc portion to alter antibody effector function has been previously described (Winter et al., U.S. Pat. Nos. 5,648,260 and 5,624,821, incorporated herein by reference). The Fc portion of an antibody mediates several important effector functions, including cytokine induction, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC), and half-life / clearance rate of antibody and antigen-antibody complexes. These effector functions may be desirable for therapeutic antibodies, but may also be unnecessary or deleterious, depending on the therapeutic objective. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC by binding to FcγR and complement C1q, respectively. The fetal Fc receptor (FcRn) is an important factor determining the circulating half-life of an antibody. In yet another embodiment, at least one amino acid residue in the constant region of an antibody (e.g., the Fc region of an antibody) is substituted to alter the antibody effector function.

[0166] One embodiment of the present invention encompasses a recombinant chimeric antigen receptor (CAR) comprising the binding region of an antibody described herein (e.g., the heavy and / or light chain CDRs of AbA). Recombinant CARs such as those described herein can be used to redirect T cell specificity to an antigen in a human leukocyte antigen (HLA)-independent manner. Thus, the CARs of the present invention can be used in immunotherapy to facilitate engineering a human subject's own immune cells to recognize and attack the subject's tumor (see, e.g., U.S. Patent Nos. 6,410,319; 8,389,282; 8,822,647; 8,906,682; 8,911,993; 8,916,381; 8,975,071; and U.S. Patent Application Publication No. US20140322275, each of which is incorporated herein by reference, for purposes of CAR technology). This type of immunotherapy, referred to as adoptive immunotherapy (ACT), can be used to treat cancer in a subject in need thereof.

[0167] The anti-EGFR CARs of the invention preferably comprise an extracellular antigen-binding domain specific for EGFR (e.g., EGFRvIII), a transmembrane domain used to anchor the CAR to a T cell, and one or more intracellular signaling domains. In one embodiment of the invention, the CAR comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In one embodiment of the invention, the CAR comprises a costimulatory domain (e.g., a costimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137)). In certain embodiments of the invention, the CAR comprises an scFv comprising a CDR or variable region described herein (e.g., a CDR or variable region from an AbA antibody), a transmembrane domain, a costimulatory domain (e.g., a functional signaling domain from CD28 or 4-1BB), and a signaling domain comprising a functional signaling domain from CD3 (e.g., CD3-zeta).

[0168] In certain embodiments, the invention encompasses T cells (also referred to as CAR T cells) comprising a CAR comprising the antigen-binding region (e.g., CDR) of an antibody described herein or an scFv described herein.

[0169] In certain embodiments of the invention, the CAR comprises a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 39, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 38, and a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 37, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 36, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 35.

[0170] In certain embodiments of the invention, the CAR comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 10, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 8, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 7, and a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 6.

[0171] One embodiment of the present invention encompasses a labeled anti-EGFR antibody or antibody portion thereof, wherein the antibody has been derivatized or linked to one or more functional molecules (e.g., another peptide or protein). For example, a labeled antibody can be obtained by functionally linking (by chemical coupling, genetic fusion, noncovalent association, or other means) an antibody or antibody portion of the invention to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detectable substance, a drug, a protein or peptide that can mediate the association of the antibody or antibody portion with another molecule (e.g., a streptavidin core region or a polyhistidine tag), and / or a cytotoxic or therapeutic agent selected from the group consisting of an antimitotic agent, an antitumor antibiotic, an immunomodulatory agent, a gene therapy vector, an alkylating agent, an antiangiogenic agent, an antimetabolite, a boron-containing agent, a chemotherapeutic agent, a hormonal agent, an antihormonal agent, a corticosteroid, a photosensitive therapeutic agent, an oligonucleotide, a radionuclide agent, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radiosensitizer, and combinations thereof.

[0172] Useful detectable substances with which antibodies or antibody portions thereof can be derivatized include fluorescent compounds. Examples of detectable fluorescent substances include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and the like. Antibodies may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, and glucose oxidase. When derivatized with a detectable enzyme, the antibody is detected by adding other reagents that the enzyme uses to produce a detectable reaction product. For example, when horseradish peroxidase is present as the detectable substance, hydrogen peroxide and diaminobenzidine are added to yield a detectable colored reaction product. Antibodies may also be derivatized with biotin and detected by indirect measurement of avidin or streptavidin binding.

[0173] In one embodiment, the antibodies of the invention are conjugated to an imaging agent. Examples of imaging agents that can be used in the compositions and methods described herein include, but are not limited to, radiolabels (e.g., indium), enzymes, fluorescent labels, luminescent labels, bioluminescent labels, magnetic labels, and biotin.

[0174] In one embodiment, the antibody or ADC is administered using, but not limited to, indium ( 111 It is linked to a radioactively labeled substance such as Fluorescent Protein (FGP) or Fluorescent Protein (FGP). 111 Indium can be used to label the antibodies or ADCs described herein for the identification of EGFR-positive tumors. In certain embodiments, the anti-EGFR antibodies (or ADCs) described herein are chelating to the EGFR-positive tumors via a bifunctional chelator that is a bifunctional cyclohexyldiethylenetriaminepentaacetic acid (DTPA) chelate. 111 I (see U.S. Patent Nos. 5,124,471, 5,434,287, and 5,286,850, each of which is incorporated herein by reference).

[0175] Another embodiment of the present invention provides a glycosylated binding protein comprising one or more sugar residues in the anti-EGFR antibody or its antigen-binding portion. Fetal in vivo protein production may undergo further processing known as post-translational modification. In particular, sugar (glycosyl) residues can be added enzymatically, a process known as glycosylation. The resulting protein has covalently attached oligosaccharide side chains and is called a glycosylated protein or glycoprotein. Antibodies are glycoproteins with one or more sugar residues in the Fc domain and variable domain. While sugar residues in the Fc domain significantly affect the effector function of the Fc domain, they have little effect on the antigen binding or half-life of the antibody (R. Jefferis, Biotechnol. Prog. 21 (2005), pp. 11-16). On the other hand, glycosylation in the variable domain may affect the antigen-binding activity of the antibody. Glycosylation of variable domains can negatively affect antibody binding affinity, possibly due to steric hindrance (Co, MS, et al., Mol. Immunol. (1993) 30:1361-1367), or may increase affinity for antigen (Wallick, SC, et al., Exp. Med. (1988) 168:1099-1109; Wright, A., et al., EMBO J. (1991) 10:2717-2723).

[0176] One aspect of the present invention relates to the generation of glycosylation site mutants in which the O-linked or N-linked glycosylation site of a binding protein is mutated. Such mutants can be generated by those skilled in the art using standard techniques known to those skilled in the art. Glycosylation site mutants that retain biological activity but have increased or decreased binding activity are also an object of the present invention.

[0177] In yet another embodiment, the glycosylation of an anti-EGFR antibody or antigen-binding portion of the invention is modified. For example, an aglycosylated antibody can be generated (i.e., the antibody is not glycosylated). Glycosylation can be modified, for example, to increase the affinity of the antibody for its antigen. Such glycosylation can be performed, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made to remove one or more variable region glycosylation sites, thereby eliminating glycosylation at those sites. Such aglycosylation can increase the affinity of the antibody for its antigen. Such approaches are described in further detail in PCT Publication WO2003016466A2 and U.S. Patent Nos. 5,714,350 and 6,350,861, the disclosures of each of which are incorporated herein by reference in their entirety.

[0178] Additionally or alternatively, modified anti-EGFR antibodies of the invention can be engineered with altered glycosylation types, such as hypofucosylated antibodies with fewer fucosyl residues or antibodies with more bisecting GlcNAc structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such glycosylation modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation mechanism. Cells with altered glycosylation mechanisms have been previously described in the art and can be used as host cells to express the recombinant antibodies of the invention to produce antibodies with altered glycosylation. See, for example, Shields, R. Let al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, as well as European Patent No. EP 1,176,195; PCT Publication No. WO 03 / 035835; and WO 99 / 54342 80, the disclosures of each of which are incorporated herein by reference in their entirety.

[0179] Protein glycosylation depends on the amino acid sequence of the protein of interest and the host cell in which the protein is expressed. Different organisms produce different glycosylation enzymes (e.g., glycosyltransferases and glycosidases) and may have different substrates (nucleotide sugars) available. Due to these factors, protein glycosylation patterns and glycosyl residue compositions may vary depending on the host system in which a particular protein is expressed. Glycosyl residues useful in the present invention include, but are not limited to, glucose, galactose, mannose, fucose, n-acetylglucosamine, and sialic acid. Glycosylated binding proteins preferably contain glycosyl residues that result in a glycosylation pattern consistent with the human pattern.

[0180] Different protein glycosylation can result in different protein properties. For example, the potency of a therapeutic protein produced in a microbial host, such as yeast, and glycosylated using the yeast endogenous pathway may be reduced compared to the same protein expressed in mammalian cells, such as a CHO cell line. Such glycoproteins may also be immunogenic in humans and may have a shorter in vivo half-life after administration. Specific receptors in humans and other animals may recognize particular glycosyl residues and promote rapid clearance of the protein from the bloodstream. Other adverse effects include alterations in protein folding, solubility, protease susceptibility, trafficking, transport, compartmentalization, secretion, recognition by other proteins or factors, antigenicity, or allergenicity. Thus, a practitioner may preferentially select therapeutic proteins with a particular glycosylation composition and pattern (e.g., the same or at least similar to that expressed in human cells or species-specific cells of the intended target animal).

[0181] Expression of glycosylated proteins different from those of the host cell can be achieved by genetically modifying the host cell to express heterologous glycosylation enzymes. Using recombinant technology, practitioners can generate antibodies or antigen-binding portions thereof that exhibit human protein glycosylation. For example, yeast strains have been genetically modified to express non-native glycosylation enzymes, and glycosylated proteins (glycoproteins) produced by these yeast strains have been modified to exhibit protein glycosylation identical to that of animal cells, particularly human cells (U.S. Patent Publication Nos. 20040018590 and 20020137134 and PCT Publication WO2005100584A2).

[0182] Antibodies may be produced by any of a number of techniques. For example, expression vectors encoding the heavy and light chains can be transfected into and expressed from host cells using standard techniques. The various forms of the term "transfection" encompass a variety of commonly used techniques for introducing foreign DNA into prokaryotic or eukaryotic host cells, including electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although antibodies can be expressed in either prokaryotic or eukaryotic host cells, they are preferably expressed in eukaryotic cells, with mammalian host cells being most preferred, because eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete correctly folded, immunologically active antibody.

[0183] Preferred mammalian host cells for expressing recombinant antibodies of the invention include Chinese hamster ovary (CHO) cells (including dhfr-deficient CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, in combination with a DHFR selection marker, e.g., as described by R.J. Kaufman and P.A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or more preferably, secretion of the antibody into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0184] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. Variations on the above procedures are, of course, within the scope of the present invention. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of either the light and / or heavy chains of an antibody of the present invention. Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that are not necessary for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also included in the antibodies of the present invention. Furthermore, bivalent antibodies in which one heavy chain and one light chain are an antibody of the present invention and the other heavy and light chains are specific for an antigen other than the antigen of interest can be produced by crosslinking an antibody of the present invention to a second antibody using standard chemical crosslinking methods.

[0185] In a preferred system for recombinant expression of the antibodies of the invention or their antigen-binding portions, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-deficient CHO cells by calcium phosphate transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high-level transcription of the genes. The recombinant expression vector also incorporates a DHFR gene, which allows for selection of CHO cells transfected with the vector using methotrexate selection / amplification. The selected transformed host cells are cultured to express the antibody heavy and light chains, and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. The invention further provides a method for synthesizing recombinant antibodies of the invention by culturing host cells in an appropriate culture medium until the recombinant antibody is synthesized. Recombinant antibodies of the invention can be produced using nucleic acid molecules corresponding to the amino acid sequences disclosed herein. In one embodiment, the nucleic acid molecules set forth in SEQ ID NOs: 86 and / or 87 are used to generate the recombinant antibody. The method can further comprise isolating the recombinant antibody from the culture medium.

[0186] III. Anti-EGFR Antibody Drug Conjugates (ADCs) The anti-EGFR antibodies described herein may be conjugated with a drug moiety to form anti-EGFR antibody-drug conjugates (ADCs). Antibody-drug conjugates (ADCs) can selectively deliver one or more drug moieties to a target tissue, such as a tumor-associated antigen (e.g., a tumor expressing EGFR), thereby increasing the therapeutic efficacy of the antibody in treating a disease (e.g., cancer). Thus, in certain embodiments, the present invention provides anti-EGFR ADCs for use in therapy (e.g., treating cancer).

[0187] The anti-EGFR ADCs of the present invention comprise an EGFR antibody (i.e., an antibody that specifically binds to EGFR) linked to one or more drug moieties. The specificity of the ADC is determined by the specificity of the antibody (i.e., the anti-EGFR antibody). In one embodiment, the anti-EGFR antibody is linked to one or more cytotoxic drugs that are delivered in vivo to transformed cancer cells that express EGFR.

[0188] Examples of drugs that can be used in the anti-EGFR ADCs of the invention, as well as examples of linkers that can be used to attach one or more drugs to an antibody, are listed below. The terms "drug," "agent," and "drug moiety" are used interchangeably herein. The terms "linked" and "conjugated" are also used interchangeably herein to refer to the covalent attachment of an antibody to a drug moiety.

[0189] In certain embodiments, the ADC has the following formula (Formula I): Ab-(LD) n (I) where Ab is an antibody (e.g., an anti-EGFR antibody AbA) and (LD) is a linker-drug moiety. The linker-drug moiety is composed of a linker, L-, and a drug moiety, -D, which has, for example, a cytostatic, cytotoxic, or other therapeutic activity against a target cell (e.g., a cell expressing EGFR), where n is an integer from 1 to 20. In some embodiments, n is 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The DAR of the ADC is equivalent to "n" as described in Formula I. In one embodiment, the ADC has the formula Ab-(LD) n where Ab is an anti-EGFR antibody (e.g., AbA), L is a linker, e.g., valine-citrulline (vc), D is a drug (e.g., an auristatin such as MMAF or MMAE), and n is 2 to 4 (equivalent to a DAR of 2 to 4). The drugs (D in Formula I) and linkers (L in Formula I) and alternative ADC structures that can be used in the ADCs of the invention are described in further detail below.

[0190] A. Anti-EGFR ADC: Examples of Drugs for Conjugation Anti-EGFR antibodies can be used in ADCs to target one or more drugs to cells of interest (e.g., cancer cells that express EGFR). The anti-EGFR ADCs of the invention provide targeted therapy, for example, by delivering one or more drugs to specific cells, which can reduce the side effects frequently observed with anti-cancer treatments.

[0191] Auristatin The anti-EGFR antibodies (e.g., AbA antibodies) of the present invention may be conjugated to at least one auristatin. Auristatins represent a group of dolastatin analogs generally shown to have anticancer effects by interfering with microtubule dynamics and GTP hydrolysis, thereby inhibiting cell division. For example, auristatin E (U.S. Pat. No. 5,635,483) is a synthetic analog of the marine natural product dolastatin 10, a compound that inhibits tubulin polymerization by binding to the same site on tubulin as the anticancer drug vincristine (GR Pettit, Prog. Chem. Org. Nat. Prod., 70:1-79 (1997)). Dolastatin 10, auristatin PE, and auristatin E are linear peptides consisting of four amino acids, three of which are unique to the dolastatin class of compounds. Specific embodiments of the auristatin subclass of antimitotic agents include, but are not limited to, monomethyl auristatin D (MMAD or auristatin D derivatives), monomethyl auristatin E (MMAE or auristatin E derivatives), monomethyl auristatin F (MMAF or auristatin F derivatives), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), and 5-benzoylvaleric acid AE ester (AEVB). The synthesis and structures of auristatin derivatives are described in U.S. Patent Application Publication Nos. 2003-0083263, 2005-0238649, and 2005-0009751; International Patent Publication No. WO04 / 010957; International Patent Publication No. WO02 / 088172, each of which is incorporated herein by reference, and U.S. Patent Nos. 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,86 Nos. 0; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414.

[0192] In one embodiment, an anti-EGFR antibody (e.g., AbA) of the invention is conjugated to at least one MMAE (monomethyl auristatin E). Monomethyl auristatin E (MMAE, vedotin) inhibits cell division by blocking tubulin polymerization. Due to its high toxicity, it cannot be used as a drug. Recent developments in cancer therapy involve linking MMAE to a monoclonal antibody (mAb) that recognizes specific marker expression in cancer cells and directs MMAE to the cancer cells. In one embodiment, the linker connecting MMAE to the anti-EGFR antibody is stable in extracellular fluid (i.e., the medium or environment outside the cell), but is cleaved by cathepsin upon binding of the ADC to a specific cancer cell antigen and entry into the cancer cell, releasing the toxic MMAE and activating a potent antimitotic mechanism.

[0193] In one embodiment, an anti-EGFR antibody (e.g., AbA) described herein is conjugated to at least one MMAF (monomethyl auristatin F). Monomethyl auristatin F (MMAF) inhibits cell division by blocking tubulin polymerization. Due to its C-terminal charged phenylalanine residue, MMAF exhibits reduced cytotoxicity compared to its uncharged counterpart, MMAE. Due to its high toxicity, MMAF cannot be used as a drug, but it can be linked to a monoclonal antibody (mAb) targeted to cancer cells. In one embodiment, the linker to the anti-EGFR antibody is stable in extracellular fluids but is cleaved by cathepsin upon entry of the conjugate into tumor cells, activating a mitotic inhibitory mechanism.

[0194] The structures of MMAF and MMAE are shown below.

[0195] [ka]

[0196] Further, an example of AbA-vcMMAE is shown in Figure 11. In particular, Figure 11 illustrates a situation where an antibody (e.g., AbA) is linked to a single drug, thus resulting in a DAR of 1. In certain embodiments, the ADC has a DAR of 2-8, or alternatively, 2-4.

[0197] Other binding agents Examples of drugs that can be used in ADCs, i.e., drugs that can be conjugated to the anti-EGFR antibodies of the present invention, include antimitotic agents, antitumor antibiotics, immunomodulatory agents, gene therapy vectors, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemotherapeutic agents, hormones, glucocorticoids, photosensitive therapeutic agents, oligonucleotides, radioisotopes, radiosensitizers, topoisomerase inhibitors, tyrosine kinase inhibitors, and combinations thereof.

[0198] 1. Mitotic inhibitors In one aspect, anti-EGFR antibodies may be conjugated with one or more antimitotic agents to form ADCs for treating cancer. As used herein, the term "antimitotic agent" refers to a cytotoxic and / or therapeutic agent that blocks mitosis, or cell division, a biological process particularly important in cancer cells. Antimitotic agents disrupt microtubules to prevent cell division, often by acting on microtubule polymerization or microtubule depolymerization. Thus, in one embodiment, the anti-EGFR antibodies of the invention are conjugated with one or more antimitotic agents that disrupt microtubule formation by inhibiting tubulin polymerization. In one embodiment, the antimitotic agent used in the ADCs of the invention is Ixempra (ixabepilone). Mitotic inhibitors that can be used in the anti-EGFR ADCs of the invention are listed below. The auristatins mentioned above are also included in the category of antimitotic agents.

[0199] Dolastatin An anti-EGFR antibody of the invention can be conjugated to at least one dolastatin to form an ADC. Dolastatins are short peptide compounds isolated from the Indian Ocean sea hare Dolabella auricularia (see Pettit et al., J. Am. Chem. Soc., 1976, 98, 4677). Exemplary dolastatins include dolastatin 10 and dolastatin 15. Dolastatin 15 is a seven-subunit depsipeptide derived from Dolabella auricularia and is a potent mitotic inhibitor structurally related to dolastatin 10, a five-subunit peptide derived from the same organism. Thus, in one embodiment, an anti-EGFR ADC of the invention comprises an anti-EGFR antibody as described herein and at least one dolastatin. The auristatin is a synthetic derivative of dolastatin 10.

[0200] b. maytansinoid The anti-EGFR antibodies of the invention can be conjugated to at least one maytansinoid to form an ADC. Maytansinoids are potent antitumor agents originally isolated from members of the higher plant families Celastraceae, Rhamnaceae, and Euphorbiaceae, and from certain species of liverworts (Kupchan et al., J. Am. Chem. Soc. 94:1354-1356

[1972] ; Wani et al., J. Chem. Soc. Chem. Commun. 390:

[1973] ; Powell et al., J. Nat. Prod. 46:660-666

[1983] ; Sakai et al., J. Nat. Prod. 51:845-850

[1988] ; and Suwanborirux et al., Experientia 46:117-120

[1990] ). Maytansinoids have been suggested to inhibit mitosis by inhibiting the polymerization of the microtubule protein tubulin, thereby disrupting the formation of microtubules (see, e.g., U.S. Pat. No. 6,441,163 and Remillard et al., Science, 189, 1002-1005 (1975)). Maytansinoids have been shown to inhibit tumor cell growth in vitro using cell culture models and in vivo using experimental animal systems. Furthermore, the cytotoxicity of maytansinoids is 1,000 times greater than that of conventional chemotherapeutic agents such as methotrexate, daunorubicin, and vincristine (see, e.g., U.S. Pat. No. 5,208,020).

[0201] Maytansinoids include maytansine, maytansinol, C3 esters of maytansinol, and other maytansinol analogs and derivatives (see, e.g., U.S. Pat. Nos. 5,208,020 and 6,441,163, each incorporated herein by reference). C3 esters of maytansinol can be naturally occurring or synthetically derived. Furthermore, both natural and synthetic C3 maytansinol esters can be classified as C3 esters with simple carboxylic acids or C3 esters with derivatives of N-methyl-L-alanine, the latter being more cytotoxic than the former. Synthetic maytansinoid analogs are described, for example, in Kupchan et al., J. Med. Chem., 21, 31-37 (1978).

[0202] Maytansinoids suitable for use in the ADCs of the invention can be isolated from natural sources, synthetically produced, or semisynthetically produced. Furthermore, maytansinoids can be modified in any suitable manner, so long as sufficient cytotoxicity is maintained in the final conjugate molecule. In this regard, maytansinoids lack suitable functional groups to which antibodies can be linked. Linking moieties are preferably utilized to link maytansinoids to antibodies to form conjugates, as discussed in more detail in Section III.B. The structure of an example maytansinoid, mertansine (DM1), is shown below.

[0203] [ka]

[0204] Representative examples of maytansinoids include, but are not limited to, DM1(N 2 '-Deacetyl-N 2 '-(3-mercapto-1-oxopropyl)maytansine; also known as mertansine, maytansinoid drug 1; ImmunoGen, Inc.; see also Chari et al. (1992) Cancer Res 52:127.), DM2, DM3 (N 2 '-Deacetyl-N 2Maytansinol (a synthetic maytansinoid analog) is a synthetic maytansinoid analogue. Examples of maytansinoids include DM4 ((4-methyl-4-mercapto-1-oxopentyl)maytansine), DM5 ((4-methyl-4-mercapto-1-oxopentyl)maytansine), and DM6 ((4-methyl-4-mercapto-1-oxopentyl)maytansine). Other examples of maytansinoids are described in U.S. Pat. No. 8,142,784, which is incorporated herein by reference.

[0205] Ansamitocins are a group of maytansinoid antibiotics isolated from various bacterial sources. These compounds have potent antitumor activity. Representative examples include, but are not limited to, ansamitocin P1, ansamitocin P2, ansamitocin P3, and ansamitocin P4.

[0206] In one embodiment of the invention, the anti-EGFR antibody is conjugated to at least one DM1. In one embodiment, the EGFR antibody is conjugated to at least one DM2. In one embodiment, the anti-EGFR antibody is conjugated to at least one DM3. In one embodiment, the anti-EGFR antibody is conjugated to at least one DM4.

[0207] D. plant alkaloids The anti-EGFR antibodies of the invention may be conjugated to at least one plant alkaloid (e.g., a taxane or vinca alkaloid). Plant alkaloids are chemotherapeutic agents produced by certain plants. Vinca alkaloids are produced from the periwinkle (Catharanthus rosea) plant, and taxanes are produced from the bark of the Pacific yew (Taxus). Both vinca alkaloids and taxanes are also known as anti-microtubule agents and are described in more detail below.

[0208] Taxanes The anti-EGFR antibodies described herein may be conjugated to at least one taxane. As used herein, the term "taxane" refers to a class of antineoplastic agents that have a microtubule-active mechanism and a structure containing a taxane ring structure and a stereospecific side chain required for cytostatic activity. The term "taxane" also encompasses various known derivatives, including both hydrophilic and hydrophobic derivatives. Taxane derivatives include, but are not limited to, the galactose and mannose derivatives described in International Patent Application No. WO 99 / 18113; the piperazino and other derivatives described in WO 99 / 14209; the taxane derivatives described in WO 99 / 09021, WO 98 / 22451, and U.S. Pat. No. 5,869,680; the 6-thio derivatives described in WO 98 / 28288; the sulfenamide derivatives described in U.S. Pat. No. 5,821,263; and the taxol derivatives described in U.S. Pat. No. 5,415,869. Taxane compounds have previously been disclosed in U.S. Patent Nos. 5,641,803, 5,665,671, 5,380,751, 5,728,687, 5,415,869, 5,407,683, 5,399,363, 5,424,073, 5,157,049, 5,773,4 64, 5,821,263, 5,840,929, 4,814,470, 5,438,072, 5,403,858, 4,960,790, 5,433,364, 4,942,184, 5,362,831, 5,705,503, and 5,278,324. Other examples of taxanes include, but are not limited to, docetaxel (Taxotere; Sanofi Aventis), paclitaxel (Abraxane or Taxol; Abraxis Oncology), and nanoparticulate paclitaxel (ABI-007 / Abraxene; Abraxis Bioscience).

[0209] In one embodiment, an anti-EGFR antibody of the invention is conjugated to at least one docetaxel.In one embodiment, an anti-EGFR antibody of the invention is conjugated to at least one paclitaxel.

[0210] Vinca alkaloids In one embodiment, the anti-EGFR antibody is conjugated to at least one vinca alkaloid. Vinca alkaloids are a class of cell-cycle-specific drugs that act on tubulin to disrupt the formation of microtubules, thereby inhibiting the ability of cancer cells to divide. Examples of vinca alkaloids that can be used in the ADCs of the invention include, but are not limited to, vindesine sulfate, vincristine, vinblastine, and vinorelbine.

[0211] 2. Antitumor antibiotics The anti-EGFR antibodies of the present invention may be conjugated to one or more anti-tumor antibiotics for the treatment of cancer. As used herein, the term "antineoplastic antibiotic" refers to antineoplastic drugs produced by microorganisms that inhibit cell growth by interfering with DNA. In many cases, anti-tumor antibiotics break down DNA strands or slow or stop DNA synthesis. Examples of anti-tumor antibiotics that can be included in the anti-EGFR ADCs of the present invention include, but are not limited to, actinomycin (e.g., pyrrolo[2,1-c][1,4]benzodiazepine), anthracyclines, calicheamicin, and duocarmycin, and are described in more detail below.

[0212] Actinomycin The anti-EGFR antibodies of the present invention may be conjugated to at least one actinomycin. Actinomycins are a subclass of antitumor antibiotics isolated from the bacterium Streptomyces. Representative examples of actinomycins include, but are not limited to, actinomycin D (Cosmegen [also known as actinomycin, dactinomycin, actinomycin IV, actinomycin C1], Lundbeck, Inc.), anthramycin, ticamycin A, DC-81, mazethramycin, neothramycin A, neothramycin B, polothramycin, prothracarcin B, SG2285, sivanomycin, sibiromycin, and tomaymycin. In one embodiment, the anti-EGFR antibodies of the present invention are conjugated to at least one pyrrolobenzodiazepine (PBD). Examples of PBDs include, but are not limited to, anthramycin, ticamycin A, DC-81, mazethramycin, neothramycin A, neothramycin B, polothramycin, prothracarcin B, SG2000 (SJG-136), SG2202 (ZC-207), SG2285 (ZC-423), sivanomycin, sibiromycin, and tomaymycin. Thus, in one embodiment, the anti-EGFR antibody of the invention is conjugated to at least one actinomycin (e.g., actinomycin D) or at least one PBD (e.g., a pyrrolobenzodiazepine (PBD) dimer).

[0213] The structure of PBDs is described, for example, in U.S. Patent Application Publication Nos. 2013 / 0028917 and 2013 / 0028919, and WO2011 / 130598A1, the disclosures of each of which are incorporated herein by reference in their entirety. The general structure of a PBD is shown below:

[0214] [ka]

[0215] PBDs vary in the number, type, and position of substituents on both the aromatic ring A and the pyrrolo ring C, as well as the degree of saturation of the C ring. The B ring generally contains an imine (N=C), carbinolamine (NH-CH(OH)), or carbinolamine methyl ether (NH-CH(OMe)) at the N10-C11 position, which is the electrophilic center involved in DNA alkylation. All known natural products have an (S) configuration at the chiral C11α position, resulting in a right-twisted structure when viewed from the C ring toward the A ring. The PBDs exemplified herein can be conjugated to the anti-EGFR antibodies of the present invention. Other examples of PBDs that can be conjugated to the anti-EGFR antibodies of the invention are described, for example, in U.S. Patent Application Publication Nos. 2013 / 0028917A1 and 2013 / 0028919A1, U.S. Patent No. 7,741,319B2, and WO2011 / 130598A1 and WO2006 / 111759A1, the disclosures of each of which are incorporated herein by reference in their entirety.

[0216] A representative PBD dimer having Formula II below can be conjugated to an anti-EGFR antibody of the invention.

[0217] [ka] In the formula, R 2 is Formula III:

[0218] [ka] where A is C 5-7 is an aryl group, and X is —O—, —S—, —C(O)O—, —C(O)—, —NH(C═O)—, and —N(R N )-[where R N is H, C 1-4 Alkyl and (C2H4O) m and CH3, where m is 1 to 3. (i)Q 1 is a single bond and Q 2 is a single bond and -Z-(CH2) n- (wherein Z is selected from the group consisting of a single bond, O, S, and NH, and n is 1 to 3); or (ii) Q 1 is -CH=CH- and Q 2 is a single bond; R 12 are halo, nitro, cyano, C 1-12 Alkoxy, C 3-20 Heterocycloalkoxy, C 5-20 Aryloxy, heteroaryloxy, alkylalkoxy, arylalkoxy, alkylaryloxy, heteroarylalkoxy, alkylheteroaryloxy, C 1-7 Alkyl, C 3-7 Heterocyclyl and bisoxy-C 1-3 C optionally substituted with one or more substituents selected from the group consisting of alkylene 5-10 is an aryl group; R 6 and R 9 are independently selected from the group consisting of H, R, OH, OR, SH, SR, NH, NHR, NRR′, nitro, MeSn, and halo; wherein R and R′ are independently optionally substituted C 1-12 Alkyl group, C 3-20 Heterocyclyl groups and C 5-20 aryl groups; R 7 is selected from the group consisting of H, R, OH, OR, SH, SR, NH, NHR, NHRR', nitro, MeSn, and halo; (a)R 10 is H and R 11 OH, OR A where R A is C 1-4 is alkyl; or (b)R 10 and R 11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are attached; or (c)R 10 is H and R 11 SO zM, where z is 2 or 3; R 11 C may contain one or more heteroatoms in the chain selected from the group consisting of O, S, NH and aromatic rings. 3-12 is an alkylene group; Y and Y' are selected from the group consisting of O, S, and NH; R 6’ , R 7’ , R 9’ are R respectively 6 , R 7 and R 9 and R 10’ and R 11’ is R 10 and R 11 and each M is a monovalent pharmaceutically acceptable cation, or two M groups together are a divalent pharmaceutically acceptable cation.

[0219] As used herein, the term "optionally substituted" means that the parent group can be unsubstituted or substituted.

[0220] Unless otherwise specified, the term "substituted," as used herein, means that a parent group bears one or more substituents. The term "substituent" is used herein in its conventional sense to refer to a chemical moiety that is covalently bonded to, or optionally fused to, a parent group. A wide variety of substituents are well known, as are methods for their formation and introduction into various parent groups.

[0221] C 1-12 Alkyl: As used herein, "C 1-12 The term "alkyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 12 carbon atoms, and may be aliphatic or alicyclic, saturated or unsaturated (e.g., partially unsaturated or fully unsaturated). Thus, the term "alkyl" includes the subclasses alkenyl, alkynyl, cycloalkyl, etc., as described below.

[0222] Examples of saturated alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), propyl (C3), butyl (C4), pentyl (C5), hexyl (C6), and heptyl (C7).

[0223] Examples of saturated straight chain alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), n-butyl (C4), n-pentyl (amyl) (C5), n-hexyl (C6), and n-heptyl (C7).

[0224] Examples of saturated branched alkyl groups include isopropyl (C3), isobutyl (C4), sec-butyl (C4), tert-butyl (C4), isopentyl (C5), and neopentyl (C5).

[0225] C 3-20 Heterocyclyl: As used herein, "C 3-20 The term "heterocyclyl" means a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, said moiety having 3 to 20 ring atoms, of which 1 to 10 are ring heteroatoms. Preferably, each ring has 3 to 7 ring atoms, of which 1 to 4 are ring heteroatoms.

[0226] In this context, subscripts (e.g., C 3-20 , C 3-7 , C 5-6 etc.) represents the number of ring atoms or range of ring atoms, regardless of whether they are carbon atoms or heteroatoms. For example, "C" as used herein 5-6 The term "heterocyclyl" refers to a heterocyclyl group having 5 or 6 ring atoms.

[0227] N1: aziridine (C3), azetidine (C4), pyrrolidine (tetrahydropyrrole) (C5), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C5), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C5), piperidine (C6), dihydropyridine (C6), tetrahydropyridine (C6), azepine (C7); O1: oxirane (C3), oxe S1: thiirane (C3), thietane (C4), thiolane (tetrahydrothiophene) (C5), thiane (tetrahydrothiopyran) (C6), thiepane (C7); O2: dioxane O3: trioxane (C6); N2: imidazolidine (C5), pyrazolidine (diazolidine) (C5), imidazoline (C5), pyrazoline (dihydropyrazole) (C5), piperazine (C6); N1O1: tetrahydrooxazole (C5), dihydrooxazole (C5), tetrahydroisoxazole (C5), di ... N1S1: thiazoline (C5), thiazolidine (C5), thiomorpholine (C6); N2O1: oxadiazine (C6); O1S1: oxathiol (C5) and oxathiane (thioxane) (C6); and N1O1S1: oxathiazine (C6).

[0228] Examples of substituted monocyclic heterocyclyl groups include those derived from cyclic sugars, such as furanoses (C5) such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranose, and pyranoses (C6) such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.

[0229] C 5-20 Aryl: As used herein, "C 5-20The term "aryl" means a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has 3 to 20 ring atoms. Preferably, each ring has 5 to 7 ring atoms.

[0230] In this context, subscripts (e.g., C 3-20 , C 5-7 , C 5-6 etc.) represents the number of ring atoms or range of ring atoms, regardless of whether they are carbon atoms or heteroatoms. For example, "C" as used herein 5-6 The term "aryl" refers to an aryl group having 5 or 6 ring atoms.

[0231] In one embodiment, the anti-EGFR antibody of the invention has the formula:

[0232] [ka] The above structure represents the PBD dimer SG2202 (ZC-207), which is conjugated to the anti-EGFR antibody of the present invention via a linker L. SG2202 (ZC-207) is disclosed, for example, in U.S. Patent Application Publication No. 2007 / 0173497, the disclosure of which is incorporated herein by reference in its entirety.

[0233] In another embodiment, the PBD dimer SGD-1882 is conjugated to an anti-EGFR antibody of the invention via a drug linker, as shown in Figure 21. SGD-1882 is disclosed in Sutherland et al. (2013) Blood 122(8):1455 and U.S. Patent Application Publication No. 2013 / 0028919, the entire disclosures of which are incorporated herein by reference. As shown in Figure 21, the PBD dimer SGD-1882 can be conjugated to the antibody via a mc-val-ala-dipeptide linker (collectively referred to as SGD-1910 in Figure 21). In certain embodiments, an anti-EGFR antibody such as those disclosed herein is conjugated to a PBD dimer as shown in Figure 21. Accordingly, in another embodiment, the invention encompasses an anti-EGFR antibody such as those disclosed herein conjugated to a PBD dimer via a mc-val-ala-dipeptide linker, as shown in Figure 21. In certain embodiments, the invention encompasses anti-EGFR antibodies comprising a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 6, conjugated to a PBD, such as, but not limited to, the PBD dimer depicted in Figure 21. In certain embodiments, the invention encompasses anti-EGFR antibodies comprising a heavy chain variable region of AbA represented by the amino acid sequence set forth in SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5, conjugated to a PBD, such as, but not limited to, the exemplary PBD dimer depicted in Figure 21.

[0234] B anthracycline The anti-EGFR antibody of the present invention may be conjugated to at least one anthracycline. Anthracyclines are a subclass of antitumor antibiotics isolated from the bacterium Streptomyces. Representative examples include, but are not limited to, daunorubicin (Cerubidine, Bedford Laboratories), doxorubicin (Adriamycin, Bedford Laboratories; also known as doxorubicin hydrochloride, hydroxydaunorubicin, and Rubex), epirubicin (Ellence, Pfizer), and idarubicin (Idamycin; Pfizer Inc.). Thus, in one embodiment, the anti-EGFR antibody of the present invention is conjugated to at least one anthracycline (e.g., doxorubicin).

[0235] C. calicheamicin The anti-EGFR antibodies of the present invention may be conjugated to at least one calicheamicin. Calicheamicin is a family of enediyne antibiotics derived from the soil organism Micromonospora echinospora. Calicheamicin binds to the minor groove of DNA, causing double-stranded DNA breaks and resulting in cell death 100 times more potent than other chemotherapeutic agents (Damle et al. (2003) Curr Opin Pharmacol 3:386). The preparation of calicheamicins that can be used as drug conjugates in the present invention is described in U.S. Patent Nos. 5,712,374; 5,714,586; 5,739,116; 5,767,285; 5,770,701; 5,770,710; 5,773,001; and 5,877,296. Structural analogs of calicheamicin that can be used include, but are not limited to, γ1 I , α2 I , α3 I , N-acetyl-γ1 I , PSAG and θ I1 (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998) and U.S. Patent Nos. 5,712,374; 5,714,586; 5,739,116; 5,767,285; 5,770,701; 5,770,710; 5,773,001; and 5,877,296). Accordingly, in one embodiment, an anti-EGFR antibody of the invention is conjugated to at least one calicheamicin.

[0236] d. duocarmycin The anti-EGFR antibody of the present invention may be conjugated to at least one duocarmycin. Duocarmycins are a subclass of antitumor antibiotics isolated from the bacterium Streptomyces (see Nagamura and Saito (1998) Chemistry of Heterocyclic Compounds, Vol. 34, No. 12). Duocarmycins bind to the minor groove of DNA and alkylate the nucleobase adenine at the N3 position (Boger (1993) Pure and Appl Chem 65(6):1123; and Boger and Johnson (1995) PNAS USA 92:3642). Synthetic analogs of duocarmycin include, but are not limited to, adozelesin, bizeresin, and carzelesin. Thus, in one embodiment, the anti-EGFR antibody of the present invention is conjugated to at least one duocarmycin.

[0237] e. Other antitumor antibiotics In addition to the above, other antitumor antibiotics that can be used in the anti-EGFR ADCs of the present invention include bleomycin (Blenoxane, Bristol-Myers Squibb), mitomycin, and plicamycin (also known as mithramycin).

[0238] 3. Immunomodulators In one aspect, the anti-EGFR antibodies of the invention may be conjugated to at least one immunomodulator. As used herein, the term "immunomodulator" refers to a substance capable of stimulating or modulating an immune response. In one embodiment, the immunomodulator is an immunostimulatory agent that enhances a subject's immune response. In another embodiment, the immunomodulator is an immunosuppressant that prevents or suppresses a subject's immune response. The immunomodulator can modulate myeloid cells (monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) or lymphoid cells (T cells, B cells, and natural killer (NK) cells) and any of their differentiated cells. Representative examples include, but are not limited to, Bacillus Calmette-Guerin (BCG) and levamisole (Ergamisol). Other examples of immunomodulators that can be used in the ADCs of the invention include, but are not limited to, cancer vaccines, cytokines, and immunomodulatory gene therapy.

[0239] Cancer vaccine The anti-EGFR antibodies of the present invention may be conjugated to a cancer vaccine. As used herein, the term "cancer vaccine" refers to a composition (e.g., a tumor antigen or cytokine) that induces a tumor-specific immune response. By administering a cancer vaccine, or in the present case, by administering an ADC comprising an anti-EGFR antibody and a cancer vaccine, a response is elicited from the subject's own immune system. In a preferred embodiment, the immune response results in the eradication of tumor cells (e.g., primary or metastatic tumor cells) in the body. Cancer vaccines generally involve the administration of a specific antigen or group of antigens, for example, present on the surface of specific cancer cells or on the surface of a specific infectious agent known to promote cancer formation. In some embodiments, the use of cancer vaccines is prophylactic, while in other embodiments, the use is therapeutic. Non-limiting examples of cancer vaccines that can be used with the anti-EGFR ADCs of the invention include recombinant bivalent human papillomavirus (HPV) vaccine types 16 and 18 vaccine (Cervarix, GlaxoSmithKline), recombinant tetravalent human papillomavirus (HPV) types 6, 11, 16, and 18 vaccine (Gardasil, Merck & Company), and sipuleucel-T (Provenge, Dendreon). Thus, in one embodiment, the anti-EGFR antibodies of the invention are conjugated to at least one cancer vaccine that is an immunostimulatory or immunosuppressant.

[0240] B cytokines The anti-EGFR antibodies of the present invention may be conjugated to at least one cytokine. The term "cytokine" generally refers to a protein released by a cell population and acting on other cells as an intracellular mediator. Cytokines directly stimulate immune effector cells and stromal cells at the tumor site and enhance tumor cell recognition by cytotoxic effector cells (Lee and Margolin (2011) Cancers 3:3856). Numerous animal tumor model studies have demonstrated that cytokines have broad antitumor activity, leading to the application of numerous cytokine therapy approaches for cancer treatment (Lee and Margolin, supra). Recently, a number of cytokines, including GM-CSF, IL-7, IL-12, IL-15, IL-18, and IL-21, have entered clinical trials in patients with advanced cancer (Lee and Margolin, supra).

[0241] Examples of cytokines that can be used in the ADCs of the invention include, but are not limited to, parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatocyte growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor; Müllerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factors such as NGF; Examples of cytokines include platelet growth factors; transforming growth factors (TGFs); insulin-like growth factors I and II; erythropoietin (EPO); bone morphogenetic factors; interferons, such as interferons α, β, and γ; colony-stimulating factors (CSFs); granulocyte-macrophage CSF (GM-CSF); granulocyte-CSF (G-CSF); interleukins (ILs), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, and IL-12; tumor necrosis factors; and other polypeptide factors, including LIF and Kit ligand (KL). As used herein, the term cytokine encompasses proteins derived from natural sources or recombinant cell culture and biologically active equivalents of native-sequence cytokines. Accordingly, in one embodiment, the invention provides ADCs comprising an anti-EGFR antibody described herein and a cytokine.

[0242] c. Colony stimulating factor (CSF) The anti-EGFR antibodies of the invention may be conjugated to at least one colony-stimulating factor (CSF). Colony-stimulating factors (CSFs) are growth factors that encourage bone marrow to produce red blood cells. Because some cancer treatments (e.g., chemotherapy) affect white blood cells (which play a role in fighting infection), colony-stimulating factors can be administered to help maintain white blood cell levels and strengthen the immune system. Colony-stimulating factors may also be used after bone marrow transplantation to encourage the new bone marrow to begin producing white blood cells. Representative examples of CSFs that can be used in the anti-EGFR ADCs of the invention include, but are not limited to, erythropoietin (Epoetin), filgrastim (Neopogen (also known as granulocyte colony-stimulating factor (G-CSF)); Amgen, Inc.), sargramostim (Leukine (granulocyte-macrophage colony-stimulating factor and GM-CSF); Genzyme Corporation), promegapoietin, and oprelvekin (recombinant IL-11; Pfizer, Inc.). Accordingly, in one embodiment, the invention provides an ADC comprising an anti-EGFR antibody described herein and a CSF.

[0243] 4. Gene therapy The anti-EGFR antibodies of the invention may be conjugated to at least one nucleic acid (directly or indirectly via a carrier) for use in gene therapy. Gene therapy generally refers to the introduction of genetic material into cells to treat a disease with the genetic material. Gene therapy, as it relates to immunomodulators, is used to stimulate a subject's natural healing ability to suppress cancer cell growth or kill cancer cells. In one embodiment, the anti-EGFR ADCs of the invention comprise a nucleic acid encoding a functional therapeutic gene used to replace a mutated or otherwise impaired (e.g., truncated) gene associated with cancer. In another embodiment, the anti-EGFR ADCs of the invention comprise a nucleic acid encoding or otherwise enabling the production of a therapeutic protein for treating cancer. The nucleic acid encoding the therapeutic gene may be conjugated directly to the anti-EGFR antibody or may be conjugated to the anti-EGFR antibody via a carrier. Examples of carriers that can be used to deliver nucleic acids for gene therapy include, but are not limited to, viral vectors and liposomes.

[0244] 5. Alkylating agents The anti-EGFR antibodies of the invention may be conjugated to one or more alkylating agents. Alkylating agents are a class of anti-neoplastic compounds that attach alkyl groups to DNA. Examples of alkylating agents that can be used in the ADCs of the invention include, but are not limited to, alkylsulfonates, ethylenimines, methylamine derivatives, epoxides, nitrogen mustards, nitrosoureas, triazines, and hydrazines.

[0245] a. Alkyl sulfonate The anti-EGFR antibodies of the invention may be conjugated to at least one alkyl sulfonate. Alkyl sulfonates have the general formula: R-SO-OR 1 wherein R and R 1 is typically an alkyl or aryl group. Representative examples of alkyl sulfonates include, but are not limited to, busulfan (Myleran, GlaxoSmithKline; Busulfex IV, PDL BioPharma, Inc.).

[0246] b. Nitrogen mustard The anti-EGFR antibodies of the invention may be conjugated to at least one nitrogen mustard. Representative examples of this subclass of anti-cancer compounds include, but are not limited to, chlorambucil (Leukeran, GlaxoSmithKline), cyclophosphamide (Cytoxan, Bristol-Myers Squibb; Neosar, Pfizer, Inc.), estramustine (estramustine phosphate sodium or Estracyt, Pfizer, Inc.), ifosfamide (Ifex, Bristol-Myers Squibb), mechlorethamine (Mustargen, Lundbeck Inc.), and melphalan (Alkeran or L-Pam or phenylalanine mustard; GlaxoSmithKline).

[0247] C. nitrosourea The anti-EGFR antibodies of the present invention may be conjugated to at least one nitrosourea. Nitrosoureas are a subclass of lipid-soluble alkylating agents. Representative examples include, but are not limited to, carmustine (BCNU [also known as BiCNU, N,N-bis(2-chloroethyl)-N-nitrosourea or 1,3-bis(2-chloroethyl)-1-nitrosourea], Bristol-Myers Squibb), fotemustine (also known as Muphoran), lomustine (CCNU or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, Bristol-Myers Squibb), nimustine (also known as ACNU), and streptozocin (Zanosar, Teva Pharmaceuticals).

[0248] d. Triazines and hydrazines The anti-EGFR antibodies of the present invention may be conjugated to at least one triazine or hydrazine. Triazines and hydrazines are subclasses of nitrogen-containing alkylating agents. In certain embodiments, these compounds can spontaneously degrade or be metabolized to produce alkyldiazonium intermediates that facilitate the transfer of the alkyl group to nucleic acids, peptides, and / or polypeptides, resulting in mutagenic, carcinogenic, or cytotoxic effects. Representative examples include, but are not limited to, dacarbazine (DTIC-Dome, Bayer Healthcare Pharmaceuticals Inc.), procarbazine (Mutalane, Sigma-Tau Pharmaceuticals, Inc.), and temozolomide (Temodar, Schering Plough).

[0249] e. Other alkylating agents The anti-EGFR antibodies of the invention may be conjugated to at least one ethyleneimine, methylamine derivative, or epoxide. Ethylenimines are a subclass of alkylating agents that typically contain at least one aziridine ring. Epoxides are a subclass of alkylating agents characterized as cyclic ethers with only three ring atoms.

[0250] Representative examples of ethylenimines include, but are not limited to, thiotepa (Thioplex, Amgen), diaziquone (also known as aziridinylbenzoquinone (AZQ)), and mitomycin C. Mitomycin C is a natural product containing an aziridine ring and is thought to induce cytotoxicity by cross-linking with DNA (Dorr RT, et al. Cancer Res. 1985;45:3510; Kennedy KA, et al. Cancer Res. 1985;45:3541). Representative examples of methylamine derivatives and analogs thereof include, but are not limited to, altretamine (Hexalen, MGI Pharma, Inc.), also known as hexamethylamine, and hexastat. Representative examples of epoxides of this class of anticancer compounds include, but are not limited to, dianhydrogalactitol. Dianhydrogalactitol (1,2:5,6-dianhydrodulcitol) is chemically related to aziridine and generally promotes alkyl group migration by a similar mechanism. Dibromodulcitol is hydrolyzed to dianhydrogalactitol, and is therefore a prodrug of epoxides (Sellei C, et al. Cancer Chemother Rep. 1969;53:377).

[0251] 6. Anti-angiogenic drugs In one aspect, the anti-EGFR antibodies described herein are conjugated to at least one anti-angiogenic agent. Anti-angiogenic agents inhibit the growth of new blood vessels. Anti-angiogenic agents exert their effects in a variety of ways. In certain embodiments, these agents prevent growth factors from reaching their targets. For example, vascular endothelial growth factor (VEGF) is one of the key proteins involved in initiating angiogenesis by binding to specific receptors on the cell surface. Thus, certain anti-angiogenic agents that interfere with the interaction of VEGF with its corresponding receptor prevent VEGF from initiating angiogenesis. In other embodiments, these agents interfere with intracellular signaling cascades. For example, when specific receptors on the cell surface are triggered, a cascade of other chemical signals is initiated, promoting blood vessel growth. Thus, certain enzymes (e.g., certain tyrosine kinases) known to facilitate intracellular signaling cascades that contribute to cell proliferation are targets for cancer therapy. In other embodiments, these agents interfere with intercellular signaling cascades. In yet other embodiments, these agents disable specific targets that activate and promote cell proliferation or directly interfere with the growth of vascular cells. Over 300 substances have been discovered with anti-angiogenic properties, many with direct and indirect inhibitory effects.

[0252] Representative examples of anti-angiogenic agents that can be used in the ADCs of the invention include, but are not limited to, angiostatin, ABX EGF, C1-1033, PKI-166, EGF vaccine, EKB-569, GW2016, ICR-62, EMD55900, CP358, PD153035, AG1478, IMC-C225 (Erbitux), ZD1839 (Iressa), OSI-774, erlotinib (tarceva), angiostatin, arrestin, endostatin, BAY12-9566 alone and in combination with fluorouracil or doxorubicin, canstatin, carboxyamidotriozole alone and in combination with paclitaxel, EMD121974, S-24, vitaxin, dimethylxanthenone acetic acid, IM862, interleukin-12, interleukin-2, NM-3, HuMV833, PTK787, Rh uMab, Angiozyme (Ribozyme), IMC-1C11, Neovastat, Marimastat, Prinomastat, BMS-275291, COL-3, MM1270, SU101, SU6668, SU11248, SU5416, combination with paclitaxel, combination with gemcitabine, cisplatin alone and in combination with irinotecan, cisplatin alone and radiation, tecogalan, combination with temozolomide and PEG-interferon α2b, tetrathiomolybdate, TNP-470, thalidomide, CC-5013 alone and in combination with taxotere, tumstatin, 2-methoxyestradiol, VEGF trap, mTOR inhibitors (deforolimus, everolimus (Afinitor, Novartis) Pharmaceutical Corporation, and temsirolimus (Torisel, Pfizer, Inc.)), tyrosine kinase inhibitors (e.g., erlotinib (Tarceva, Genentech, Inc.), imatinib (Gleevec, Novartis Pharmaceutical Corporation), gefitinib (Iressa, AstraZeneca Pharmaceuticals), dasatinib (Sprycel, Brystol-Myers Squibb), sunitinib (Sutent, Pfizer, Inc.)), nilotinib (Tasigna, Novartis Pharmaceutical Corporation), lapatinib (Tykerb, GlaxoSmithKline Pharmaceuticals), sorafenib (Nexavar, Bayer and Onyx), and phosphoinositide 3-kinase (PI3K).

[0253] 7. Antimetabolites The anti-EGFR antibodies of the invention may be conjugated to at least one antimetabolite. Antimetabolites are a type of chemotherapy therapeutic agent that closely mimics normal substances within cells. When cells incorporate antimetabolites into their intracellular metabolism, the result is negative for the cell, for example, the cell is unable to divide. Antimetabolites are classified according to the substance they interfere with. Examples of antimetabolites that can be used in the ADCs of the invention include, but are not limited to, antifolates (e.g., methotrexate), pyrimidine antagonists (e.g., 5-fluorouracil, floxuridine, cytarabine, capecitabine, and gemcitabine), purine antagonists (e.g., 6-mercaptopurine and 6-thioguanine), and adenosine deaminase inhibitors (e.g., cladribine, fludarabine, nelarabine, and pentostatin), as described in more detail below.

[0254] Folate antagonists The anti-EGFR antibodies of the present invention may be conjugated to at least one antifolate. Antifolates are a subclass of antimetabolites that are structurally similar to folic acid. Representative examples include, but are not limited to, methotrexate, 4-aminofolic acid (also known as aminopterin and 4-aminopteroic acid), lometrexol (LMTX), pemetrexed (Alimpta, Eli Lilly and Company), and trimetrexate (Neutrexin, Ben Venue Laboratories, Inc.).

[0255] B. Purine antagonists The anti-EGFR antibodies of the invention may be conjugated to at least one purine antagonist. Purine analogs are a subclass of antimetabolites that are structurally similar to a group of compounds known as purines. Representative examples of purine antagonists include, but are not limited to, azathioprine (Azasan, Salix; Imuran, GlaxoSmithKline), cladribine (Leustatin [also known as 2-CdA], Janssen Biotech, Inc.), mercaptopurine (Purinethol [also known as 6-mercaptoethanol], GlaxoSmithKline), fludarabine (Fludara, Genzyme Corporation), pentostatin (Nipent [also known as 2'-deoxycoformycin (DCF)]), and 6-thioguanine (Lanvis [also known as thioguanine], GlaxoSmithKline).

[0256] C. pyrimidine antagonist The anti-EGFR antibodies of the invention may be conjugated to at least one pyrimidine antagonist. Pyrimidine antagonists are a subclass of antimetabolites that are structurally similar to a group of compounds known as pyrimidines. Representative examples of pyrimidine antagonists include, but are not limited to, azacitidine (Vidaza, Celgene Corporation), capecitabine (Xeloda, Roche Laboratories), cytarabine (also known as cytosine arabinoside and arabinosylcytosine, Bedford Laboratories), decitabine (Dacogen, Eisai Pharmaceuticals), 5-fluorouracil (Adrucil, Teva Pharmaceuticals; Efudex, Valeant Pharmaceuticals, Inc.), 5-fluoro-2'-deoxyuridine 5'-phosphate (FdUMP), 5-fluorouridine triphosphate, and gemcitabine (Gemzar, Eli Lilly and Company).

[0257] 8. Boron-containing agents The anti-EGFR antibodies of the present invention may be conjugated to at least one boron-containing agent. Boron-containing agents are a class of cancer therapeutic compounds that interfere with cell proliferation. Representative examples of boron-containing agents include, but are not limited to, borophycin and bortezomib (Velcade, Millenium Pharmaceuticals).

[0258] 9. Chemical protectants The anti-EGFR antibodies of the present invention may be conjugated to at least one chemoprotective agent. Chemoprotective agents are a class of compounds that help protect the body from certain toxic effects of chemotherapy. Chemoprotective agents can be administered with various chemotherapy agents to protect healthy cells from the toxic effects of the chemotherapy agent while allowing the administered chemotherapy agent to treat cancer cells. Representative chemoprotective agents include, but are not limited to, amifostine (Ethyol, Medimmune, Inc.), which is used to reduce nephrotoxicity associated with cumulative administration of cisplatin; dexrazoxane (Totect, Apricus Pharma; Zinecard), which is used to treat extravasation resulting from administration of anthracyclines (Totect) and cardiac complications resulting from administration of the antitumor antibiotic doxorubicin (Zinecard); and mesna (Mesnex, Bristol-Myers Squibb), which is used to prevent hemorrhagic cystitis during chemotherapy treatment with ifosfamide.

[0259] 10. Hormones The anti-EGFR antibodies of the present invention may be conjugated to at least one hormonal agent. Hormonal agents (including synthetic hormones) are compounds that interfere with the production or activity of endogenously produced hormones of the endocrine system. In certain embodiments, these compounds interfere with cell proliferation or produce cytotoxic effects. Non-limiting examples include androgens, estrogens, medroxyprogesterone acetate (Provera, Pfizer, Inc.), and progestins.

[0260] 11. Antihormonal agents The anti-EGFR antibodies of the invention may be conjugated to at least one antihormonal agent. An "antihormonal" agent is an agent that suppresses the production and / or interferes with the function of certain endogenous hormones. In one embodiment, the antihormonal agent interferes with the growth of various cancer cells by interfering with the activity of hormones selected from the group consisting of androgen, estrogen, progesterone, and gonadotropin-releasing hormone. Representative examples of antihormonal agents include, but are not limited to, aminoglutethimide, anastrozole (Arimidex, AstraZeneca Pharmaceuticals), bicalutamide (Casodex, AstraZeneca Pharmaceuticals), cyproterone acetate (Cyprostat, Bayer PLC), degarelix (Firmagon, Ferring Pharmaceuticals), exemestane (Aromasin, Pfizer Inc.), flutamide (Drogenil, Schering-Plough Ltd), fulvestrant (Faslodex, AstraZeneca Pharmaceuticals), goserelin (Zolodex, AstraZeneca Pharmaceuticals), letrozole (Femara, Novartis Pharmaceuticals Corporation), leuprolide (Prostap), Lupron, medroxyprogesterone acetate (Provera, Pfizer Inc.), megestrol acetate (Megace, Bristol-Myers Squibb Company), tamoxifen (Nolvadex, AstraZeneca Pharmaceuticals), and triptorelin (Decapetyl, Ferring).

[0261] 12. Corticosteroids The anti-EGFR antibodies of the invention may be conjugated to at least one corticosteroid. Corticosteroids can be used in the ADCs of the invention to reduce inflammation. Examples of corticosteroids include, but are not limited to, glucocorticoids such as prednisone (Deltasone, Pharmacia & Upjohn Company, a subsidiary of Pfizer, Inc.).

[0262] 13. Photosensitive therapeutic agents The anti-EGFR antibodies of the present invention may be conjugated to at least one photosensitive therapeutic agent. Photosensitive therapeutic agents include compounds that can be configured to kill administered cells by irradiating them with electromagnetic radiation of a specific wavelength. Therapeutic compounds absorb electromagnetic radiation of a wavelength that penetrates tissue. In preferred embodiments, the compounds are administered in a non-toxic form that can produce photochemical effects that are toxic to cells or tissues upon sufficient activation. In other preferred embodiments, these compounds are retained in cancerous tissue and readily excreted from normal tissue. Non-limiting examples include various pigments and dyes.

[0263] 14. Oligonucleotides The anti-EGFR antibodies of the present invention may be conjugated to at least one oligonucleotide. Oligonucleotides consist of short nucleic acid strands that act by interfering with the processing of genetic information. In certain embodiments, the oligonucleotides used in ADCs are unmodified single- and / or double-stranded DNA or RNA molecules; in other embodiments, these therapeutic oligonucleotides are chemically modified single- and / or double-stranded DNA or RNA molecules. In one embodiment, the oligonucleotides used in ADCs are relatively short (19-25 nucleotides) and hybridize to unique nucleic acid sequences across the entire pool of nucleic acid targets present in cells. Important oligonucleotide technologies include antisense oligonucleotides (including RNA interference (RNAi)), aptamers, CpG oligonucleotides, and ribozymes.

[0264] a. antisense oligonucleotide The anti-EGFR antibodies of the present invention may be conjugated to at least one antisense oligonucleotide. The antisense oligonucleotide is designed to bind to RNA by Watson-Crick hybridization. In some embodiments, the antisense oligonucleotide is complementary to a polynucleotide encoding a region, domain, portion, or segment of EGFR. In some embodiments, the antisense oligonucleotide comprises about 5 to about 100 nucleotides, about 10 to about 50 nucleotides, about 12 to about 35 nucleotides, or about 18 to about 25 nucleotides. In some embodiments, the oligonucleotide is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% homologous to a region, portion, domain, or segment of the EGFR gene. In some embodiments, substantial sequence homology exists over at least 15, 20, 25, 30, 35, 40, 50, or 100 contiguous nucleotides of the EGFR gene. In a preferred embodiment, these antisense oligonucleotides are 12 to 25 nucleotides in length, with the majority of antisense oligonucleotides being 18 to 21 nucleotides in length. There are several mechanisms that can be utilized to inhibit RNA function when an oligonucleotide binds to a target RNA (Crooke ST. (1999). Biochim. Biophys. Acta, 1489, 30-42). The most well-defined antisense mechanism involves cleavage of the target RNA by endogenous cellular nucleases, such as RNase H and nucleases associated with RNA interference mechanisms. Alternatively, oligonucleotides that inhibit target gene expression through non-catalytic mechanisms, such as modulation of splicing or translational arrest, can also be potent and selective modulators of gene function.

[0265] Another RNase-dependent antisense mechanism that has recently attracted much attention is RNAi (Fire et al. (1998). Nature, 391, 806-811; Zamore PD. (2002). Science, 296, 1265-1269). RNA interference (RNAi) is a post-translational process in which double-stranded RNA inhibits gene expression in a sequence-specific manner. In certain embodiments, RNAi is achieved by introducing relatively long double-stranded RNA (dsRNA), but in preferred embodiments, this RNAi is achieved by introducing short double-stranded RNA, such as short interfering RNA (siRNA) and / or microRNA (miRNA). In yet another embodiment, RNAi can be achieved by introducing a plasmid that produces dsRNA complementary to a target gene. In each of the above embodiments, the double-stranded RNA is designed to disrupt gene expression of a specific target sequence in a cell. In general, this mechanism regulates gene expression by converting dsRNA into short RNAs that guide ribonucleases to homologous mRNA targets (abstract, Ruvkun, Science 2294:797 (2001)), followed by degradation of the corresponding endogenous mRNA. In particular, dsRNA has been reported to have antiproliferative properties, making it potentially useful for therapeutic purposes (Aubel et al., Proc. Natl. Acad. Sci., USA 88:906 (1991)). For example, synthetic dsRNA has been shown to suppress tumor growth in mice (Levy et al. Proc. Nat. Acad. Sci. USA, 62:357-361 (1969)), is effective in treating leukemic mice (Zeleznick et al., Proc. Soc. Exp. Biol. Med. 130:126-128 (1969)), and inhibits chemically induced tumor formation in mouse skin (Gelboin et al., Science 167:205-207 (1970)). Thus, in a preferred embodiment, the present invention provides for the use of antisense oligonucleotides in ADCs for the treatment of breast cancer.In another embodiment, the present invention provides compositions and methods for disrupting target cell expression of EGFR at the mRNA level using dsRNA, as compositions and methods for initiating antisense oligonucleotide therapy. As used above, dsRNA refers to naturally occurring RNA, partially purified RNA, recombinantly produced RNA, synthetic RNA, and modified RNA that differs from naturally occurring RNA by including non-standard nucleotides, non-nucleotide materials, nucleotide analogs (e.g., locked nucleic acids (LNA)), deoxyribonucleotides, and any combination thereof. The RNA of the present invention need only be sufficiently similar to naturally occurring RNA that it can be mediated by the antisense oligonucleotides described herein.

[0266] B aptamer The anti-EGFR antibodies of the present invention may be conjugated to at least one aptamer. Aptamers are nucleic acid molecules selected from a random pool based on their ability to bind to other molecules. Like antibodies, aptamers can bind to target molecules with very strong affinity and specificity. In many embodiments, aptamers adopt complex, sequence-dependent three-dimensional structures that allow them to interact with target proteins, forming tightly bound complexes similar to antibody-antigen interactions and interfering with the function of the protein. Aptamers are particularly noteworthy for their ability to bind strongly and specifically to target proteins, making them suitable for use in molecular targeted therapy.

[0267] CpG oligonucleotides The anti-EGFR antibody of the present invention may be conjugated to at least one CpG oligonucleotide. Bacterial and viral DNA are known to be potent activators of both innate and specific immunity in humans. These immunological characteristics are believed to be related to unmethylated CpG dinucleotide motifs present in bacterial DNA. Because these motifs are rare in humans, the human immune system has evolved the ability to recognize these motifs as early signs of infection and subsequently initiate an immune response. Therefore, oligonucleotides containing this CpG motif can be used to initiate an anti-tumor immune response.

[0268] d. ribozyme The anti-EGFR antibody of the present invention may be conjugated to at least one ribozyme. Ribozymes are catalytic RNA molecules approximately 40 to 155 nucleotides in length. Ribozymes are capable of recognizing and cleaving specific RNA molecules, making them promising candidates for therapeutic drugs. A representative example is angiozyme.

[0269] 15. Radionuclide drugs (radioisotopes) The anti-EGFR antibody of the present invention may be conjugated to at least one radionuclide agent. A radionuclide agent comprises a moiety characterized by an unstable nucleus capable of radioactive decay. The success of radionuclide therapy is based on the radionuclide being present in the cancer cells at a sufficient concentration and for a long time to persist in the cancer cells. Other factors to consider include the half-life of the radionuclide, the energy of the emitted particles, and the maximum range of the emitted particles. In a preferred embodiment, the therapeutic agent is 111 In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, I25 I, I31 I, 32 P, 33 P, 47 Sc, 111 Ag, 67 Ga, 142Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, I09 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au and 211 The radionuclides are preferably selected from the group consisting of Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, I-125, Ho-161, Os-189m, and Ir-192. The decay energies of useful beta particle-emitting nuclides are preferably Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213, and Fm-255. Useful alpha particle-emitting radionuclides preferably have decay energies of 2,000 to 10,000 keV, more preferably 3,000 to 8,000 keV, and most preferably 4,000 to 7,000 keV. Other potentially usable radioisotopes include: 11 C. 13 N, 15 O. 75 Br, 198 Au, 224 Ac, 126 I, 133 I, 77 Br, 113m In, 95 Ru, 97 Ru, I03 Ru, 105 Ru, 107 Hg,203 Hg, 121m Te, 122m Te, 125m Te, 165 Tm, I67 Tm, 168 Tm, 197 Pt, 109 Pd, 105 Rh, 142 Pr, 143 Pr, 161 Tb, !66 Ho, 199 Au, 57 Co, 58 Co, 51 Cr, 59 Fe, 75 Se, 201 Tl, 225 Ac, 76 Br, I69 Examples include Yb.

[0270] 16.Radiosensitizers The anti-EGFR antibodies of the present invention may be conjugated to at least one radiosensitizer. As used herein, the term "radiosensitizer" is defined as a molecule, preferably a low-molecular-weight molecule, administered to an animal in a therapeutically effective amount to enhance the electromagnetic radiation sensitivity of cells to which the radiosensitizer is administered and / or to promote the treatment of diseases treatable with electromagnetic radiation. Radiosensitizers are drugs that increase the sensitivity of cancer cells to radiation treatment while generally having minimal effect on normal cells. Thus, radiosensitizers can be used in combination with radiolabeled antibodies or ADCs. The addition of a radiosensitizer can enhance efficacy compared to the administration of a radiolabeled antibody or antibody fragment alone. Radiosensitizers are described in D.M. Goldberg (ed.), Cancer Therapy with Radiolabeled Antibodies, CRC Press (1995). Examples of radiosensitizers include gemcitabine, 5-fluorouracil, taxanes, and cisplatin.

[0271] Radiosensitizers can be activated by electromagnetic radiation such as X-rays. Representative examples of X-ray activated radiosensitizers include, but are not limited to, metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C, RSU1069, SR4233, E09, RB6145, nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cisplatin, and their therapeutically active analogs and derivatives. Alternatively, photodynamic therapy (PDT) can be used to activate the radiosensitizer. Representative examples of radiosensitizers for photodynamic therapy include, but are not limited to, hematoporphyrin derivatives, Photofrin®, benzoporphyrin derivatives, NPe6, tin etioporphyrin (SnET2), pheophorbide a, bacteriochlorophyll a, naphthalocyanines, phthalocyanines, zinc phthalocyanines, and analogs and derivatives thereof that are effective as therapeutic agents.

[0272] 17. Topoisomerase inhibitors The anti-EGFR antibodies of the present invention may be conjugated to at least one topoisomerase inhibitor. Topoisomerase inhibitors are chemotherapeutic agents designed to interfere with the action of topoisomerase enzymes (topoisomerase I and II), which control changes in DNA structure by catalyzing the cleavage and rejoining of the phosphodiester backbone of DNA strands during the normal cell cycle. Representative examples of DNA topoisomerase I inhibitors include, but are not limited to, camptothecin and its derivatives irinotecan (CPT-11, Camptosar, Pfizer, Inc.) and topotecan (Hycamtin, GlaxoSmithKline Pharmaceuticals). Representative examples of DNA topoisomerase II inhibitors include, but are not limited to, amsacrine, daunorubicin, doxorubicin, epipodophyllotoxin, ellipticine, epirubicin, etoposide, razoxane, and teniposide.

[0273] 18. Tyrosine kinase inhibitors The anti-EGFR antibodies of the present invention may be conjugated to at least one tyrosine kinase inhibitor. Tyrosine kinases are intracellular enzymes that function to attach phosphate groups to the amino acid tyrosine. Preventing protein tyrosine kinases from functioning can suppress tumor growth. Examples of tyrosine kinases that can be used in the ADCs of the present invention include, but are not limited to, axitinib, bosutinib, cediranib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, lestaurtinib, nilotinib, semaxanib, sunitinib, and vandetanib.

[0274] 19. Other drugs Examples of other agents that can be used in the ADCs of the invention include, but are not limited to, abrin (e.g., abrin A chain), alpha toxin, Aleurites fordii proteins, amatoxin, crotin, curcin, dianthin proteins, diphtheria toxin (e.g., diphtheria A chain and non-binding active fragments of diphtheria toxin), deoxyribonuclease (Dnase), gelonin, mitogelin, modeccin A chain, momordica charantia inhibitor, neomycin, onconase, phenomycin, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), pokeweed antiviral protein, Pseudomonas endotoxin, Pseudomonas exotoxin (e.g., Pseudomonas aeruginosa), aeruginosa exotoxin A chain), restrictocin, ricin A chain, ribonuclease (RNase), soapwort (sapaonaria officinalis) inhibitor, saporin, alpha-sarcin, Staphylococcal enterotoxin A, tetanus toxin, cisplatin, carboplatin, and oxaliplatin (Eloxatin, Sanofi Aventis), proteasome inhibitors (e.g., PS-341 [bortezomib or Velcade]), HDAC inhibitors (vorinostat (Zolinza, Merck & Company, Inc.), belinostat, entinostat, mocetinostat, and panobinostat), COX-2 inhibitors, substituted ureas, heat shock protein inhibitors (e.g., geldanamycin and its many analogs), adrenocortical suppressants, and trichothecenes (see, e.g., WO 93 / 21232). Other medications include asparaginase (Espar, Lundbeck Inc.), hydroxyurea, levamisole, mitotane (Lysodren, Bristol-Myers Squibb), and tretinoin (Renova, Valeant Pharmaceuticals Inc.).

[0275] The drug moieties listed above as groups that can be used in the anti-EGFR ADCs of the invention are not limiting in that the exemplified drugs may fall into more than one category; for example, ansamitocin is both an antimitotic and an antitumor antibiotic.

[0276] All stereoisomers of the drug moiety, ie, any combination of R and S configurations at the chiral carbon of D, are included in the compounds of the invention.

[0277] The anti-EGFR antibodies described herein and the above-described agents (i.e., the agent not bound to the antibody) may be used in combination therapy. In one embodiment, an anti-EGFR antibody or ADC is used in combination therapy with one of the above-described agents to treat cancer, where the agent is administered to a subject before, simultaneously with, or after administration of the anti-EGFR antibody or ADC.

[0278] B. Anti-EGFR ADC: Linker Examples Anti-EGFR ADCs comprise an anti-EGFR antibody and at least one drug, with the antibody and at least one drug being linked by a linker. As used herein, the term "linker" refers to a chemical moiety, which may be bivalent, trivalent, or higher, used to attach an antibody to a drug moiety. The linker may contain a single linking component or multiple components.

[0279] For example, the linker can include a spacer, which is a moiety that extends the drug linkage, e.g., to avoid blocking the active site of the antibody or to improve the solubility of the ADC. Other examples of linker components include stretcher units and amino acid units.

[0280] Two methods are widely used to conjugate drugs to antibodies: reductive alkylation of interchain cysteine ​​disulfides via enzyme-noncleavable maleimide linkers or simple cleavable disulfide linkers, and acylation of lysines with cleavable linear amino acids.

[0281] In one embodiment, a linker covalently attaches an antibody to a drug moiety. ADCs are prepared using a linker with a reactive functional group for conjugating an antibody and a drug. For example, a cysteine ​​thiol or amine (e.g., the N-terminus or an amino acid side chain such as lysine) of an antibody can form a bond with a functional group on a linker.

[0282] In one embodiment, the linker has a functional group capable of reacting with a free cysteine ​​present on the antibody to form a covalent bond. Non-limiting examples of such reactive functional groups include activated esters such as maleimides, haloacetamides, α-haloacetyls, succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, acid anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. See, for example, Klussman, et al. (2004), Bioconjugate Chemistry 15(4):765-773, page 766 for conjugation methods.

[0283] In certain embodiments, a linker has a functional group capable of reacting with an electrophilic group present on an antibody. Examples of such electrophilic groups include, but are not limited to, aldehyde groups and ketone carbonyl groups. In certain embodiments, the heteroatom of the reactive functional group of the linker can react with an electrophilic group on an antibody to form a covalent bond with an antibody unit. Non-limiting examples of such reactive functional groups include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0284] Examples of linker components include 6-maleimidocaproyl, maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" to "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), and 4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("MCC").

[0285] In one embodiment, an anti-EGFR antibody is conjugated to an auristatin (e.g., MMAE) via a linker comprising maleimidocaproyl ("mc"), valine-citrulline (val-cit or "vc"), and PABA (also known as an "mc-vc-PABA linker"). Maleimidocaproyl functions as a linker to the anti-EGFR antibody and is non-cleavable. Val-cit is a dipeptide that is the amino acid unit of the linker, allowing proteases, particularly the protease cathepsin B, to cleave the linker. Thus, the val-cit component of the linker provides a means for releasing the auristatin from the ADC upon exposure to the intracellular environment. Within the linker, p-aminobenzyl alcohol (PABA) functions as a spacer and is self-immolative, thereby allowing for the release of MMAE. The structure of the mc-vc-PABA-MMAE linker is shown in Figure 11.

[0286] Suitable linkers include, for example, cleavable linkers and non-cleavable linkers. The linker can be a "cleavable linker" that facilitates drug release. Non-limiting examples of cleavable linkers include acid-sensitive linkers (e.g., containing hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photosensitive linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020). Cleavable linkers are generally susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include peptide linkers that are cleavable by intracellular proteases, such as lysosomal or endosomal proteases. In an exemplary embodiment, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit) or phenylalanine-lysine (phe-lys) linker.

[0287] Preferably, the linker is sufficiently stable extracellularly to be therapeutically effective. Prior to intracellular transport or delivery, the ADC preferably remains stable and intact, i.e., the antibody remains attached to the drug moiety. A linker that is stable outside the target cell can be cleaved at a predetermined, effective rate once inside the cell. Thus, an effective linker (i) maintains the specific binding of the antibody; (ii) enables delivery, e.g., intracellular delivery, of the drug moiety; and (iii) maintains the therapeutic effect, e.g., cytotoxic effect, of the drug moiety.

[0288] In one embodiment, the linker is cleavable under intracellular conditions such that cleavage of the linker releases the drug from the antibody into the intracellular environment sufficiently to be therapeutically effective. In some embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a certain pH value. Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, acid-sensitive linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in the lysosome can be used (see, e.g., U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as those found in blood, but are unstable below pH 5.5 or 5.0, the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to a therapeutic agent via an acylhydrazone bond) (see, e.g., U.S. Pat. No. 5,622,929).

[0289] In other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene), SPDB, and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel ed., Oxford U.S. Press, 1987). See also U.S. Pat. No. 4,880,935.)

[0290] In some embodiments, the linker is cleavable by a cleaving agent (e.g., an enzyme) present in the intracellular environment (e.g., within a lysosome, endosome, or caveolae). The linker is, for example, a peptide linker cleavable by an intracellular peptidase or protease enzyme (including, but not limited to, a lysosomal or endosomal protease). In some embodiments, the peptide linker is at least two amino acids in length or at least three amino acids in length. Cleaving agents include cathepsins B and D and plasmin, both of which are known to hydrolyze dipeptide drug derivatives to release the active drug inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Peptide linkers cleavable by enzymes present in cells expressing EGFR are most common. Examples of such linkers are described, for example, in U.S. Patent No. 6,214,345, the entire disclosure of which is incorporated herein by reference for all purposes. In a specific embodiment, the intracellular protease-cleavable peptide linker is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with a val-cit linker.) One advantage of using intracellular proteolytic release of a therapeutic agent is that the conjugation of the therapeutic agent generally results in lower concentrations, and the serum stability of the conjugate is generally high.

[0291] In other embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0292] In yet other embodiments, the linker unit is non-cleavable, e.g., antibody degradation releases the drug. See U.S. Publication No. 20050238649, the entire disclosure of which is incorporated herein by reference. ADCs containing non-cleavable linkers can be designed so that the ADC remains substantially outside the cell, interacts with selected receptors on the surface of target cells, and initiates (or prevents) specific intracellular signaling pathways upon ADC binding.

[0293] In certain embodiments, the linker is a substantially hydrophilic linker (e.g., PEG4Mal and sulfo-SPDB), which can be used to reduce the extent to which the drug is extruded from resistant cancer cells by MDR (multidrug resistance) or functionally similar transporters.

[0294] In other embodiments, the linker, upon cleavage, functions to directly or indirectly inhibit cell growth and / or cell proliferation. For example, in certain embodiments, the linker, upon cleavage, can function as an intercalating agent, inhibiting macromolecular biosynthesis (e.g., DNA replication, RNA transcription, and / or protein synthesis).

[0295] In other embodiments, the linker is designed to facilitate bystander killing (killing of neighboring cells) through diffusion of the linker-drug and / or drug alone to neighboring cells. In other embodiments, the linker promotes cellular internalization.

[0296] The presence of a sterically hindered disulfide can increase the stability of a particular disulfide bond and enhance the potency of an ADC. Thus, in one embodiment, the linker comprises a sterically hindered disulfide bond. A sterically hindered disulfide refers to a disulfide bond that exists within a particular molecular environment, typically characterized by atoms within the same molecule or compound being arranged in a particular spatial configuration or orientation that prevents or at least partially inhibits reduction of the disulfide bond. Thus, the presence of bulky (or sterically hindering) chemical moieties and / or bulky amino acid side chains in close proximity to a disulfide bond prevents or at least partially inhibits participation of the disulfide bond in potential interactions that lead to disulfide bond reduction.

[0297] Notably, the above linker types are not mutually exclusive. For example, in one embodiment, the linker used in the anti-EGFR ADCs described herein is a non-cleavable linker that facilitates cellular internalization.

[0298] In certain embodiments, the ADC has the following formula (Formula I): Ab-(LD) n (I) or a pharmaceutically acceptable salt or solvate thereof, wherein Ab is an antibody (e.g., anti-EGFR antibody AbA), and (LD) is a linker-drug moiety. The linker-drug moiety is composed of a linker, L-, and a drug moiety, -D, that has, for example, a cytostatic, cytotoxic, or other therapeutic effect on target cells (e.g., cells expressing EGFR), and n is an integer from 1 to 20.

[0299] In certain embodiments, n is 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.

[0300] In certain embodiments, the -D moieties are the same. In yet other embodiments, the -D moieties are different from one another.

[0301] As noted above, the linker may be a single moiety or may include two or more components. Thus, in certain embodiments, the ADC has the following formula (II): Ab-(A a -W w -Y y -D) n (II) or a pharmaceutically acceptable salt or solvate thereof, wherein Ab is an antibody (e.g., an anti-EGFR antibody AbA), and -A a -W w -Y y - is a linker (L) comprising three or more components; -A- is an optional Stretcher unit; a is 0 or 1; each -W- is independently an amino acid unit (or a glucuronide unit in certain embodiments; see also U.S. Publication No. 2012 / 0107332A1); w is an integer from 0 to 12; -Y- is a self-immolative spacer unit; y is 0, 1, or 2; -D is a drug moiety that has, for example, a cytostatic, cytotoxic, or other therapeutic activity against a target cell (e.g., a cell expressing EGFR); and n is an integer from 1 to 20.

[0302] In certain embodiments, the linker component comprises a "stretcher unit" (A) that links the antibody to another linker component or to a drug moiety. Non-limiting examples of stretcher units are shown below (where the wavy line indicates the site of covalent attachment to an antibody, drug, or other linker component):

[0303] [ka]

[0304] When the Stretcher unit (A) is present, the antibody can be linked to the amino acid unit (-W-) (if present), the spacer unit (-Y-) (if present), or the drug (-D) (see Formula II). Useful functional groups that can be present naturally or by chemical manipulation on the anti-EGFR antibodies described herein include, but are not limited to, sulfhydryl groups, amino groups, hydroxyl groups, anomeric hydroxyl groups of carbohydrate chains, and carboxyl groups. Suitable functional groups are sulfhydryl groups and amino groups. In one example, sulfhydryl groups can be generated by reduction of intramolecular disulfide bonds in the anti-EGFR antibody. In another embodiment, sulfhydryl groups can be generated by reacting amino groups of lysine moieties in the anti-EGFR antibody with 2-iminothiolane (Traut's reagent) or other sulfhydryl group-generating reagents. In certain embodiments, the anti-EGFR antibody is a recombinant antibody and is engineered to have one or more lysine moieties. In certain other embodiments, the recombinant anti-EGFR antibody is engineered to have an additional sulfhydryl group (eg, an additional cysteine).

[0305] In one embodiment, the Stretcher unit forms a bond with a sulfur atom of the antibody. The sulfur atom can be derived from a sulfhydryl group of the antibody. Exemplary Stretcher units of this embodiment are shown within the brackets in Formulae IIIa and IIIb, as follows:

[0306] [ka] In the formula, L-, -W-, -Y-, -D, w, and y are as defined above, and R 17 Ha-C1-C 10 Alkylene-, -C1-C 10 Alkenylene-, -C1-C 10 Alkynylene-, carbocyclo-, -O-(C1-C8 alkylene)-, O-(C1-C8 alkenylene)-, -O-(C1-C8 alkynylene)-, -arylene-, -C1-C 10 Alkylene-arylene-, -C2-C 10 Alkenylene-arylene, -C2-C10 Alkynylene-arylene, arylene-C1-C 10 Alkylene-, -arylene-C2-C 10 Alkenylene, -arylene-C2-C 10 Alkynylene-, -C1-C 10 Alkylene-(carbocyclo)-, -C2-C 10 Alkenylene-(carbocyclo)-, C2-C 10 Alkynylene-(carbocyclo)-, -(carbocyclo)-C1-C 10 Alkylene-, -(carbocyclo)-C2-C 10 Alkenylene-, -(carbocyclo)-C2-C 10 Alkynylene, -heterocyclo-, -C1-C 10 Alkylene-(heterocyclo)-, -C2-C 10 Alkenylene-(heterocyclo)-, -C2-C 10 Alkynylene-(heterocyclo)-, -(heterocyclo)-C1-C 10 Alkylene-, -(heterocyclo)-C2-C 10 Alkenylene-, -(heterocyclo)-C1-C 10 Alkynylene-, -(CH2CH2O) r -or-(CH2CH2O) r and —CH—, where r is an integer from 1 to 10, and the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocyclic, carbocyclo, heterocyclo, and arylene groups, alone or as part of another group, are optionally substituted. In certain embodiments, the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocyclic, carbocyclo, heterocyclo, and arylene groups, alone or as part of another group, are unsubstituted. In certain embodiments, the group R 17 Ha-C1-C 10 Alkylene, -carbocyclo, -O-(C1-C8 alkylene), -arylene, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C10 Alkylene-(carbocyclo)-, -(carbocyclo)-C1-C 10 Alkylene, -C3-C8 heterocyclo, -C1-C 10 Alkylene-(heterocyclo)-, -(heterocyclo)-C1-C 10 Alkylene-, -(CH2CH2O) r and -(CH2CH2O)-CH2-, where r is an integer from 1 to 10, and the alkylene group is unsubstituted and the other groups are optionally substituted.

[0307] An example of a stretcher unit is shown below: 17 is a unit of formula IIIa where is —(CH 2 ) 5 — (see also US 8,309,093).

[0308] Another example of a Stretcher unit is shown below in Formula IIIa: 17 -(CH2CH2O) r -CH2- and r is 2 (see also US 8,309,093, incorporated herein by reference).

[0309] [ka]

[0310] Another example of a Stretcher unit is in Formula IIIa, R 17 is arylene- or arylene-C1-C 10 In certain embodiments, the aryl group is an unsubstituted phenyl group. Further, another example of a Stretcher unit is shown below in Formula IIIb, where R 17 is —(CH 2 ) 5 — (see also US 8,309,093, which is incorporated herein by reference).

[0311] [ka]

[0312] In certain embodiments, the Stretcher unit is linked to the anti-EGFR antibody via a disulfide bond between a sulfur atom of the anti-EGFR antibody unit and a sulfur atom of the Stretcher unit. An exemplary Stretcher unit of this embodiment is shown inside brackets in Formula IV (see below; see also U.S. Pat. No. 8,309,093, incorporated herein by reference), where R 17 , L-, -W-, -Y-, -D, w and y are as defined above.

[0313] [ka]

[0314] Unless otherwise specified by the context, the S moiety in the formula below (see also US Pat. No. 8,309,093, which is incorporated herein by reference) represents a sulfur atom in the antibody.

[0315] [ka]

[0316] In yet other embodiments, the Stretcher comprises a reactive site capable of forming a bond with a primary or secondary amino group of an antibody. Examples of these reactive sites include, but are not limited to, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, acid anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. Exemplary Stretcher units of this embodiment are shown within brackets in Formulas Va and Vb (see below (see also U.S. Pat. No. 8,309,093, incorporated herein by reference)), where R 17 , L-, -W-, -Y-, -D, w and y are as defined above.

[0317] [ka]

[0318] In certain embodiments, the Stretcher comprises a reactive site reactive to a (-CHO) group of a modified sugar chain that may be present on an antibody. For example, the sugar chain can be mildly oxidized using a reagent such as sodium periodate, and the resulting (-CHO) unit of the oxidized sugar chain can be condensed with a Stretcher containing a functional group such as a hydrazide, oxime, primary or secondary amine, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide, such as those described in Kaneko et al., 1991, Bioconjugate Chem. 2:133-41. Exemplary Stretcher units of this embodiment are shown within brackets in Formulae VIa, VIb, and VIc (see below (see also U.S. Pat. No. 8,309,093, incorporated herein by reference)), where R 17 , L-, -W-, -Y-, -D, w and y are as defined above.

[0319] [ka]

[0320] In certain embodiments, the linker component comprises an "amino acid unit" (W). In certain such embodiments, the amino acid unit allows for protease cleavage of the linker, facilitating release of the drug from the immunoconjugate upon exposure to intracellular proteases, such as lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21:778-784). Examples of amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Examples of dipeptides include, but are not limited to, valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe), phenylalanine-lysine (fk or phe-lys), phenylalanine-homolysine (phe-homolys), and N-methyl-valine-citrulline (Me-val-cit). Examples of tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). The amino acid unit can include amino acid residues that are naturally occurring amino acids and / or minor amino acids and / or non-naturally occurring amino acid analogs, such as citrulline. The amino acid unit can be designed and optimized for enzymatic cleavage by specific enzymes (e.g., tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases).

[0321] In one embodiment, the amino acid unit W is valine-citrulline (vc or val-cit). In another embodiment, the amino acid unit is phenylalanine-lysine (i.e., fk). In yet another embodiment of the amino acid unit, the amino acid unit is N-methyl-valine-citrulline. In yet another embodiment, the amino acid unit is 5-aminovaleric acid, homophenylalanine-lysine, tetraisoquinolinecarboxylate-lysine, cyclohexylalanine-lysine, isonipecotic acid-lysine, β-alanine-lysine, glycine-serine-valine-glutamine, and isonipecotic acid.

[0322] Alternatively, in certain embodiments, -W- is a glucuronide unit that links the Stretcher unit to the Spacer unit, if present, the Stretcher unit to the Drug moiety, if the Spacer unit is absent, or the Linker unit to the Drug, if the Stretcher unit and Spacer unit are absent. The glucuronide unit comprises a site that can be cleaved by a β-glucuronidase enzyme (see also US 2012 / 0107332, incorporated herein by reference). In certain embodiments, the glucuronide unit comprises a sugar moiety (Su) linked to a self-immolative group (Z) via a glycosidic bond (-O'-) of the formula: (see also US 2012 / 0107332, incorporated herein by reference).

[0323] [ka]

[0324] The glycosidic bond (-O'-) is typically a β-glucuronidase cleavage site, such as a bond cleavable by human lysosomal β-glucuronidase. The term "self-immolative group," in reference to a glucuronide unit, refers to a bifunctional or trifunctional chemical moiety that is capable of covalently linking two or three separate chemical moieties (i.e., a sugar moiety (via a glycosidic bond), a drug moiety (directly or indirectly via a Spacer unit), and, in certain embodiments, a linker (directly or indirectly via a Stretcher unit)) into a stable molecule. A self-immolative group spontaneously separates from the first chemical moiety (e.g., a Spacer unit or a Drug unit) upon cleavage of the bond to the sugar moiety.

[0325] In certain embodiments, the sugar moiety (Su) is a cyclic hexose such as a pyranose or a cyclic pentose such as a furanose. In certain embodiments, the pyranose is a glucuronide or a hexose. The sugar moiety is typically in the β-D configuration. In one particular embodiment, the pyranose is a β-D-glucuronide moiety (i.e., β-D-glucuronic acid linked to a self-immolative group -Z- via a glycosidic bond cleavable by β-glucuronidase). In certain embodiments, the sugar moiety is unsubstituted (e.g., a natural cyclic hexose or cyclic pentose). In other embodiments, the sugar moiety can be a substituted β-D-glucuronide (i.e., glucuronic acid substituted with one or more groups such as hydrogen, hydroxyl, halogen, sulfur, nitrogen, or lower alkyl).

[0326] In certain embodiments, the glucuronide unit has one of the following formulas (see also US 2012 / 0107332, incorporated herein by reference):

[0327] [ka] wherein Su is a sugar moiety, the glycosidic bond includes an oxygen bond between Su and the self-immolative group Z, and each R is independently hydrogen, halo (e.g., chloro, bromo, fluoro, etc.), -CN, -NO, or other electron-withdrawing or electron-donating group, provided that the glucuronide unit (particularly Z) self-immolates upon cleavage of the glycosidic bond. In certain embodiments, each R is independently hydrogen, halo (e.g., chloro, bromo, fluoro, etc.), -CN, or -NO.

[0328] In certain embodiments, the glucuronide unit has one of the following formulas (see also US 2012 / 0107332, incorporated herein by reference):

[0329] [ka] wherein Su is a sugar moiety, the glycosidic bond (—O′—) comprises an oxygen bond between Su and the self-immolative group Z, and each R is independently hydrogen.

[0330] In certain embodiments, the self-immolative group (Z) is covalently attached to the sugar moiety, the drug (directly or indirectly through a Spacer unit), and the linker (directly or indirectly through a Stretcher unit). In certain embodiments, the drug-linker conjugate has the formula: (See also US 2012 / 0107332, incorporated herein by reference):

[0331] [ka] wherein Su, O', Z, Y, y, D, A, and a are as defined herein. Typically, 1 to 20 such drug-linker conjugates can be linked to a linker.

[0332] In certain embodiments, the ADC comprising the glucuronide unit has one of the following formulas (see also US 2012 / 0107332, incorporated herein by reference), where S, Y, y, D, A, a, and L are as described herein.

[0333] [ka]

[0334] In certain embodiments, the ADC comprising the glucuronide unit has the formula: (see also US 2012 / 0107332, incorporated herein by reference), where Y, y, D, A, a, and L are as described herein:

[0335] [ka]

[0336] In certain embodiments, the ADC comprising the glucuronide unit has the formula: (see also US 2012 / 0107332, incorporated herein by reference), where Y, y, D, and L are as described herein:

[0337] [ka]

[0338] In certain embodiments, the ADC comprising the glucuronide unit has the formula: (see also US 2012 / 0107332, incorporated herein by reference), where Y, y, D, and L are as described herein:

[0339] [ka]

[0340] In certain embodiments, the ADC comprising the glucuronide unit has the formula: (see also US 2012 / 0107332 A1), where D is as described herein and mAb is a monoclonal antibody.

[0341] [ka]

[0342] When the Spacer unit (-Y-) is present, it links the Amino Acid unit (or Glucuronide unit, see also US2012 / 0107332, incorporated herein by reference) to the Drug moiety, if present. Alternatively, when the Amino Acid unit is absent, it links the Stretcher unit to the Drug moiety. When neither the Amino Acid unit nor the Stretcher unit is present, the Spacer unit can also link the Drug unit to the Antibody unit.

[0343] Spacer units are generally of two types: non-self-immolative or self-immolative. A non-self-immolative spacer unit is one in which some or all of the spacer unit remains attached to the drug moiety after the amino acid unit (or glucuronide unit) is cleaved from the antibody-drug conjugate, particularly by an enzyme. Examples of non-self-immolative spacer units include, but are not limited to, a (glycine-glycine) spacer unit and a glycine spacer unit, both of which are depicted in Scheme 1 below (see also U.S. Pat. No. 8,309,093, incorporated herein by reference).

[0344] [ka]

[0345] When a conjugate containing a glycine-glycine spacer unit or a glycine spacer unit is enzymatically cleaved by an enzyme (e.g., a tumor cell-associated protease, a cancer cell-associated protease, or a lymphocyte-associated protease), the glycine-glycine-drug moiety or the glycine-drug moiety becomes LA a -W w In one embodiment, an independent hydrolysis reaction occurs within the target cell, cleaving the glycine-drug moiety bond and releasing the drug.

[0346] In certain embodiments, the non-self-immolative spacer unit (-Y-) is -Gly-. In certain embodiments, the non-self-immolative spacer unit (-Y-) is -Gly-Gly-.

[0347] In one embodiment, there is provided a Drug-Linker conjugate, or a pharmaceutically acceptable salt or solvate thereof, in which the Spacer unit is absent (y=0).

[0348] Alternatively, the drug moiety can be released by conjugates containing a self-immolative spacer unit, which spontaneously separates from the second chemical moiety upon cleavage of the bond to the first moiety.

[0349] In certain embodiments, -Y y- is the phenylene moiety Q m where Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro, or -cyano, and m is an integer from 0 to 4. The alkyl, alkenyl, and alkynyl groups, alone or as part of another group, are optionally substituted.

[0350] In certain embodiments, -Y- is a PAB group and is linked to -W through the amino nitrogen atom of the PAB group. w and is directly bonded to -D via a carbonate group, a carbamate group, or an ether group. Without being bound to a particular theory or mechanism, Scheme 2 below (see also U.S. Pat. No. 8,309,093) shows a possible drug release mechanism for a PAB group directly bonded to -D via a carbamate group or a carbonate group, as described by Toki et al., 2002, J. Org. Chem. 67:1866-1872.

[0351] [ka]

[0352] In Scheme 2, Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro, or -cyano, m is an integer from 0 to 4, and p is from 1 to about 20. The alkyl, alkenyl, and alkynyl groups, alone or as part of another group, are optionally substituted.

[0353] Other examples of self-immolative spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (Hay et al., 1999, Bioorg. Med. Chem. Lett. 9:2237) and ortho- or para-aminobenzyl acetals. Spacers that undergo cyclization upon hydrolysis of the amide bond can also be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al., 1972, J. Amer. Chem. Soc. 94:5815), and 2-aminophenylpropionic acid amides (Amsberry et al., 1990, J. Org. Chem. 55:5867). Elimination of amine-containing drugs substituted alpha to glycine (Kingsbury et al., 1984, J. Med. Chem. 27:1447) is also an example of a self-immolative spacer.

[0354] In one embodiment, the spacer unit (-Y y -) is represented by formula (X)-(XII) (see below (see also US 8,309,093)), where Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro, or -cyano; and m is an integer from 0 to 4.

[0355] [ka]

[0356] Other examples of self-immolative spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (see, e.g., Hay et al., 1999, Bioorg. Med. Chem. Lett. 9:2237) and ortho- or para-aminobenzyl acetals. Spacers that undergo cyclization upon hydrolysis of the amide bond can be used, such as substituted and unsubstituted 4-aminobutyric acid amides (see, e.g., Rodrigues et al., 1995, Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (see, e.g., Storm et al., 1972, J. Amer. Chem. Soc. 94:5815), and 2-aminophenylpropionic acid amides (see, e.g., Amsberry et al., 1990, J. Org. Chem. 55:5867). Elimination of amine-containing drugs substituted at the α-position of glycine (see, e.g., Kingsbury et al., 1984, J. Med. Chem. 27:1447) is also an example of a self-immolative spacer.

[0357] Other suitable spacer units are disclosed in US Patent Application Publication No. 2005-0238649, the disclosure of which is incorporated herein by reference.

[0358] Another approach to generating ADCs is to use heterobifunctional crosslinkers to link the anti-EGFR antibody to the drug moiety. Examples of crosslinkers that can be used include N-succinimidyl 4-(5-nitro-2-pyridyldithio)pentanoate or its highly water-soluble analogs, N-succinimidyl 4-(5-nitro-2-pyridyldithio)pentanoate, N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), N-succinimidyl-4-(5-nitro-2-pyridyldithio)butyrate (SNPB), and N-sulfosuccinimidyl N-succinimidyl-4-(5-nitro-2-pyridyldithio)butyrate (SSNPB), N-succinimidyl-4-methyl-4-(5-nitro-2-pyridyldithio)pentanoate (SMNP), N-succinimidyl-4-(5-N,N-dimethylcarboxamido-2-pyridyldithio)butyrate (SCPB) or N-sulfosuccinimidyl 4-(5-N,N-dimethylcarboxamido-2-pyridyldithio)butyrate (SSCPB). The antibodies of the invention can be modified with the crosslinking agent N-succinimidyl 4-(5-nitro-2-pyridyldithio)pentanoate, N-sulfosuccinimidyl 4-(5-nitro-2-pyridyldithio)pentanoate, SPDB, SNPB, SSNPB, SMNP, SCPB, or SSCPB, and then reacted with a slight excess of a specific drug containing a thiol moiety to give ADCs in excellent yields. Preferred crosslinkers are compounds of the formula shown below (see also U.S. Pat. No. 6,913,748, incorporated herein by reference):

[0359] [ka] In the formula, R, R1, R2, and R3 are the same or different and are H, methyl, ethyl, or a linear, branched, or cyclic alkyl group having 3 to 6 carbon atoms; n is 0 or an integer of 1 to 4; X and Y are the same or different and are H, CONR4R5, or NO2, provided that X and Y are not both H at the same time; R4 and R5 are the same or different and are each H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl; and Z is SO3 - M+ or H, provided that M + represents a metal ion or a tetraalkylammonium ion, with the proviso that when X and / or Y are NO, then Z is other than H. Other heterobifunctional crosslinkers and methods for making ADCs using such crosslinkers are described in U.S. Pat. No. 6,913,748, specifically incorporated herein by reference.

[0360] In one embodiment, a charged linker (also known as a precharged linker) is used to link an anti-EGFR antibody to a drug to form an ADC. Charged linkers include linkers that assume a charged state after cellular processing. The presence of a charged group on the linker or drug of a particular ADC after cellular processing provides several advantages, including (i) increased aqueous solubility of the ADC, (ii) the ability to manipulate higher concentrations in aqueous solution, (iii) the ability to attach more drug molecules per antibody, potentially increasing potency, (iv) the ability to retain the charged conjugate species inside the target cell, increasing potency, and (v) improved sensitivity of multidrug-resistant cells, preventing export of the charged drug species from the cell. Examples of certain suitable charged or precharged crosslinkers and their synthesis are shown in Figures 1-10 of U.S. Pat. No. 8,236,319, which are incorporated herein by reference. Charged or precharged crosslinkers preferably contain sulfonate, phosphate, carboxyl, or quaternary amine substituents, which significantly increase the solubility of ADCs, especially ADCs carrying 2 to 20 drugs. Conjugates prepared from linkers containing procharged moieties generate one or more charged moieties after the conjugate is metabolized in cells.

[0361] In another embodiment, the ADCs of the invention comprise a linker having the formula: (see also U.S. Pat. No. 8,236,319, incorporated herein by reference):

[0362] [ka] wherein Y' represents a functional group that allows reaction with an antibody; Q represents a functional group that allows linkage with a drug via a disulfide, thioether, thioester, peptide, hydrazone, ester, ether, carbamate, or amide bond; R1, R2, R3, R4, R5, R6, R7, R8, R9, and R 10 are the same or different and may be H, a linear alkyl group having 1 to 6 carbon atoms, a branched or cyclic alkyl group having 3 to 6 carbon atoms, a linear, branched or cyclic alkenyl or alkynyl group having 2 to 6 carbon atoms, or any of a group including, but not limited to, SO3 - , X-SO3 - , OPO3 2- , X-OPO3 2- , PO3 2- , X-PO3 2- anions such as, but not limited to, nitrogen-containing heterocycles, N + R 11 R 12 R 13 Or XN + R 11 R 12 R 13 or phenyl, provided that R 11 , R 12 and R 13 are the same or different and are H, a linear alkyl group having 1 to 6 carbon atoms, or a branched or cyclic alkyl group having 3 to 6 carbon atoms; X is phenyl, a linear alkyl group having 1 to 6 carbon atoms, or a branched or cyclic alkyl group having 3 to 6 carbon atoms; l, m, and n are 0 or integers of 1 to 4; A is phenyl or substituted phenyl, and the substituent is a linear alkyl group having 1 to 6 carbon atoms, or a branched or cyclic alkyl group having 3 to 6 carbon atoms, or, but not limited to, SO3 - , X-SO3 - , OPO3 2- , X-OPO3 2- , PO3 2- , X-PO3 2- , CO2- and anions such as, but not limited to, nitrogen-containing heterocycles, N + R 11 R 12 R 13 Or XN + R 11 R12 R 13 wherein X is as defined above; g is 0 or 1; and Z is an optionally present group of the formula (OCH2CH2) p (wherein p is 0 or an integer of 2 to about 1000) or a polyethyleneoxy unit of the formula F1-E1-P-E2-F2, where E1 and E2 are the same or different and are C=O, O or NR14, provided that R 14 is H, a linear alkyl group having 1 to 6 carbon atoms, a branched or cyclic alkyl group having 3 to 6 carbon atoms, or a linear, branched or cyclic alkenyl or alkynyl group having 2 to 6 carbon atoms; P is a peptide unit having a length of 2 to 20 amino acids, and E1 or E2 can be linked to the peptide through the terminal nitrogen, terminal carbon, or the side chain of one of the amino acids of the peptide; F1 and F2 are the same or different and optionally have the formula (OCH2CH2) p (wherein p is 0 or an integer of 2 to about 1000), provided that when Z is other than F1-E1-P-E2-F2, R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 At least one of A, R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 At least one of is a charged substituent.

[0363] Other examples of linkers that can be used in the compositions and methods include valine-citrulline, maleimidocaproyl, aminobenzoic acid, p-aminobenzylcarbamoyl (PAB), a lysosomal enzyme-cleavable linker, maleimidocaproyl-polyethylene glycol (MC(PEG)6-OH), N-methyl-valine-citrulline, N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), and N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP) (see also US2011 / 0076232). Another linker for use in the present invention includes an avidin-biotin bond to provide an avidin-biotin-containing ADC (see U.S. Pat. No. 4,676,980; PCT Publication Nos. WO1992 / 022332A2, WO1994 / 016729A1, WO1995 / 015770A1, WO1997 / 031655A2, WO1998 / 035704A1, WO1999 / 019500A1, WO2001 / 09785A2, WO2001 / 090198A1, WO2003 / 093793A2, WO2004 / 050016A2, WO2005 / 081898A2, WO2006 / 083562A2, WO2006 / 089668A1, WO2007 / 150020A1, WO2008 / 135237A1, WO2010 / 111198A1, WO2011 / 057216A1, WO2011 / 058321A1, WO2012 / 027494A1 and EP77671B1.), certain such linkers are resistant to biotinidase cleavage. Other linkers that can be used in the present invention include cohesin / dockerin pairs to provide cohesin-dockerin-containing ADCs (see PCT Publication Nos. WO2008 / 097866A2, WO2008 / 097870A2, WO2008 / 103947A2, and WO2008 / 103953A2).

[0364] Other linkers for use in the present invention can include non-peptide polymers (examples include, but are not limited to, polyethylene glycol, polypropylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA (poly(lactic acid)), PLGA (poly(lactic-glycolic acid)), and combinations thereof, with polyethylene glycol being the preferred polymer) (see also PCT Publication No. WO2011 / 000370). Other linkers are also described in WO2004-010957, U.S. Publication No. 20060074008, U.S. Publication No. 20050238649, and U.S. Publication No. 20060024317, each of which is incorporated herein by reference in its entirety.

[0365] In ADCs containing maytansinoids, multiple positions on the maytansinoid are available for chemically attaching a linking moiety. In one embodiment, the maytansinoid comprises a linking moiety containing a reactive chemical group, the linking moiety comprising a disulfide bond, and the chemically reactive group is a C3 ester of maytansinol and its analogs, including N-succinimidyl or N-sulfosuccinimidyl esters. For example, a C3 position bearing a hydroxyl group, a C14 position modified with a hydroxymethyl group, a C15 position modified with a hydroxy group, and a C20 position bearing a hydroxy group are all useful. Most preferably, the linking moiety is attached to the C3 position of maytansinol.

[0366] Attachment of a drug to an antibody via a linker can be carried out by any technique known in the art. Many different reactions are available for covalently attaching a drug and a linker to an antibody. This can be achieved by reaction of amino acid residues on the antibody, including the amine groups of lysine, the free carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl groups of cysteine, and various portions of aromatic amino acids. One of the most widely used nonspecific covalent attachment methods is the carbodiimide reaction, which links the carboxy (amino) group of a compound to the amino (or carboxy) group of an antibody. Furthermore, bifunctional reagents such as dialdehydes and imidoesters have been used to link the amino groups of a compound to the amino groups of an antibody. The Schiff base reaction can also be used to attach a drug to an antibody. This method involves periodate oxidation of a drug containing a glycol or hydroxy group to form an aldehyde, which is then reacted with a linking agent. Conjugation occurs via the formation of a Schiff base with the amino group of the antibody. Isothiocyanates can also be used as coupling agents to covalently attach drugs to antibodies. Other techniques are known to those skilled in the art and are within the scope of the present invention.

[0367] In certain embodiments, an intermediate that is a precursor to the linker is reacted with the drug under appropriate conditions. In certain embodiments, a reactive group on the drug or the intermediate is used. The product of the reaction of the drug with the intermediate or derivatized drug is then reacted with the anti-EGFR antibody under appropriate conditions. The synthesis and structures of exemplary linkers, stretcher units, amino acid units, and self-immolative spacer units are described in U.S. Patent Application Publication Nos. 20030083263, 20050238649, and 20050009751, each of which is incorporated herein by reference.

[0368] The stability of the ADC can be measured by standard analytical techniques such as mass spectrometry, HPLC and separation / analysis techniques such as LC / MS.

[0369] IV. Purification of anti-EGFR ADCs Purification of ADCs can be performed to recover ADCs with a certain DAR. For example, HIC resin can be used to separate highly drug-loaded ADCs from ADCs with an optimal drug-to-antibody ratio (DAR) (e.g., a DAR of 4 or less). In one embodiment, a hydrophobic resin is added to an ADC mixture, allowing undesired ADCs, i.e., highly drug-loaded ADCs, to bind to the resin and be selectively removed from the mixture. In certain embodiments, separation of ADCs can be performed by contacting the ADC mixture with a sufficient amount of hydrophobic resin to bind the drug-loaded species to be removed from the ADC mixture (e.g., a mixture of ADC drug-loaded species of 4 or less and ADC drug-loaded species of 6 or more). The resin and the ADC mixture are mixed so that the ADC species to be removed (e.g., drug-loaded species of 6 or more) can bind to the resin and be removed from the other ADC species in the ADC mixture. The amount of resin used in this method is selected according to the weight ratio of the species to be removed to the resin so as to avoid significant binding to the desired drug-loaded species. Thus, methods can be used to reduce an average DAR of 5.5 to less than 4. Furthermore, the purification methods described herein can be used to isolate ADCs containing any desired range of drug-loaded species (e.g., 4 or less drug-loaded species, 3 or less drug-loaded species, 2 or less drug-loaded species, 1 or less drug-loaded species).

[0370] Certain molecular species bind to surfaces based on hydrophobic interactions between the species and the hydrophobic resin. In one embodiment, the method of the present invention relates to a purification method that relies on mixing a mixture of ADCs with a hydrophobic resin, and the amount of resin added to the mixture determines which species (e.g., ADCs with a DAR of 6 or more) bind. After production and purification of antibodies from an expression system (e.g., a mammalian expression system), the antibodies are reduced and conjugated to drugs via a conjugation reaction. The resulting ADC mixture often contains ADCs with a DAR, for example, in the range of 1 to 8. In one embodiment, the ADC mixture contains drug-loaded species of 4 or less and drug-loaded species of 6 or more. According to the method of the present invention, ADC mixtures can be purified by methods such as, but not limited to, a batch process to select and separate ADCs with a drug load of 4 or less from ADCs with a higher drug load (e.g., ADCs with a drug load of 6 or more). In particular, the purification methods described herein can be used to isolate ADCs with any desired DAR range (e.g., DAR of 4 or less, DAR of 3 or less, DAR of 2 or less).

[0371] Thus, in one embodiment, an ADC mixture comprising a drug-loaded species of 4 or less and a drug-loaded species of 6 or more is contacted with a hydrophobic resin, such that the amount of hydrophobic resin contacted with the ADC mixture is sufficient to bind the drug-loaded species of 6 or more, while not resulting in significant binding of the drug-loaded species of 4 or less, to form a resin mixture; and the hydrophobic resin is removed from the ADC mixture to obtain a composition comprising an ADC comprising an antibody conjugated to an auristatin, wherein the drug-loaded species of 6 or less constitutes less than 15%. In another embodiment, the method of the invention comprises the steps of: contacting an ADC mixture comprising a drug-loaded species of 4 or less and a drug-loaded species of 6 or more with a hydrophobic resin, such that the amount of hydrophobic resin contacted with the ADC mixture is sufficient to bind the drug-loaded species of 6 or more, while not resulting in significant binding of the drug-loaded species of 4 or less, to form a resin mixture; and removing the hydrophobic resin from the ADC mixture to obtain a composition comprising an ADC comprising an antibody conjugated to an auristatin, wherein the drug-loaded species of 6 or less constitutes less than 15% and the weight of the hydrophobic resin is 3 to 12 times that of the drug-loaded species of 6 or more in the ADC mixture.

[0372] The ADC separation methods described herein can be carried out using a batch purification method. Batch purification generally involves adding an ADC mixture to a hydrophobic resin in a vessel, mixing, and then separating the resin from the supernatant. For example, in the context of batch purification, the hydrophobic resin can be prepared in or equilibrated with a desired equilibration buffer, resulting in a slurry of the hydrophobic resin. The ADC mixture is then contacted with the slurry, allowing the specific ADC species to be separated to adsorb to the hydrophobic resin. The solution containing the desired ADC that does not bind to the hydrophobic resin material is then separated from the slurry, for example, by filtration or settling the slurry and removing the supernatant. The resulting slurry can be subjected to one or more wash steps. The salt concentration can be reduced to elute the bound ADC. In one embodiment, the method used herein utilizes 50 g or less of hydrophobic resin.

[0373] Thus, a batch method can be used to contact an ADC mixture containing a drug-loaded species of 4 or less and a drug-loaded species of 6 or more with a hydrophobic resin, such that the amount of hydrophobic resin contacted with the ADC mixture is sufficient to bind the drug-loaded species of 6 or more, but does not result in significant binding of the drug-loaded species of 4 or less, to form a resin mixture; and to remove the hydrophobic resin from the ADC mixture to obtain a composition comprising an ADC comprising an antibody conjugated to an auristatin, wherein the drug-loaded species of 6 or less constitutes less than 15%. In another embodiment, a batch method can be used to contact an ADC mixture containing a drug-loaded species of 4 or less and a drug-loaded species of 6 or more with a hydrophobic resin, such that the amount of hydrophobic resin contacted with the ADC mixture is sufficient to bind the drug-loaded species of 6 or more, but does not result in significant binding of the drug-loaded species of 4 or less, to form a resin mixture; and to remove the hydrophobic resin from the ADC mixture to obtain a composition comprising an ADC comprising an antibody conjugated to an auristatin, wherein the drug-loaded species of 6 or more constitutes less than 15% and the weight of the hydrophobic resin is 3 to 12 times that of the drug-loaded species of 6 or more in the ADC mixture.

[0374] Alternatively, in another embodiment, purification can be performed using a circulation method, in which the resin is loaded into a vessel and the ADC mixture is passed through the hydrophobic resin until the particular ADC species to be separated is removed, after which the supernatant (containing the desired ADC species) is drained from the vessel and the resin can be subjected to a washing step.

[0375] A circulation method can be used to form a resin mixture by contacting an ADC mixture containing a drug-loaded species of 4 or less and a drug-loaded species of 6 or more with a hydrophobic resin, such that the amount of hydrophobic resin contacted with the ADC mixture is sufficient to bind the drug-loaded species of 6 or more, but does not result in significant binding of the drug-loaded species of 4 or less; and to remove the hydrophobic resin from the ADC mixture to obtain a composition comprising an ADC comprising an antibody conjugated to an auristatin, wherein the drug-loaded species of 6 or less constitutes less than 15%. In another embodiment, a circulation method can be used to form a resin mixture by contacting an ADC mixture containing a drug-loaded species of 4 or less and a drug-loaded species of 6 or more with a hydrophobic resin, such that the amount of hydrophobic resin contacted with the ADC mixture is sufficient to bind the drug-loaded species of 6 or more, but does not result in significant binding of the drug-loaded species of 4 or less; and to remove the hydrophobic resin from the ADC mixture to obtain a composition comprising an ADC comprising an antibody conjugated to an auristatin, wherein the drug-loaded species of 6 or more constitutes less than 15% and the weight of the hydrophobic resin is 3 to 12 times the weight of the drug-loaded species of 6 or more in the ADC mixture.

[0376] Alternatively, a flow-through method can be used to purify an ADC mixture to arrive at a composition predominantly consisting of ADCs with a predetermined desired DAR. In a flow-through method, a resin is packed into a vessel (e.g., a column), and the ADC mixture is passed through the packed resin such that the desired ADC species pass through the resin substantially unbound, while the undesired ADC species bind to the resin. The flow-through method can be performed in a single-pass mode (the desired ADC species are obtained after a single pass through the resin in the vessel) or in a multi-pass mode (the desired ADC species are obtained after multiple passes through the resin in the vessel). The flow-through method is performed such that a selected weight of resin binds the undesired ADC population, while the desired ADCs (e.g., DARs 2-4) pass through the resin and are collected in the flow-through after one or more passes.

[0377] A flow-through method can be used in which an ADC mixture containing a drug-loaded species of 4 or less and a drug-loaded species of 6 or more is contacted with the hydrophobic resin in an amount sufficient to bind the drug-loaded species of 6 or more, but which does not result in significant binding of the drug-loaded species of 4 or less, and the drug-loaded species of 4 or less are collected after one or more passes to obtain a composition containing a desired ADC in which an antibody is conjugated to an auristatin (e.g., DAR 2 to 4), in which the drug-loaded species of 6 or more account for less than 15%. In another embodiment, the ADC mixture containing a drug-loaded species of 4 or less and a drug-loaded species of 6 or more is contacted with the hydrophobic resin by passing the ADC mixture through the resin, such that the amount of hydrophobic resin contacted with the ADC mixture is sufficient to bind the drug-loaded species of 6 or more, but does not allow significant binding of the drug-loaded species of 4 or less, and a flow-through method is used to collect the drug-loaded species of 4 or less after one or more passes, such that a composition comprising an ADC in which the drug-loaded species of 6 or more constitutes less than 15% and the weight of the hydrophobic resin is 3 to 12 times that of the drug-loaded species of 6 or more in the ADC mixture is obtained.

[0378] After the flow-through step, the resin may be washed one or more times with a wash solution to further recover ADCs in the desired DAR range (present in the wash filtrate). For example, multiple washes with decreasing conductivity can be used to further recover ADCs with the desired DAR. The eluate from the resin washes can then be combined with the filtrate from the flow-through step to improve recovery of ADCs with the desired DAR.

[0379] The batch, circulation, and flow-through methods are based on the use of a hydrophobic resin to separate high and low drug-loaded species of ADC. The hydrophobic resin contains hydrophobic groups that interact with the hydrophobicity of the ADC. The hydrophobic groups on the ADC interact with the hydrophobic groups in the hydrophobic resin. The more hydrophobic a protein is, the stronger its interaction with the hydrophobic resin.

[0380] Hydrophobic resins generally comprise a substrate (e.g., cross-linked agarose or synthetic copolymer materials) to which hydrophobic ligands (e.g., alkyl or aryl groups) are attached. Many hydrophobic resins are commercially available. Examples include, but are not limited to, Phenyl Sepharose™ 6 Fast Flow with low or high substitution (Pharmacia LKB Biotechnology, AB, Sweden); Phenyl Sepharose™ High Performance (Pharmacia LKB Biotechnology, AB, Sweden); Octyl Sepharose™ High Performance (Pharmacia LKB Biotechnology, AB, Sweden); Fractogel™ EMD Propyl or Fractogel™ EMD Phenyl columns (E. Merck, Germany); Macro-Prep™ Methyl or Macro-Prep™ t-Butyl Supports (Bio-Rad, California); WP HI-Propyl(C3)™ (JT Baker, New Jersey); and Toyopearl™ ether, hexyl, phenyl, or butyl (TosoHaas, PA). In one embodiment, the hydrophobic resin is a butyl hydrophobic resin. In another embodiment, the hydrophobic resin is a phenyl hydrophobic resin. In another embodiment, the hydrophobic resin is a hexyl hydrophobic resin, an octyl hydrophobic resin, or a decyl hydrophobic resin. In one embodiment, the hydrophobic resin is a methacrylic acid polymer with n-butyl ligands (e.g., TOYOPEARL® Butyl-600M).

[0381] Other methods for purifying ADC mixtures to obtain compositions with desirable DARs are described in U.S. Application No. 14 / 210,602 (U.S. Patent Application Publication No. US2014 / 0286968), the entire disclosure of which is incorporated herein by reference.

[0382] V. Uses of Anti-EGFR Antibodies and Anti-EGFR ADCs Preferably, the antibodies and antibody portions (and ADCs) of the invention are capable of neutralizing human EGFR activity in vivo. Accordingly, such antibodies and antibody portions of the invention can be used to inhibit hEGFR activity, e.g., in cells containing hEGFR, in a human subject or other mammalian subject having an EGFR with which the antibodies of the invention cross-react. In one embodiment, the invention provides a method for inhibiting hEGFR activity, comprising contacting hEGFR with an antibody or antibody portion of the invention, such that hEGFR activity is inhibited. For example, in a cell culture containing or suspected of containing hEGFR, an antibody or antibody portion of the invention can be added to the culture medium to inhibit hEGFR activity in the culture.

[0383] In another embodiment, the present invention relates to a method for reducing hEGFR activity in a subject, preferably a subject suffering from a disease or disorder in which EGFR activity is detrimental. The present invention provides a method for reducing EGFR activity in a subject suffering from such a disease or disorder, the method comprising administering an antibody or antibody portion of the present invention to the subject so as to reduce EGFR activity in the subject. Preferably, the EGFR is human EGFR, and the subject is a human subject. Alternatively, the subject can be a mammal expressing an EGFR capable of binding to an antibody of the present invention. Further, the subject can be a mammal into which EGFR has been introduced (e.g., by administration of EGFR or expression of an EGFR transgene). The antibodies of the present invention can be administered to human subjects for therapeutic purposes. Furthermore, the antibodies of the present invention can be administered to non-human mammals expressing an EGFR capable of binding to such antibodies for veterinary purposes or as animal models of human disease. Regarding the latter, such animal models may be useful for assessing the therapeutic efficacy of antibodies of the present invention (e.g., testing dosages and courses of administration).

[0384] As used herein, the term "disorder in which EGFR activity is detrimental" encompasses diseases and other disorders in which the presence of EGFR in a subject suffering from such a disorder is known or suspected to be involved in the pathophysiology of the disorder or to be a contributing factor in the progression of the disorder. Thus, a disorder in which EGFR activity is detrimental is one in which reduction of EGFR activity is expected to alleviate the symptoms and / or progression of the disorder. Such disorders can be determined, for example, by elevated levels of EGFR in the body fluids of a subject suffering from the disorder (e.g., elevated levels of EGFR in the subject's tumor, serum, plasma, synovial fluid, etc.), and can be detected, for example, using an anti-EGFR antibody as described above. Non-limiting examples of disorders that can be treated with the antibodies (e.g., AbAs) or antigen-binding fragments thereof of the present invention include those described below. For example, suitable disorders include, but are not limited to, various cancers, including, but not limited to, breast cancer, lung cancer, glioma, prostate cancer, pancreatic cancer, colon cancer, head and neck cancer, and kidney cancer. Other examples of cancers that can be treated using the compositions and methods disclosed herein include squamous cell carcinoma (e.g., lung squamous cell carcinoma or head and neck squamous cell carcinoma), triple-negative breast cancer, non-small cell lung cancer, colorectal cancer, and mesothelioma. In one embodiment, the antibodies and ADCs disclosed herein are used to treat solid tumors, e.g., to inhibit the growth or reduce the size of solid tumors that overexpress EGFR or are EGFR-positive. In one embodiment, the invention relates to the treatment of EGFR-amplified lung squamous cell carcinoma. In one embodiment, the antibodies and ADCs disclosed herein are used to treat EGFR-amplified head and neck squamous cell carcinoma. In another embodiment, the antibodies and ADCs disclosed herein are used to treat triple-negative breast cancer (TNBC). The diseases and disorders described herein can be treated with the anti-EGFR antibodies or ADCs of the invention, as well as pharmaceutical compositions containing such anti-EGFR antibodies or ADCs.

[0385] In certain embodiments, the antibodies and ADCs disclosed herein are administered to a subject in need of treatment to treat advanced solid tumor types that are likely to exhibit high levels of epidermal growth factor receptor (EGFR). Examples of such tumors include, but are not limited to, head and neck squamous cell carcinoma, non-small cell lung cancer, triple-negative breast cancer, colorectal cancer, and glioblastoma multiforme.

[0386] In certain embodiments, the invention encompasses methods of inhibiting or suppressing solid tumor growth in a subject having a solid tumor, the method comprising administering to the subject having the solid tumor an anti-EGFR antibody or ADC described herein, such that growth of the solid tumor is inhibited or suppressed. In certain embodiments, the solid tumor is non-small cell lung cancer or glioblastoma. In other embodiments, the solid tumor is an EGFRvIII-positive tumor or an EGFR-expressing solid tumor. In other embodiments, the solid tumor is an EGFR-amplified solid tumor or an EGFR-overexpressing solid tumor. In certain embodiments, an anti-EGFR antibody or ADC described herein is administered alone or in combination with another agent (e.g., radiation and / or temozolomide) to a subject having glioblastoma multiforme.

[0387] In certain embodiments, the present invention encompasses a method for inhibiting or suppressing solid tumor growth in a subject with a solid tumor identified as an EGFR-expressing tumor or an EGFR-overexpressing tumor (or an EGFRvIII-expressing tumor), the method comprising administering to the subject an anti-EGFR antibody or ADC described herein to inhibit or suppress growth of the solid tumor. Methods for identifying EGFR-expressing tumors (e.g., EGFR-overexpressing tumors) are known in the art and include FDA-approved tests and validation assays. For example, the EGFR pharmDx™ assay (Dako North America, Inc.) is a qualitative immunohistochemistry (IHC) kit system used to identify EGFR expression in normal and neoplastic tissues routinely scheduled for histological evaluation. EGFR pharmDx specifically detects EGFR (HER1) protein in cells expressing EGFR. Additionally, PCR assays can also be used to identify EGFR-overexpressing tumors. For example, these assays can use primers specific for a mutant EGFR gene (e.g., SEQ ID NO: 33) and / or cDNA to amplify the EGFR gene / cDNA or a portion thereof. The amplified PCR product can then be analyzed, for example, by gel electrophoresis using standard methods known in the art to determine the size of the PCR product. Such tests can be used to identify tumors that can be treated with the methods and compositions described herein.

[0388] Any gene therapy method available in the art can be used in accordance with the present invention. For a general discussion of gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215. Among the methods well known in the field of recombinant DNA technology, methods that can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990). A detailed description of various gene therapy methods is provided in US20050042664A1, which is incorporated herein by reference.

[0389] In another aspect, this application relates to a method of treating (e.g., curing, inhibiting, ameliorating, delaying or preventing onset, or preventing recurrence or recurrence) or preventing an EGFR-related disorder in a subject. The method includes administering to the subject an EGFR-binding agent (particularly an antagonist), such as an anti-EGFR antibody or fragment thereof as described herein, in an amount sufficient to treat or prevent the EGFR-related disorder. The EGFR antagonist (e.g., an anti-EGFR antibody or fragment thereof) can be administered to the subject alone or in combination with other therapies as described herein.

[0390] To treat such diseases, the antibodies or ADCs of the present invention, or antigen-binding portions thereof, can be used alone or in combination. It will be appreciated that the antibodies or antigen-binding portions thereof of the present invention can be used alone or in combination with another agent (e.g., a therapeutic agent), which will be selected by those skilled in the art for the intended purpose. For example, the other agent can be a therapeutic agent recognized in the art as being useful for treating the disease or condition to be treated by the antibodies of the present invention. The other agent can also be an agent that imparts a beneficial attribute to the therapeutic composition (e.g., an agent that alters the viscosity of the composition).

[0391] It will be further understood that the combination agent included in the present invention is a combination agent useful for its intended purpose. The drugs described below are for illustrative purposes only and are not limiting. The combination agent that is part of the present invention can be an antibody of the present invention and at least one other drug selected from the list below. The combination agent may contain two or more other drugs, for example, two or three other drugs, as long as the formed composition can perform its intended function.

[0392] Combination therapy can involve the combination and / or administration of one or more EGFR antagonists, e.g., anti-EGFR antibodies or fragments thereof, with one or more additional therapeutic agents, such as one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents (e.g., systemic anti-inflammatory agents), antifibrogenic agents, metabolic inhibitors, enzyme inhibitors and / or cytotoxic or cytostatic agents, mitotic inhibitors, antitumor antibiotics, immunomodulatory agents, gene therapy vectors, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemotherapeutic agents, hormones, antihormones, corticosteroids, photosensitive therapeutic agents, oligonucleotides, radionuclides, topoisomerase inhibitors, tyrosine kinase inhibitors, or radiosensitizers, as described in more detail herein.

[0393] In certain embodiments, the anti-EGFR binding proteins (e.g., anti-EGFR antibodies) described herein are used in combination with an anti-cancer or anti-neoplastic agent. The terms "anti-cancer agent" and "anti-neoplastic agent" refer to drugs used to treat malignant tumors, such as cancerous growths. Drug therapies may be used alone or in combination with other treatments, such as surgery or radiation therapy. Multiple classes of drugs may be used in combination depending on the type of organ affected. For example, breast cancer is generally stimulated by estrogen and can be treated with drugs that inactivate sex hormones. Similarly, prostate cancer can be treated with drugs that inactivate the male hormone androgen. Anti-cancer agents that can be used in combination with the anti-EGFR antibodies or ADCs of the invention include, among others, the following agents:

[0394] [Table 2] TIFF2025143392000037.tif242168TIFF2025143392000038.tif243166TIFF2025143392000039.tif210165

[0395] In addition to the anti-cancer agents described above, the agents described in Section II may be administered in combination with the anti-EGFR antibodies and ADCs described herein. Additionally, the anti-cancer agents described above may be used in the ADCs of the present invention.

[0396] In certain embodiments, the anti-EGFR antibodies or ADCs can be administered alone or in combination with another anti-cancer agent that acts synergistically or potentiates with the antibody to treat a disease associated with EGFR activity. Such anti-cancer agents include, for example, agents well known in the art (e.g., cytotoxins, chemotherapeutic agents, small molecules, and radiation). Examples of anti-cancer agents include, but are not limited to, Panorex (Glaxo-Welcome), Rituxan (IDEC / Genentech / Hoffman la Roche), Mylotarg (Wyeth), Campath (Millennium), Zevalin (IDEC and Schering AG), Bexxar (Corixa / GSK), Erbitux (Imclone / BMS), Avastin (Genentech), and Herceptin (Genentech / Hoffman la Roche). Other anti-cancer agents include, but are not limited to, those disclosed in U.S. Patent No. 7,598,028 and International Publication No. WO2008 / 100624, the disclosures of which are incorporated herein by reference. One or more anti-cancer agents can be administered simultaneously with, before, or after administration of an antibody, or antigen-binding portion thereof, of the invention.

[0397] In certain embodiments of the invention, the anti-EGFR antibodies or ADCs described herein can be used in combination with an apoptotic agent, such as a bcl-xl inhibitor or a Bcl-2 (B-cell lymphoma 2) inhibitor (e.g., ABT-199 (venetoclax)), to treat cancer, such as leukemia, in a subject. In one embodiment, the anti-EGFR antibodies or ADCs described herein can be used in combination with a bcl-xl inhibitor to treat cancer. In one embodiment, the anti-EGFR antibodies or ADCs described herein can be used in combination with venetoclax to treat cancer.

[0398] In certain embodiments of the invention, the anti-EGFR antibodies or ADCs described herein can be used in combination with inhibitors of NAMPT (see, for example, US 2013 / 0303509; AbbVie, Inc., incorporated herein by reference) to treat a subject in need thereof. NAMPT (also known as B-cell colony-enhancing factor (PBEF) and visfatin) is an enzyme that catalyzes the phosphoribosylation of nicotinamide and is the rate-limiting enzyme in one of two pathways that salvage NAD. In one embodiment of the invention, the anti-EGFR antibodies and ADCs described herein are administered in combination with an NAMPT inhibitor to treat cancer in a subject.

[0399] In certain embodiments of the invention, the anti-EGFR antibodies or ADCs described herein can be used in combination therapy with SN-38, the active metabolite of the topoisomerase inhibitor irinotecan.

[0400] In other embodiments of the invention, the anti-EGFR antibodies or ADCs described herein can be used in combination with PARP (poly ADP-ribose polymerase) inhibitors (e.g., veliparib) to treat cancers, including breast cancer, ovarian cancer, and non-small cell lung cancer.

[0401] Other examples of additional therapeutic agents that can be co-administered and / or formulated with the anti-EGFR antibodies or anti-EGFR ADCs described herein include, but are not limited to, one or more of the following: inhaled steroids; beta-agonists (e.g., short-acting or long-acting beta-agonists); leukotriene or leukotriene receptor antagonists; combination medications such as ADVAIR; IgE inhibitors, e.g., anti-IgE antibodies (e.g., XOLAIR®, omalizumab); phosphodiesterase inhibitors (e.g., PDE4 inhibitors); xanthines; anticholinergics; mast cell stabilizers such as cromolyn; IL-4 inhibitors; IL-5 inhibitors; eotaxin / CCR3 inhibitors; antagonists of histamine or its receptors, including H1, H2, H3, and H4; and antagonists of prostaglandin D or its receptors (DP1 and CRTH2). Such combinations can be used, for example, to treat asthma and other respiratory disorders. Other examples of additional therapeutic agents that can be co-administered and / or formulated with the anti-EGFR antibodies or anti-EGFR ADCs described herein include, but are not limited to, one or more of temozolomide, ibrutinib, duvelisib, and idelalisib. Other examples of therapeutic agents that can be co-administered and / or formulated with one or more anti-EGFR antibodies or fragments thereof include, among others, TNF antagonists (e.g., soluble fragments of TNF receptors, such as p55 or p75 human TNF receptors or derivatives thereof, e.g., 75 kD TNFR-IgG (75 kD TNF receptor-IgG fusion protein, ENBREL)); TNF enzyme antagonists, such as TNF-converting enzyme (TACE) inhibitors; muscarinic receptor antagonists; TGF-β antagonists; interferon gamma; pirfenidone; chemotherapeutic agents, such as methotrexate, leflunomide, or sirolimus (rapamycin) or analogs thereof (e.g., CCI-77); COX2 and cPLA2 inhibitors; NSAIDs; immunomodulators; p38 inhibitors, TPL-2, MK-2, and NFkB inhibitors.

[0402] Other preferred co-administration agents are cytokine suppressive anti-inflammatory drugs (CSAIDs); other human cytokines or growth factors (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, IL-21, IL-31, interferons, EMAP-II, GM-CSF, FGF, EGF, PDGF, and endothelin-1), and antibodies or antagonists of the receptors for these cytokines and growth factors. The antibodies of the invention, or antigen-binding portions thereof, can be used in combination with antibodies against cell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA, CTLA-4, PD-1, or their ligands, including CD154 (gp39 or CD40L).

[0403] Preferred combinations of therapeutic agents can intervene at various points in the inflammatory cascade, and preferred examples include TNF antagonists such as chimeric humanized or human TNF antibodies, adalimumab (HUMIRA; D2E7; PCT Publication No. WO 97 / 29131 and U.S. Patent No. 6,090,382, which are incorporated herein by reference), CA2 (Remicade™), CDP571, and soluble p55 or p75 TNF receptors, their derivatives (p75TNFR1gG (Enbrel™) or p55TNFR1gG (Lenercept)), and TNF-converting enzyme (TACE) inhibitors; IL-1 inhibitors (e.g., interleukin-1 converting enzyme inhibitors, IL-1RA) may also be effective for the same reasons. Another preferred combination agent is interleukin-4.

[0404] Pharmaceutical compositions of the invention can contain a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antibody portion of the invention. A "therapeutically effective amount" means an amount effective to achieve a desired therapeutic result, at the time of administration and for a period of time necessary. A therapeutically effective amount of an antibody or antibody portion can be determined by one of skill in the art and will vary depending on the individual's disease state, age, sex, and weight, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount such that the beneficial effects of treatment outweigh any toxic or detrimental effects of the antibody or antibody portion. A "prophylactically effective amount" means an amount effective to achieve a desired prophylactic result, at the time of administration and for a period of time necessary. Generally, since prophylactic administration is used in subjects who are pre-disease or in an early stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0405] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, or several divided doses may be administered over time, or the dose may be proportionally increased or decreased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity. As used herein, dosage unit form refers to physically discrete units suited as unitary doses for the mammalian subject to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect together with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are determined by and directly depend on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect sought to be achieved, and (b) the limitations inherent in the technology for formulating such active compounds, taking into account the treatment of hypersensitivity in the individual.

[0406] A non-limiting example of a therapeutically or prophylactically effective dose range for an ADC, antibody, or antibody portion of the invention is 0.1 to 20 mg / kg, more preferably 1 to 10 mg / kg. In one embodiment, the dose of the antibodies and ADCs described herein is 1 to 6 mg / kg, including individual doses described herein, such as 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, and 6 mg / kg. In another embodiment, the dose of the antibodies and ADCs described herein is 1-200 μg / kg, including individual doses described herein, such as 1 μg / kg, 2 μg / kg, 3 μg / kg, 4 μg / kg, 5 μg / kg, 10 μg / kg, 20 μg / kg, 30 μg / kg, 40 μg / kg, 50 μg / kg, 60 μg / kg, 80 μg / kg, 100 μg / kg, 120 μg / kg, 140 μg / kg, 160 μg / kg, 180 μg / kg, and 200 μg / kg. Dosage values ​​will vary depending on the type and severity of the condition to be alleviated. It will be further understood that for any particular subject, specific dosage regimens will be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the compositions of the invention.

[0407] In one embodiment, an anti-EGFR antibody (e.g., AbA) or antigen-binding portion thereof described herein is administered as an ADC to a subject in need thereof (e.g., a subject with cancer) at a dose of 0.1 to 30 mg / kg. In another embodiment, the anti-EGFR antibody (e.g., AbA) or antigen-binding portion thereof is administered as an ADC to a subject in need thereof (e.g., a subject with cancer) at a dose of 1 to 15 mg / kg. In another embodiment, the anti-EGFR antibody (e.g., AbA) or antigen-binding portion thereof is administered as an ADC to a subject in need thereof (e.g., a subject with cancer) at a dose of 1 to 10 mg / kg. In another embodiment, the anti-EGFR antibody (e.g., AbA) or antigen-binding portion thereof is administered as an ADC to a subject in need thereof (e.g., a subject with cancer) at a dose of 2 to 3 mg. In another embodiment, the anti-EGFR antibody (e.g., AbA) or antigen-binding portion thereof is administered as an ADC to a subject in need thereof (e.g., a subject with cancer) at a dose of 1 to 4 mg / kg.

[0408] In one embodiment, an anti-EGFR antibody (e.g., AbA) or antigen-binding portion thereof described herein is administered as an ADC to a subject in need thereof (e.g., a subject with cancer) at a dose of 1 to 200 μg / kg. In another embodiment, the anti-EGFR antibody (e.g., AbA) or antigen-binding portion thereof is administered as an ADC to a subject in need thereof (e.g., a subject with cancer) at a dose of 5 to 150 μg / kg. In another embodiment, the anti-EGFR antibody (e.g., AbA) or antigen-binding portion thereof is administered as an ADC to a subject in need thereof (e.g., a subject with cancer) at a dose of 5 to 100 μg / kg. In another embodiment, the anti-EGFR antibody (e.g., AbA) or antigen-binding portion thereof is administered as an ADC to a subject in need thereof (e.g., a subject with cancer) at a dose of 5 to 90 μg / kg. In another embodiment, the anti-EGFR antibody (e.g., AbA) or antigen-binding portion thereof is administered as an ADC to a subject in need thereof (e.g., a subject with cancer) at a dose of 5 to 80 μg / kg. In another embodiment, the anti-EGFR antibody (e.g., AbA) or its antigen-binding portion is administered as an ADC to a subject in need thereof (e.g., a subject with cancer) at a dose of 5 to 70 μg / kg. In another embodiment, the anti-EGFR antibody (e.g., AbA) or its antigen-binding portion is administered as an ADC to a subject in need thereof (e.g., a subject with cancer) at a dose of 5 to 60 μg / kg. In another embodiment, the anti-EGFR antibody (e.g., AbA) or its antigen-binding portion is administered as an ADC to a subject in need thereof (e.g., a subject with cancer) at a dose of 10 to 80 μg / kg.

[0409] In one embodiment, a subject in need thereof (e.g., a subject with cancer) is administered an anti-EGFR ADC described herein (e.g., AbA-vc-MMAE) at a dose of 1 to 6 mg / kg. In another embodiment, a subject in need thereof (e.g., a subject with cancer) is administered an anti-EGFR ADC described herein (e.g., AbA-vc-MMAE) at a dose of 5 to 4 mg / kg. In another embodiment, a subject in need thereof (e.g., a subject with cancer) is administered an anti-EGFR ADC described herein (e.g., AbA-vc-MMAE) at a dose of 1.8 to 2.4 mg / kg. In another embodiment, a subject in need thereof (e.g., a subject with cancer) is administered an anti-EGFR ADC described herein (e.g., AbA-vc-MMAE) at a dose of 1 to 4 mg / kg. In another embodiment, a subject in need thereof (e.g., a subject with cancer) is administered an anti-EGFR ADC described herein (e.g., AbA-vc-MMAE) at a dose of about 1 mg / kg. In another embodiment, a subject in need thereof (e.g., a subject with cancer) is administered an anti-EGFR ADC described herein (e.g., AbA-vc-MMAE) at a dose of 3 to 6 mg / kg. In another embodiment, a subject in need thereof (e.g., a subject with cancer) is administered an anti-EGFR ADC described herein (e.g., AbA-vc-MMAE) at a dose of 3 mg / kg. In another embodiment, a subject in need thereof (e.g., a subject with cancer) is administered an anti-EGFR ADC described herein (e.g., AbA-vc-MMAE) at a dose of 2 to 3 mg / kg. In another embodiment, a subject in need thereof (e.g., a subject with cancer) is administered an anti-EGFR ADC described herein (e.g., AbA-vc-MMAE) at a dose of 6 mg / kg.

[0410] In another embodiment, a subject in need thereof (e.g., a subject with cancer) is administered an anti-EGFR antibody (ADC) described herein conjugated to a drug (e.g., a PBD) at a dose of 1 to 200 μg / kg. In another embodiment, a subject in need thereof (e.g., a subject with cancer) is administered an anti-EGFR ADC described herein at a dose of 5 to 100 μg / kg. In another embodiment, a subject in need thereof (e.g., a subject with cancer) is administered an anti-EGFR ADC described herein at a dose of 5 to 90 μg / kg. In another embodiment, a subject in need thereof (e.g., a subject with cancer) is administered an anti-EGFR ADC described herein at a dose of 5 to 80 μg / kg. In another embodiment, a subject in need thereof (e.g., a subject with cancer) is administered an anti-EGFR ADC described herein at a dose of 5 to 70 μg / kg. In another embodiment, a subject in need thereof (e.g., a subject with cancer) is administered an anti-EGFR ADC described herein at a dose of 5 to 60 μg / kg.

[0411] The above doses are believed to be useful for administering the anti-EGFR ADCs or antibodies disclosed herein.

[0412] In another aspect, the present application provides an in vitro method for detecting the presence or absence of EGFR in a sample (e.g., a biological sample, such as serum, plasma, tissue, biopsy, etc.). The method can be used to diagnose a disorder (e.g., cancer). The method includes the steps of (i) contacting the sample or a control sample with an anti-EGFR antibody or fragment thereof as described herein; and (ii) detecting the formation of a complex between the anti-EGFR antibody or fragment thereof and the sample or the control sample, and determining the presence of EGFR in the sample if there is a statistically significant difference in the formation of the complex in the sample compared to the control sample.

[0413] The anti-human EGFR antibodies or portions thereof (and ADCs thereof) of the present invention can bind to human EGFR and thus can be used to detect human EGFR (e.g., in biological samples such as serum or plasma) using conventional immunoassays such as enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), or immunohistochemistry. In one aspect, the present invention provides a method for detecting human EGFR in a biological sample, comprising the steps of contacting the biological sample with an antibody or portion thereof of the present invention and detecting either the antibody (or antibody portion) bound to human EGFR or the unbound antibody (or antibody portion), thereby detecting human EGFR in the biological sample. To facilitate detection of the bound or unbound antibody, the antibody is directly or indirectly labeled with a detectable substance. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, -galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material is luminol; and examples of suitable radioactive materials include 3 H, 14 C. 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho or 153 Examples include Sm.

[0414] Instead of labeling the antibody, human EGFR can be quantified in biological fluids by a competitive immunoassay using a detectably labeled rhEGFR standard and an unlabeled anti-human EGFR antibody. In this assay, the biological sample is mixed with the labeled rhEGFR standard and the anti-human EGFR antibody, and the amount of labeled rhEGFR standard bound to the unlabeled antibody is measured. The amount of human EGFR in the biological sample is inversely proportional to the amount of labeled rhEGFR standard bound to the anti-EGFR antibody. Similarly, human EGFR can be quantified in biological samples by a competitive immunoassay using a detectably labeled rhEGFR standard and an unlabeled anti-human EGFR antibody.

[0415] In yet another aspect, the present application provides a method for in vivo detection of the presence of EGFR (e.g., in vivo imaging in a subject). The method can be used to diagnose a disorder (e.g., an EGFR-related disorder). The method includes the steps of (i) administering to a subject or a control subject an anti-EGFR antibody or fragment thereof as described herein under conditions that allow binding of the antibody or fragment to EGFR; and (ii) detecting the formation of a complex between the antibody or fragment and EGFR, and determining the presence of EGFR if there is a statistically significant difference in complex formation in the subject compared to the control subject.

[0416] VI. Pharmaceutical Compositions The present invention also provides pharmaceutical compositions containing an antibody or antigen-binding portion thereof, or ADC of the present invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing an antibody or ADC of the present invention are used, without limitation, for the diagnosis, detection, or monitoring of a disorder, for the prevention, treatment, management, or amelioration of a disorder or one or more symptoms thereof, and / or for research purposes. In a specific embodiment, the composition contains one or more antibodies of the present invention. In another embodiment, the pharmaceutical composition contains one or more antibodies or ADCs of the present invention and one or more prophylactic or therapeutic agents, other than an antibody or ADC of the present invention, for treating a disorder in which EGFR activity is detrimental. Preferably, the prophylactic or therapeutic agent is one known to be useful in, or already used or currently being used for, the prevention, treatment, management, or amelioration of a disorder or one or more symptoms thereof. In accordance with these embodiments, the composition can further contain a carrier, diluent, or excipient.

[0417] The antibodies and antibody portions or ADCs of the present invention can be formulated into pharmaceutical compositions suitable for administration to a subject. Typically, such pharmaceutical compositions contain an antibody or antibody portion of the present invention and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and absorption delaying agents, and the like. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it is preferable to add an isotonic agent, such as a sugar, a polyalcohol (e.g., mannitol, sorbitol), or sodium chloride to the composition. The pharmaceutically acceptable carrier may further contain minor amounts of additives such as wetting agents, emulsifiers, preservatives, or buffers that enhance the shelf life or effectiveness of the antibody, antibody portion, or ADC.

[0418] In one embodiment, the present invention relates to a lyophilized formulation comprising an anti-EGFR antibody-drug conjugate, sucrose, polysorbate 80, and histidine, wherein the formulation has a pH of about 5 to 7, and the anti-EGFR antibody-drug conjugate is an anti-EGFR antibody or an antigen-binding portion thereof conjugated to monomethyl auristatin E (MMAE). In one embodiment, the present invention further provides an anti-EGFR ADC as described herein in which the anti-EGFR antibody or an antigen-binding portion thereof is conjugated to an auristatin (e.g., MMAE), a saccharide (e.g., sucrose), a surfactant (e.g., a polysorbate such as polysorbate 80), and histidine. In one embodiment, the lyophilized formulation contains 1 to 20 mg of histidine, about 320 to 410 mg of a saccharide, about 0.1 to 0.9 mg of a surfactant, and about 1 to 150 mg of an anti-EGFR ADC as described herein in which the anti-EGFR antibody or an antigen-binding portion thereof is conjugated to an auristatin (e.g., MMAE). The present invention further provides an aqueous formulation containing about 1 to 100 mg / ml of an anti-EGFR ADC in which an anti-EGFR antibody or an antigen-binding portion thereof as described herein is conjugated to an auristatin (e.g., MMAE), about 1 to 10 mg / mL of histidine, about 50 to 90 mg / ml of a sugar (e.g., sucrose), and about 0.01 to 0.2 mg / ml of a surfactant (e.g., polysorbate 80).

[0419] A variety of delivery systems are known and can be used to administer one or more antibodies or ADCs of the invention, or a combination of one or more antibodies of the invention and a prophylactic or therapeutic agent useful for preventing, managing, treating, or ameliorating a disorder or one or more symptoms thereof. These delivery systems include, for example, liposomes, microparticles, microcapsules, encapsulation in recombinant cells capable of expressing the antibody or antibody fragment, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), and construction of a nucleic acid as part of a retroviral or other vector. Methods for administering the prophylactic or therapeutic agents of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural administration, intratumoral administration, and mucosal administration (e.g., intranasal and oral routes). Additionally, pulmonary administration can be utilized, for example, by use of an inhaler or nebulizer and formulation with an aerosolizing agent. See, e.g., U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078, and PCT Publication Nos. WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903, the disclosures of each of which are incorporated herein by reference in their entirety. In one embodiment, an antibody, combination therapy, or composition of the invention is administered using Alk...

Claims

1. An anti-human epidermal growth factor receptor (anti-hEGFR) antibody, or an antigen-binding portion thereof, comprising: a) binds to an epitope within the amino acid sequence CGADSYEMEEDGVRKC (SEQ ID NO: 45) or competes with a second anti-hEGFR antibody for binding to epidermal growth factor receptor variant III (EGFRvIII) (SEQ ID NO: 33) in a competitive binding assay, wherein said second anti-EGFR antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5; b) Approximately 1 x 10 as measured by surface plasmon resonance －６ Dissociation constant (K ｄ ) binds to EGFR(1-525) (SEQ ID NO: 47); and c) inhibiting tumor growth in an in vivo human non-small cell lung cancer (NSCLC) xenograft assay by at least about 50% tumor growth inhibition % (TGI%) compared to a human IgG antibody that is not specific for EGFR in the NSCLC xenograft assay, wherein the human IgG antibody that is not specific for EGFR is administered at the same dose and frequency as the anti-hEGFR antibody, or antigen-binding portion thereof; The antibody or antigen-binding portion thereof.

2. When measured by surface plasmon resonance, it is about 1 × 10 －６ M ~ approx. 1 x 10 －１０ K of M ｄ The antibody or antigen-binding portion thereof according to claim 1, which binds to EGFR(1-525) (SEQ ID NO: 47) at the

3. When measured by surface plasmon resonance, it is about 1 × 10 －６ M ~ approx. 1 x 10 －７ K of M ｄ The antibody or antigen-binding portion thereof according to claim 1, which binds to EGFR(1-525) (SEQ ID NO: 47) at the

4. When measured by surface plasmon resonance, it was found to be approximately 8.2 × 10 －９ K below M ｄ 4. The antibody or antigen-binding portion thereof according to claim 1, which binds to EGFRvIII (SEQ ID NO: 33) at the nucleotide sequence indicated by ...

5. When measured by surface plasmon resonance, it was found to be approximately 8.2 × 10 －９ M ~ approx. 6.3 x 10 －１０ K of M ｄ 4. The antibody or antigen-binding portion thereof according to claim 1, which binds to EGFRvIII (SEQ ID NO: 33) at the nucleotide sequence indicated by ...

6. When measured by surface plasmon resonance, it was found to be approximately 8.2 × 10 －９ M ~ approx. 2.0 x 10 －９ K of M ｄ 4. The antibody or antigen-binding portion thereof according to claim 1, which binds to EGFRvIII (SEQ ID NO: 33) at the nucleotide sequence indicated by ...

7. 7. The antibody or antigen-binding portion thereof of any one of claims 1 to 6, which inhibits tumor growth by at least about 60% in an in vivo human non-small cell lung cancer (NSCLC) xenograft assay compared to a human IgG antibody non-specific for EGFR.

8. 7. The antibody or antigen-binding portion thereof of any one of claims 1 to 6, which inhibits tumor growth by at least about 70% in an in vivo human non-small cell lung cancer (NSCLC) xenograft assay compared to a human IgG antibody non-specific for EGFR.

9. 7. The antibody or antigen-binding portion thereof of any one of claims 1 to 6, which inhibits tumor growth by at least about 80% in an in vivo human non-small cell lung cancer (NSCLC) xenograft assay compared to a human IgG antibody non-specific for EGFR.

10. The antibody or antigen-binding portion thereof according to any one of claims 1 to 9, wherein the antibody or antigen-binding portion thereof comprises a heavy chain CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 12, a heavy chain CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 11, and a heavy chain CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 10; and a light chain CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 8, a light chain CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 7, and a light chain CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO:

6.

11. The antibody or antigen-binding portion thereof according to any one of claims 1 to 9, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

5.

12. An antibody or antigen-binding portion thereof according to any one of claims 1 to 9, comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 15 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

13.

13. a light chain CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 40, a light chain CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 39, and a light chain CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 38; a heavy chain CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 37, a heavy chain CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 36, and a heavy chain CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 35; An anti-hEGFR antibody or antigen-binding portion thereof comprising:

14. 1. An anti-hEGFR antibody, or an antigen-binding portion thereof, comprising: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78; and a light chain variable region comprising an amino acid sequence selected from the group consisting of: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, and 79.

15. a heavy chain CDR set (CDR1, CDR2, and CDR3) selected from the group consisting of SEQ ID NOs: 10, 11, and 12; SEQ ID NOs: 16, 17, and 18; SEQ ID NOs: 10, 11, and 19; SEQ ID NOs: 20, 11, and 12; SEQ ID NOs: 21, 3, and 22; SEQ ID NOs: 16, 17, and 19; SEQ ID NOs: 2, 3, and 4; SEQ ID NOs: 10, 3, and 12; SEQ ID NOs: 80, 11, and 18; SEQ ID NOs: 80, 3, and 18; SEQ ID NOs: 20, 3, and 12; SEQ ID NOs: 80, 11, and 12; and SEQ ID NOs: 81, 11, and 22; 1. An anti-hEGFR antibody, or antigen-binding portion thereof, comprising a light chain CDR set (CDR1, CDR2, and CDR3) selected from the group consisting of SEQ ID NOs: 6, 7, and 8; SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 26, 27, and 28; SEQ ID NOs: 29, 30, and 31; SEQ ID NOs: 6, 7, and 84; SEQ ID NOs: 82, 83, and 31; and SEQ ID NOs: 82, 27, and 85, The anti-hEGFR antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof does not simultaneously comprise a heavy chain CDR set of SEQ ID NOs: 2, 3, and 4 and a light chain CDR set of SEQ ID NOs: 6, 7, and 8.

16. An antibody or antigen-binding portion thereof according to any one of claims 1 to 15, comprising a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 41 and / or a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO:

43.

17. 16. The antibody or antigen-binding portion thereof of any one of claims 1 to 15, wherein the antibody or antigen-binding portion thereof comprises a heavy chain immunoglobulin constant region selected from the group consisting of a human IgG constant region, a human IgM constant region, a human IgE constant region, and a human IgA constant region.

18. 18. The antibody or antigen-binding portion thereof of claim 17, wherein the IgG constant region is selected from the group consisting of an IgG1 constant region, an IgG2 constant region, an IgG3 constant region, and an IgG4 constant region.

19. 19. The antibody or antigen-binding portion thereof of any one of claims 1 to 18, wherein the antibody is a multispecific antibody.

20. The antibody or antigen-binding portion thereof may be Fab, Fab', F(ab') ２ 20. The antibody or antigen-binding portion thereof of any one of claims 1 to 19, wherein the antibody or antigen-binding portion thereof is selected from the group consisting of a Fv, a disulfide-linked Fv, a scFv, a single domain antibody, and a diabody.

21. 21. The antibody or antigen-binding portion thereof of claim 1, wherein the antibody or antigen-binding portion thereof is conjugated to an imaging agent.

22. 22. The antibody or antigen-binding portion thereof of claim 21, wherein the imaging agent is selected from the group consisting of a radioactive label, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin.

23. 23. The antibody or antigen-binding portion thereof of claim 22, wherein the radiolabeled substance is indium.

24. 24. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof of any one of claims 1 to 23 and a pharmaceutically acceptable carrier.

25. An antibody-drug conjugate (ADC) comprising the antibody or antigen-binding portion thereof described in any one of claims 1 to 23 conjugated to at least one drug.

26. 26. The ADC of claim 25, wherein the at least one drug is selected from the group consisting of an anti-apoptotic agent, an antimitotic agent, an antitumor antibiotic, an immunomodulatory agent, a gene therapy nucleic acid, an alkylating agent, an anti-angiogenic agent, an antimetabolite, a boron-containing agent, a chemotherapeutic agent, a hormonal agent, an antihormonal agent, a corticosteroid, a photosensitive therapeutic agent, an oligonucleotide, a radionuclide agent, a radiosensitizer, a topoisomerase inhibitor, and a tyrosine kinase inhibitor.

27. 27. The ADC of claim 26, wherein the antimitotic agent is selected from the group consisting of dolastatins, auristatins, maytansinoids, and plant alkaloids.

28. 28. The ADC of claim 27, wherein the auristatin is monomethyl auristatin F (MMAF) or monomethyl auristatin E (MMAE).

29. 28. The ADC of claim 27, wherein the maytansinoid is selected from the group consisting of DM1, DM2, DM3, and DM4.

30. 27. The ADC of claim 26, wherein the antitumor antibiotic is selected from the group consisting of actinomycin, anthracycline, a calicheamicin, and a duocarmycin.

31. 31. A pharmaceutical composition comprising an ADC mixture comprising a plurality of ADCs according to any one of claims 25 to 30 and a pharmaceutically acceptable carrier.

32. 32. The pharmaceutical composition of claim 31, wherein the ADC mixture has an average drug-to-antibody ratio (DAR) of 2 to 4.

33. 32. The pharmaceutical composition of claim 31, wherein the ADC mixture comprises ADCs each having a DAR of 2 to 8.

34. 34. A method of treating a subject with cancer, comprising administering to said subject a pharmaceutical composition of any one of claims 24 or 31 to 33 so as to treat said subject with cancer.

35. 35. The method of claim 34, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, glioblastoma, prostate cancer, pancreatic cancer, colon cancer, head and neck cancer, and kidney cancer.

36. 35. The method of claim 34, wherein the cancer is squamous cell carcinoma.

37. 37. The method of claim 36, wherein the squamous cell carcinoma is lung squamous cell carcinoma or head and neck squamous cell carcinoma.

38. 35. The method of claim 34, wherein the cancer is triple-negative breast cancer.

39. 35. The method of claim 34, wherein the cancer is non-small cell lung cancer.

40. 40. The method of any one of claims 34 to 39, wherein the cancer is characterized by EGFR overexpression.

41. 34. A method for inhibiting or suppressing solid tumor growth in a subject having a solid tumor, the method comprising administering to the subject having the solid tumor a pharmaceutical composition described in any one of claims 24 or 31 to 33, so as to inhibit or suppress the solid tumor growth.

42. 34. A method of inhibiting or suppressing solid tumor growth in a subject having a solid tumor, comprising administering to the subject having the solid tumor an effective amount of an antibody or ADC of any one of claims 1 to 23 or 25 to 30 so as to inhibit or suppress the solid tumor growth.

43. 43. The method of claim 41 or 42, wherein the solid tumor is an EGFR-expressing solid tumor or an EGFRvIII-positive solid tumor.

44. 43. The method of claim 41 or 42, wherein the solid tumor is an EGFR-overexpressing solid tumor.

45. 45. The method of any one of claims 41 to 44, wherein the solid tumor is non-small cell lung cancer or glioblastoma.

46. 46. ​​The method of any one of claims 34 to 45, wherein the antibody, ADC or pharmaceutical composition is administered in combination with another drug or therapy.

47. 47. The method of claim 46, wherein the additional agent is selected from the group consisting of an anti-PD1 antibody, an anti-CTLA-4 antibody, temozolomide, a bcl-xl inhibitor, ibrutinib, duvelisib, idelalisib, venetoclax, and a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor.

48. 47. The method of claim 46, wherein the additional therapy is radiation.

49. 47. The method of claim 46, wherein the additional agent is a chemotherapeutic agent.

50. 24. An isolated nucleic acid encoding the antibody or antigen-binding portion thereof of any one of claims 1 to 23.

51. A vector comprising the nucleic acid of claim 50.

52. 52. A host cell comprising the vector of claim 51.

53. 53. The host cell of claim 52, which is a prokaryotic or eukaryotic cell.

54. 54. The host cell of claim 53, wherein the eukaryotic cell is selected from the group consisting of an animal cell, a protozoan cell, a plant cell, and a fungal cell.

55. 55. The host cell of claim 54, wherein the animal cell is selected from the group consisting of a mammalian cell, an insect cell, and an avian cell.

56. 56. The host cell of claim 55, wherein the mammalian cell is selected from the group consisting of a CHO cell, a COS cell, and an Sp2 / 0 cell.

57. An anti-hEGFR antibody drug conjugate (ADC) comprising an anti-hEGFR antibody conjugated to an auristatin, wherein the antibody comprises a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10; and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 8, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO:

6.

58. 58. The ADC of claim 57, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

5.

59. 59. The ADC of claim 57 or 58, wherein the antibody comprises an IgG heavy chain immunoglobulin constant region.

60. 60. The ADC of claim 59, wherein the IgG is an IgG1 or IgG4 heavy chain immunoglobulin constant region.

61. 61. The ADC of any one of claims 57 to 60, wherein the auristatin is monomethyl auristatin F (MMAF) or monomethyl auristatin E (MMAE).

62. 59. The ADC of claim 57 or 58, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising the amino acid sequence of SEQ ID NO:

13.

63. 62. The ADC of claim 61, wherein the antibody is covalently attached to the auristatin by a linker comprising maleimidocaproyl, valine-citrulline, p-aminobenzyl alcohol (mc-vc-PABA).

64. 64. The ADC of any one of claims 57 to 63, wherein the ADC comprises a radiolabeled substance.

65. 65. The ADC of claim 64, wherein the radiolabel is indium.

66. 66. A pharmaceutical composition comprising the ADC of any one of claims 57 to 65 and a pharmaceutically acceptable carrier.

67. 66. A pharmaceutical composition comprising an ADC mixture comprising the ADC of any one of claims 57 to 65, wherein the ADC mixture has an average drug-to-antibody ratio (DAR) in the range of 2 to 4.

68. 1. A pharmaceutical composition comprising an anti-hEGFR antibody drug conjugate (ADC) mixture and a pharmaceutically acceptable carrier, wherein the ADC mixture has an average drug-to-antibody ratio (DAR) of 2 to 4, and the ADC is an anti-hEGFR antibody comprising: a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a heavy chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10; and a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 8, a light chain CDR2 domain comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 6, conjugated to monomethyl auristatin E (MMAE).

69. 69. The pharmaceutical composition of claim 68, wherein the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID NO:9 and the light chain variable region of the anti-EGFR antibody comprises the amino acid sequence set forth in SEQ ID NO:

5.

70. 70. The pharmaceutical composition of claim 68 or 69, wherein the antibody comprises an IgG heavy chain immunoglobulin constant region.

71. 71. The pharmaceutical composition of claim 70, wherein the IgG is an IgG1 or IgG4 heavy chain immunoglobulin constant region.

72. 70. The pharmaceutical composition of claim 68 or 69, wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 15 and a light chain comprising the amino acid sequence of SEQ ID NO:

13.

73. 73. The pharmaceutical composition of any one of claims 68 to 72, wherein the MMAE is attached to the antibody by a linker comprising maleimidocaproyl, val-cit, or PABA.

74. 74. A method of treating a subject with cancer, comprising administering to said subject a pharmaceutical composition according to any one of claims 68 to 73 so as to treat said subject with cancer.

75. 75. The method of claim 74, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, glioblastoma, prostate cancer, pancreatic cancer, colon cancer, head and neck cancer, and kidney cancer.

76. 75. The method of claim 74, wherein the cancer is squamous cell carcinoma.

77. 77. The method of claim 76, wherein the squamous cell carcinoma is lung squamous cell carcinoma or head and neck squamous cell carcinoma.

78. 75. The method of claim 74, wherein the cancer is triple-negative breast cancer.

79. 75. The method of claim 74, wherein the cancer is non-small cell lung cancer.

80. 80. The method of any one of claims 74 to 79, wherein the cancer is characterized by EGFR overexpression.

81. 74. A method for inhibiting or suppressing solid tumor growth in a subject having a solid tumor, comprising administering to the subject having the solid tumor a pharmaceutical composition described in any one of claims 66 to 73, such that the solid tumor growth is inhibited or suppressed.

82. 82. The method of claim 81, wherein the solid tumor is non-small cell lung cancer or glioblastoma.

83. 82. The method of claim 81, wherein the solid tumor is squamous cell carcinoma.

84. 84. The method of any one of claims 81 to 83, wherein the solid tumor is an EGFRvIII-positive solid tumor or an EGFR-expressing solid tumor.

85. 84. The method of any one of claims 81 to 83, wherein the solid tumor overexpresses EGFR.

86. 86. The method of any one of claims 74 to 85, wherein the pharmaceutical composition is administered in combination with another drug or therapy.

87. 87. The method of claim 86, wherein the additional agent is selected from the group consisting of an anti-PD1 antibody, an anti-CTLA-4 antibody, temozolomide, a bcl-xl inhibitor, ibrutinib, duvelisib, idelalisib, venetoclax, and a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor.

88. 87. The method of claim 86, wherein the additional therapy is radiation.

89. 87. The method of claim 86, wherein the additional agent is a chemotherapeutic agent.

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