CN116333118B

CN116333118B - Anti-EGF receptor antibody, preparation method and application thereof

Info

Publication number: CN116333118B
Application number: CN202111542303.2A
Authority: CN
Inventors: J·Y·李
Original assignee: Laitekang Suzhou Biopharmaceutical Co ltd
Current assignee: Laitekang Suzhou Biopharmaceutical Co ltd
Priority date: 2021-12-16
Filing date: 2021-12-16
Publication date: 2024-04-19
Anticipated expiration: 2041-12-16
Also published as: CN116333118A

Abstract

The present invention provides an anti-human Epidermal Growth Factor Receptor (EGFR) antibody or antigen binding fragment thereof. The invention further provides bispecific or multispecific molecules, immunoconjugates comprising the antibodies or antigen-binding fragments thereof, and nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof, vectors and host cells comprising the nucleic acid molecules. The invention also provides methods of making the antibodies or antigen-binding fragments thereof, pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof, and methods and uses of the antibodies or antigen-binding fragments thereof, or pharmaceutical compositions thereof, in the treatment of diseases, the invention provides candidate specific antibody drugs for the treatment and/or prevention of anti-tumor or other diseases or disorders (e.g., inflammatory and autoimmune diseases) against EGFR.

Description

Anti-EGF receptor antibody, preparation method and application thereof

Technical Field

The present invention relates to anti-Epidermal Growth Factor Receptor (EGFR) antibodies or antigen binding fragments thereof, polynucleotides encoding the antibodies or antigen binding fragments thereof, expression vectors and host cells, pharmaceutical compositions comprising the antibodies or antigen binding fragments thereof, and uses or methods for treating a subject.

Background

The epidermal growth factor receptor (EPIDERMAL GROWTH FACTOR RECEPTOR, EGFR), also known as ErbB-1 or HER1, is one of the members of the ErbB receptor tyrosine kinase superfamily, which is a multifunctional transmembrane glycoprotein encoded by the proto-oncogene c-erbB and having a molecular weight of about 170kDa, is composed primarily of an extracellular region involved in ligand binding, a transmembrane region, and an intracellular region having tyrosine kinase activity (Modjtahedi et al, br. J. Cancer 73:228-235, 1996;Herbst and Shin,Cancer 94:1593-1611, 2002). Known ligands for EGFR include Epidermal Growth Factor (EGF), transforming growth factor alpha (Transforming Growth Factor-alpha, TGF-alpha), epidermal regulator/EREG and HBEGF/heparin binding EGF, bi-regulating factors, betacellulin/BTC, epithelial cell mitoproteins (epi)/EPGN, EGFR triggering EGFR homodimerization or heterodimerization upon ligand binding, resulting in autophosphorylation of EGFR intracellular tyrosine kinase active regions, phosphorylated EGFR recruitment adaptor proteins (e.g., GRB 2) in turn activating downstream signaling pathways, in turn regulating various cell physiological processes, e.g., regulating biological functions such as proliferation, differentiation, apoptosis, inflammatory response and angiogenesis of cells by RAS-RAF-MEK-ERK) signaling pathways (e.g., RAS-RAF-MEK-ERK), regulating cell proliferation and cytoskeletal recombination, transport, cell adhesion and motility, vascular regeneration and action by PI3 kinase-AKT signaling pathways (PI 3K), and regulating vascular endothelial cell proliferation and vascular regeneration and vascular proliferation by means of F-K signaling pathways (e.g., RAF-EGFR) signaling pathways (e.g., RAF-EGF-EGFR) signaling pathways (e.35: 35, 2003-35, 35F-35, and so on). Ligand binding through EGFR can also activate NF- κ -B signaling cascades, thereby participating in inflammatory responses, immune responses, and modulating apoptosis and stress responses in the body. EGFR binding to the ligand may also directly phosphorylate other proteins (e.g., RGS 16), thereby activating its gtpase activity and possibly signaling EGF receptors to G-protein coupled receptors. EGFR binding to ligands also phosphorylates MUC1 and increases its interaction with SRC and CTNNB 1/beta-catenin, etc., thereby modulating biological functions such as cell growth and cell adhesion.

Studies have shown that there is either high or abnormal expression of EGFR in a variety of solid tumors (e.g., non-small cell lung cancer, colon cancer, kidney cancer, ovarian cancer, head and neck cancer, esophageal cancer, prostate cancer, pancreatic cancer, breast cancer, glioma, bladder cancer, kidney cancer, etc. (Woodburn JR, pharmacol thier 1999;82:241-50;Yarden Y,Eur J Cancer 2001;37:S3-S8; mendelsohn J et al, oncogene2000; 19:6550-65)), possible mechanisms of EGFR signaling abnormality include EGFR overexpression, EGFR mutation, enhancement of the function of the autocrine loop, increased ligand expression, disruption of the receptor down-regulation mechanism, activation of abnormal signaling pathways, etc. (Roza Zandi et al, cellular Signalling,2007, 19:2013-2023). Overexpression and/or proliferation of EGFR on tumor cell membranes is associated with poor prognosis, reduced survival and higher tumor invasiveness in tumor patients. In addition, EGFR is also expressed in normal tissues (e.g., epithelial tissues of the skin, liver, and gastrointestinal tract), but at much lower levels than tumor tissues (Herbst and Shin, cancer 94:1593-1611 (2002)).

Currently, monoclonal antibodies targeting the extracellular domain portion of EGFR include Cetuximab (also known as IMC-225, erbitux), panitumumab (Panitumumab, also known as ABX-EGF), cetuximab (Necitumumab, also known as IMC-11F 8) and Matuzumab (EMD 72000), which block the binding of EGFR receptors to ligands and thus inhibit ligand-mediated activation of EGFR tyrosine kinase or interfere with EGFR dimerization, and as a result of blocking receptor-ligand binding, the physiological processes of tumor cells regulated by EGFR signaling pathways in tumor cells or in stromal cells in the tumor microenvironment are disrupted, thereby inhibiting tumor cell growth, inducing apoptosis, reducing the production of matrix metalloproteinases and vascular epithelial growth factors, thereby exerting an anti-tumor effect (Fan Z et al, 1994; abanell J et al, 2001; prewett M et al, 1996, hub et al SM, 1999, fan et al, 1993a Z et al). In addition, cetuximab and cetuximab also induce EGFR internalization and degradation, triggering EGFR ⁺ tumor cells to develop antibody-dependent cytotoxicity (anti-DEPENDENT CELL-mediated cytotoxicity, ADCC) (Kawaguchi Y et al, 1996; kimura H et al, 2007). ADCC is dependent on the interaction between cellular fcγr and monoclonal antibodies, triggering an innate immune response that includes NK cells, monocytes, macrophages, activated T lymphocytes, and granulocytes. Receptor internalization down-regulates the number of cell surface receptors available and can therefore affect EGFR activation. Although panitumumab is a fully human monoclonal antibody prepared by transgenic mouse technology, the mechanism of action is competitive binding to EGFR on tumor cells, blocking EGFR binding to ligands EGF and TGFa, inducing EGFR internalization, eliminating the cell effects mediated by EGFR, panitumumab can significantly reduce the immunogenicity of human murine chimeric antibodies when applied in humans, panitumumab is an IgG2 subtype antibody, ADCC and/or CDC activity of IgG2 is significantly reduced compared to IgG1, and stability of IgG2 subtype antibodies is poor, which may be why panitumumab has no significant advantage compared to cetuximab in terms of clinical efficacy.

Although currently marketed EGFR-targeting antibodies and some EGFR antibodies are in clinical trial research, the current antibodies are not always effective in the treatment of egfr+ tumors, e.g., cetuximab and panitumumab are only effective in treating KRAS wild-type tumors expressed by EGFR, but have no inhibitory effect on KRAS mutated tumors, thus limiting the clinical use of cetuximab and panitumumab. Even though patients who have a therapeutic response to cetuximab or panitumumab and benefit from the antibody may develop resistance to the antibody over a period of time, one of the reasons for developing resistance is one or more mutations in the EGFR extracellular domain. In addition, since various normal tissues express EGFR, anti-EGFR antibodies target EGFR-expressing normal tissues also when anti-tumor therapy is performed, resulting in side effects such as skin injury and/or allergic reactions, and sometimes serious side effects may pose a life hazard to patients.

Given that currently the problem of treatment of more solid tumors with high or abnormal expression of EGFR has not been solved, it is desirable in the art to develop more new anti-EGFR antibodies to meet the therapeutic needs.

Disclosure of Invention

The invention provides an anti-EGFR antibody, a preparation method thereof and application thereof in EGFR over-expression or abnormal expression.

In one aspect, the invention provides an antibody or antigen binding fragment capable of specifically binding EGFR.

In some embodiments, the anti-EGFR antibody, or antigen-binding fragment thereof, specifically binds to the extracellular domain of human EGFR. In certain embodiments, the anti-EGFR antibody, or antigen-binding fragment thereof, specifically binds human EGFR with an EC50 value of no more than 1nM, preferably no more than 0.5nM, as measured by ELISA. In some embodiments, the anti-EGFR antibody or antigen-binding fragment thereof has a binding activity that is slightly less than cetuximab. In certain embodiments, the anti-EGFR antibody or antigen-binding fragment thereof binds to human EGFR with high affinity, its binding and dissociation constants are detected by binding kinetics, and the affinity constant (KD) value is no more than 5 x 10 ^-8 M, preferably the KD value is about 10 ^-8 M, as calculated by fitting.

In some embodiments, the anti-EGFR antibody or antigen binding fragment thereof is not cross-reactive with other human ErbB receptor tyrosine kinases, such as HER2, HER3, and/or HER 4.

In some embodiments, the anti-EGFR antibody or antigen-binding fragment thereof has an inhibitory effect on the proliferation activity of tumor cells expressing EGFR. In some embodiments, the anti-EGFR antibody or antigen-binding fragment thereof has an IC50 value for tumor cells of no more than 1nM, preferably no more than 0.1nM, as detected by ELISA.

In some embodiments, the anti-EGFR antibody, or antigen-binding fragment thereof, comprises HCDR1 as shown in SEQ ID No.1, as shown in SEQ ID NO:2, and HCDR2 as set forth in SEQ ID NO:3, or any one, two or three heavy chain complementarity determining region (HCDR) amino acid sequences of HCDR 3. In some embodiments, the anti-EGFR antibody, or antigen-binding fragment thereof, comprises HCDR1 as shown in SEQ ID No.1, as shown in SEQ ID NO:2, and HCDR2 as set forth in SEQ ID NO:3 or an amino acid sequence at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identical to said HCDR1, HCDR2 and HCDR3, respectively.

In some embodiments, the anti-EGFR antibody or antigen binding fragment thereof comprises an LCDR1 as shown in SEQ ID NO. 4, an LCDR2 as shown in SEQ ID NO. 5, and any one, two or three light chain complementarity determining region (LCDR) amino acid sequences of LCDR3 as shown in SEQ ID NO. 6. In some embodiments, the anti-EGFR antibody or antigen binding fragment thereof comprises LCDR1 as set forth in SEQ ID NO. 4, LCDR2 as set forth in SEQ ID NO. 5, and LCDR3 as set forth in SEQ ID NO. 6, or has at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identical amino acid sequence to the LCDR1, LCDR2 and LCDR3, respectively, and retains specific binding activity to human EGFR.

In some embodiments, the anti-EGFR antibody or antigen binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID NO. 7, or has an amino acid sequence at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identical to the heavy chain variable region, and retains a specific binding activity to human EGFR.

In some embodiments, the anti-EGFR antibody or antigen binding fragment thereof comprises a light chain variable region as set forth in SEQ ID NO. 8, or has an amino acid sequence at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identical to the light chain variable region and retains a specific binding activity to human EGFR.

In another aspect, the invention provides nucleic acid molecules encoding the anti-EGFR antibodies or antigen-binding fragments thereof of the invention, as well as expression vectors comprising the nucleic acids and host cells comprising the nucleic acids or expression vectors. The invention also relates to methods of using the host cells to make the anti-EGFR antibodies or antigen-binding fragments thereof of the invention.

In another aspect, the invention also provides a method of producing an anti-EGFR antibody, or antigen-binding fragment thereof, of the invention using a host cell comprising a nucleic acid or expression vector, the method comprising (1) expressing the antibody in the host cell, and (2) isolating the antibody from the host cell or cell culture thereof.

In another aspect, the invention provides bispecific or multispecific molecules, immunoconjugates, chimeric antigen receptors, engineered T cell receptors, or oncolytic viruses comprising the anti-EGFR antibodies or antigen binding fragments thereof of the invention.

In some embodiments, the bispecific or multispecific molecule comprises another functional molecule selected from an Fc receptor, an antibody specific for a cancer-associated antigen (TAA) or an immune checkpoint protein antigen, or an antigen-binding fragment thereof. In some embodiments, the bispecific molecule or multispecific molecule may further comprise a third binding specificity, such as an anti-Enhancement Factor (EF) moiety.

In some embodiments, the immunoconjugate comprises a therapeutic agent, or a detectable label (e.g., a radioisotope, luminescent label, fluorescent label, or enzyme substrate label) coupled to an antibody or antigen-binding fragment thereof of the invention.

In another aspect, the invention provides a pharmaceutical composition comprising an anti-EGFR antibody, or antigen-binding fragment thereof, of the invention, or a bispecific or multispecific antibody, immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or oncolytic virus comprising an antibody, or antigen-binding fragment thereof, of the invention, and a pharmaceutically acceptable carrier.

In another aspect, the invention provides a kit comprising an effective amount of a pharmaceutical composition of an anti-EGFR antibody, or antigen-binding fragment thereof, of the invention, and one or more other therapeutic agents selected from the group consisting of chemotherapeutic agents, anti-cancer agents, and immunomodulatory agents.

In another aspect, the invention provides a kit comprising an anti-EGFR antibody, or antigen-binding fragment thereof, of the invention, or a pharmaceutical composition comprising an anti-EGFR antibody, or antigen-binding fragment thereof, of the invention, or an immunoconjugate of the antibody, or antigen-binding fragment thereof, of the invention, with a detectable label.

In another aspect, the invention provides the use of an anti-EGFR antibody or antigen binding fragment thereof of the present invention in the manufacture of a pharmaceutical composition or formulation for the treatment and/or prevention of EGFR related diseases or disorders, in the manufacture of a diagnostic agent for the diagnosis of EGFR related diseases or disorders. In some embodiments, the disease or disorder is cancer or other EGFR-related disease. In some embodiments, the cancer includes, but is not limited to, melanoma, lung cancer (non-small cell lung cancer or small cell lung cancer), colorectal cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, renal cancer or cell carcinoma, liver cancer, esophageal cancer, gall bladder cancer, pancreatic cancer, gastric cancer, thyroid cancer, bladder cancer, head and neck cancer, glioblastoma, skin cancer, and other solid and/or metastatic cancers. In some embodiments, the other EGFR-related disease includes, but is not limited to, autoimmune disease, psoriasis, or inflammatory arthritis (e.g., rheumatoid arthritis, systemic lupus erythematosus-related arthritis, psoriatic arthritis), or other cell proliferative diseases other than cancer (including adrenocortical hyperplasia (cushing's disease), congenital adrenocortical hyperplasia, endometrial hyperplasia, benign prostatic hyperplasia, breast hyperplasia, intimal hyperplasia, focal epithelial hyperplasia (Heck's disease), sebaceous gland hyperplasia, compensatory liver hyperplasia, and any other cell proliferative disease).

In another aspect, the invention provides a method of treating and/or preventing an EGFR-related disease or disorder, the method comprising administering to a subject an effective amount of an anti-EGFR antibody, or antigen-binding fragment thereof, or a pharmaceutical composition, or kit of parts, of the invention. In some embodiments, the anti-EGFR antibodies, or antigen-binding fragments thereof, pharmaceutical compositions, or kits of the invention may be administered alone, or in combination with one or more other therapeutic agents, including chemotherapeutic agents, anti-cancer agents, immunomodulatory agents, or therapeutic agents known in the art, including, but not limited to, surgery, chemotherapy, cytotoxic agents, photodynamic therapy, immunomodulation, or radiation therapy, as disclosed herein. In some embodiments, the anti-EGFR antibodies, or antigen-binding fragments thereof, pharmaceutical compositions, or kits of the invention may be administered simultaneously with one or more other therapeutic agents, or may be administered separately from the other therapeutic agents.

In another aspect, the invention provides a method of modulating EGFR activity in an EGFR-overexpressing cell, comprising eliciting one or more biological activities in vivo or in vitro, e.g., inhibiting EGF or TGF- α -induced autophosphorylation in an EGFR-expressing cell, using an anti-EGFR antibody, or antigen-binding fragment thereof, or a pharmaceutical composition or kit thereof, of the invention; inhibiting activation of EGFR cells expressed by induction of autocrine EGF or TGF-alpha; or inhibit the growth or proliferation of EGFR-expressing cells.

In another aspect, the invention provides a method for detecting the level of EGFR in a test sample, comprising contacting the test sample with an antibody or antigen binding fragment thereof of the invention, and determining the level of EGFR in the test sample, or detecting the level of EGFR in the test sample using a kit of the invention.

In another aspect, the invention provides a method of detecting, diagnosing or monitoring an EGFR-related disease or disorder in a subject, the method comprising: (1) Detecting the content or expression level of EGFR in a test sample obtained from a subject using a kit of the invention, or contacting the test sample obtained from the subject with an anti-EGFR antibody or antigen-binding fragment thereof of the invention, detecting the content or expression level of EGFR in the test sample; (2) Diagnosing the subject for the presence or absence of an EGFR-related disease or disorder or a condition of the disease or disorder.

Other features and advantages of the present disclosure will be apparent from the following drawings and detailed description, which should not be construed as limiting the scope of the application, and variations which will be apparent to those skilled in the art are intended to be included within the spirit of the application and the scope of the appended claims. All references, including publications, patents, and patent applications cited in this disclosure are incorporated by reference in their entirety.

Drawings

Figure 1 elisa assay for binding activity of anti-EGFR antibodies and control antibodies (cetuximab) to the antigen hEGFR, respectively.

Figure 2 elisa method to detect cross-reactivity of anti-EGFR antibodies and control antibodies (cetuximab) with HER2, HER3 and HER4, respectively.

FIG. 3 detection of the effect of anti-EGFR antibodies and control antibodies (cetuximab) on proliferation activity of A431 cell lines.

Detailed Description

Definition of the definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.

Herein, "EGFR" may be derived from any vertebrate source, including mammals, such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats). Exemplary sequences for human EGFR include full length human EGFR protein Genbank accession No.: NP-005219.2), the specific sequences are as follows:

MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA

The amino acid sequence of the human EGFR extracellular domain structural protein (ECD) used in the present invention is as follows:

LEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCP

Herein, the term "EGFR" is intended to encompass any form of EGFR, e.g., (1) naturally untreated EGFR molecules, "full length" EGFR chains or naturally occurring EGFR variants, including, e.g., splice variants or allelic variants; (2) any form of EGFR produced after intracellular treatment; or (3) a full length, fragment (e.g., truncated form, extracellular/transmembrane domain) or modified form thereof (e.g., mutant form, glycosylated/pegylated, his-tag/immunofluorescent fusion form) of an EGFR subunit produced by recombinant means.

In this context, the term "affinity" or "binding affinity" refers to the inherent binding capacity of an interaction between a molecule (e.g., a receptor) and its counterpart (e.g., a ligand or antibody), i.e., the strength of the sum of all non-covalent interactions. As used herein, unless otherwise stated, a "binding affinity" is an intrinsic binding affinity that is used to reflect a 1:1 interaction between members of a binding pair (e.g., a receptor and a ligand or antibody). The affinity of a molecule X for its partner Y can generally be expressed in terms of an equilibrium dissociation constant (K _D), which is the ratio of the dissociation rate constant and the association rate constant (K dissociation (K _dis or K _Off) and K association (Ka or Kon), respectively.

Herein, "antibody" refers to a protein comprising one or more polypeptides encoded substantially or partially by immunoglobulin genes or fragments of immunoglobulin genes. Putative immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, which in turn define immunoglobulin classes IgG, igM, igA, igD and IgE, respectively. Antibodies can be of any isotype/class (e.g., igG, igM, igA, igD and IgE) or of any subclass (e.g., igG1, igG2, igG3, igG4, igA1, igA 2). Typical immunoglobulin (e.g., antibody) building blocks include tetramers. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" (about 50-70 kD) chain. Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used structurally and functionally. The N-terminus of each chain defines a variable (V) region or domain of about 100 to 110 amino acids or more that is primarily responsible for antigen recognition. As used herein, "antibody" is intended to be the broadest sense and includes a variety of antibody structures that exhibit the desired antigen binding activity, and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments.

Antibodies exist as intact immunoglobulins or as a variety of well-characterized fragments produced by digestion with peptidases. Herein, the term "antigen binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment") refers to an antibody fragment formed from an antibody portion containing one, or two, or more CDRs (complementarity determining regions), or any other antibody fragment that binds an antigen (e.g., IL-2 protein) but does not have the complete antibody structure. The antigen binding fragment may bind the same antigen as the whole antibody. In certain embodiments, an antigen binding fragment may contain one, or two, or more CDRs from a particular human antibody, grafted to a framework region from one, or two, or more different human antibodies. Antigen binding fragments include, but are not limited to, fab ', F (ab ') 2, fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv) 2, bispecific dsFv (dsFv-dsFv '), disulfide stabilized diabodies (dsdiabody), single chain antibody molecules (scFv), scFv dimers (diabodies), diabodies (BsFv), camelylated single domain antibodies (camelized single domain antibody), nanobodies, domain antibodies, and diabodies. For example, a "Fab" fragment of an antibody refers to an antibody fragment that consists of one light chain (including the light chain variable region and the light chain constant region) disulfide-bonded to the variable region of one heavy chain and CH 1. "Fab'" fragment refers to a Fab fragment which comprises part of the hinge region. "F (ab') 2" refers to the dimer of Fab. The "Fc" fragment of an antibody is an antibody fragment in which CH2 and CH3 of the heavy chain are linked by disulfide bonds. The Fc fragment of an antibody is responsible for a number of different effector functions, such as determining the half-life of the antibody in vivo serum, mediating immune responses, e.g., antibody-dependent cell-mediated cytotoxicity (anti-body-DEPENDENT CELL-mediated cytotoxicity, ADCC), activating complement-dependent cytotoxicity (Complement Dependent Cytotoxicity, CDC) or antibody-dependent cell-mediated phagocytosis (Antibody Dependent Cellular Phagocytosis, ADCP), but not involved in antigen binding. The "Fv" portion of an antibody refers to the smallest antibody fragment that contains the complete antigen binding site. Fv fragments consist of a variable region of the light chain and a variable region of the heavy chain. "Single chain antibody" or "Single chain Fv antibody (scFv)" refers to an engineered antibody/single protein chain format in which the light chain variable region and the heavy chain variable region are paired to form a monovalent molecule, i.e., scFv, either directly linked to the heavy chain variable region or linked by one peptide chain using recombinant methods (Huston JS et al, proc NATL ACAD SCI USA, 85:5879 (1988)). "(dsFv) 2" contains three peptide chains: refers to two VH groups connected by a single polypeptide linker and bound to two VL groups by disulfide bonds. A "bispecific ds bifunctional antibody" comprises VL1-VH2 (linked by a polypeptide linker) and VH1-VL2 (also linked by a polypeptide linker), which are joined by disulfide bonds between VH1 and VL 1. "bispecific dsFv" or "dsFv-dsFv" comprise three polypeptide chains: VH1-VH2 fragments wherein the heavy chains are linked by a polypeptide linker (e.g., a long elastic linker) and are bound to VL1 and VL2 fragments, respectively, by disulfide bonds, each pair of heavy and light chains paired by disulfide bonds having different antigen specificities. "scFv dimer" is a diabody or diabody (BsFv) comprising two VH-VL (linked by a polypeptide linker) fragments that dimerize, wherein the VH of one fragment cooperates with the VL of the other fragment to form two binding sites that can be targeted to bind the same antigen (or antigen-binding epitope) or different antigens (or antigen-binding epitopes). In other embodiments, the "scFv dimer" is a bispecific bifunctional antibody comprising VL1-VH2 (linked by a polypeptide linker) and VH1-VL2 (linked by a polypeptide linker) that are linked to each other, wherein VH1 and VL1 cooperate, VH2 and VL2 cooperate, and each cooperating pair has a different antigen specificity. "Single chain antibody Fv-Fc (scFv-Fc)" refers to an engineered antibody consisting of scFv and an antibody Fc fragment. "camelized single-domain antibody (Camelized single domain antibody)", "heavy chain antibody" or "HCAb (heavy-chain antibodies, HCAb)" refer to antibodies comprising two VH domains but no light chain (Riechmann L. And Muyldermans S.,J Immunol Methods.Dec 10;231(1-2):25-38(1999);Muyldermans S.,J Biotechnol.Jun;74(4):277-302(2001);WO94/04678;WO94/25591;U.S.Patent No.6,005,079). heavy chain antibodies originally found in camelidae (including camel, dromedary and llama),. Although the light chain is deleted, camelized antibody (camelized antibodies) has the full function of antigen binding (Hamers-Casterman C. Et al., nature. Jun 3;363 (6428): 446-8 (1993) ",. Nguyen VK. et al. ,"Heavy-chain antibodies in Camelidae：a case of evolutionary innovation,"Immunogenetics.Apr;54(1):39-47(2002);Nguyen VK., et cetera), immunology. Main.; 109 (1): 93-101 (2003)). Heavy chain antibodies' variable region (VHH domain) is the least currently known antigen binding unit (VL-NolteF. Et al., FASEB J. Nov;21 (3490-8),. 2005) is a fragment comprising a light chain polypeptide or two VH-binding fragment (VH-3) that is a fragment of either of the same heavy-chain or two VH-domain (VH-domain antibody fragment or two variable region binding chains can be formed by the same heavy-chain polypeptide (VH-heavy-domain or two VH-heavy-chain fragment) and the same polypeptide domain binding domain (VH-binding domain or two VH-fragment) as in the case, proc NATL ACAD SCI usa. Jul 15;90 6444-8 (1993); EP404097; WO 93/11161). The linker between the two domains is so short that the two domains on the same strand cannot mate with each other, forcing the two domains to mate with the complementary domains of the other strand, forming two antibody binding sites. The two antibody binding sites may be targeted to bind the same or different antigens (or antigen binding epitopes).

The anti-EGFR antibodies of the invention include all or antigen-binding fragments thereof (as defined above) of full-length antibodies, and optionally comprise all or part of an antibody variable region capable of competitively binding EGFR with an EGFR ligand, such as EGF, and optionally include one, or two, or more regions encoded by the V gene and/or D gene and/or J gene.

Herein, the term "immunoglobulin molecule" refers to a protein having a naturally occurring antibody structure. For example, an immunoglobulin of the IgG class is a heterotetrameric glycoprotein of about 150,000 daltons, which is composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminal to the C-terminal, each heavy chain has a variable region (VH, also known as a variable heavy domain or heavy chain variable domain) followed by 3 constant domains (CH 1, CH2 and CH3, also known as heavy chain constant regions). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL, also known as a variable light domain or light chain variable region), followed by a constant light domain (also known as a light chain constant region, CL). The heavy chains of immunoglobulins can be classified as alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG) or mu (IgM), some of which can be further classified into subclasses such as gamma 1 (IgG 1), gamma 2 (IgG 2), gamma 3 (IgG 3), gamma 4 (IgG 4), alpha 1 (IgA 1) and alpha 2 (IgA 2). Based on the amino acid sequence of its constant domain, the light chain of immunoglobulins can be divided into what are known as kappa (kappa) and lambda (lambda). Immunoglobulins generally consist of two Fab molecules and an Fc domain linked by an immunoglobulin hinge region.

Herein, the terms "light chain variable region (V _L)" and variable heavy chain (V _H) refer to polypeptides comprising V _L or V _H, respectively. Pairing of V _H and V _L together forms a single antigen binding site. Endogenous V _L is encoded by gene segments V (variable) and J (linkage), and endogenous V _H is encoded by V, D (diversity) and J. V _L or V _H each comprise a hypervariable region CDR (complementarity determining region) and a Framework Region (FR). The term "variable region" or "V region" is used interchangeably and refers to a heavy chain variable region or a light chain variable region comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. The V region may be naturally occurring, recombinant or synthetic. The antibody light chain variable region and/or antibody heavy chain variable region may sometimes be collectively referred to herein as an "antibody variable region" or "antibody chain". As provided and further described herein, an "antibody light chain variable region" or "antibody heavy chain variable region" and/or "antibody chain" optionally comprises a polypeptide sequence that incorporates cysteine residues.

The terms "Complementarity Determining Region (CDR)" or "hypervariable region (HVR)" are used interchangeably herein to refer to each region of an antibody variable region that is highly variable in sequence and/or forms a structurally defined loop ("hypervariable loop"). CDRs are the target protein binding sites of antibodies, which are structurally complementary to epitopes of the target protein, and thus have the specificity of binding to such target protein. Typically, a natural four-chain antibody comprises six CDRs, three in V _H (HCDR 1, HCDR2 and HCDR 3) and three in V _L (LCDR 1, LCDR2 and LCDR 3). The remaining VL or VH regions, i.e., framework Regions (FR), of the CDRs have fewer amino acid sequence variations (Kuby, immunology [ Immunology ], 4 th edition, chapter 4, w.h.freeman & Co. [ w.h. frieman company ], new york, 2000).

The positions of the CDRs and FRs may be determined using a variety of definition methods well known in the art, e.g., kabat, chothia, IMGT and contacts (see, e.g., kabat et al 1991,Sequences of Proteins of Immunological Interest [ immunologically relevant protein sequences ], fifth edition, U.S. Pat. No. HEALTH AND human services [ U.S. health and public service ], NIH publication No. 91-3242, johnson et al, nucleic Acids Res [ nucleic acids research ],29:205-206 (2001); chothia and Lesk, J.mol.biol. [ journal of molecular biology ],196:901-917 (1987); chothia et al, nature [ Nature ],342:877-883 (1989); chothia et al, J.mol.biol. [ J.Mol., nucleic acids research ],227:799-817 (1992; al-Lazikani et al, J.mol.biol. [ J.Mol., no. Lefranc, M. -P., [ Immunol., 7,132-136 (1999), lefranc, M. -P.et al, J.mol.biol.262 (1996): 732-745.). Definition of antigen binding sites is also described in Ruiz et al, nucleic Acids Res. [ nucleic acids research ],28:219-221 (2000), and Lefran, M.P., nucleic Acids Res., [ nucleic acids research ],29:207-209 (2001); (IMGT) No. Lefranc, M., [ Immunol., 7,132-136 (1999)), lefranc, M. -P.Eomp. Biol. [ and Immunol. ] [ comparative Immunol. ],27,55 (2003.), [ 2003, J.219-37.), [ J.38:, 203:121-153 (1991); and Rees et al, in: sternberg M.J.E. (eds.), protein Structure Prediction [ protein Structure prediction ], oxford University Press [ oxford university Press ], oxford [ oxford ],141-172 (1996). The present invention encompasses any of the definition methods for determining the CDRs in an anti-EGFR antibody or antigen-binding fragment thereof of the invention, and table 1 shows the amino acid residues of the antibody CDRs determined using the different definition methods. The exact number of amino acid residues covering a particular CDR varies with the sequence of the CDR. Where the amino acid sequence of the antibody variable region is specified, one skilled in the art can determine the CDRs of the antibody by conventional methods including, but not limited to, the definition.

TABLE 1 CDR's determined by different definition methods

CDR	Kabat	Chothia	IMGT	Contact
					HCDR1	31-35	26-32	27-38	30-35
HCDR2	50-65	52-56	56-65	47-58
					HCDR3	95-102	95-102	105-117	93-101
LCDR1	24-34	24-34	27-38	30-36
					LCDR2	50-56	50-56	56-65	46-55
LCDR3	89-97	89-97	105-117	89-96

Furthermore, kabat et al define a numbering system for variable region sequences that is applicable to any antibody. One of ordinary skill in the art can explicitly apply this "Kaba numbering" system to the variable region sequences of any antibody without relying on any experimental data other than the antibody sequence itself to determine the variable region sequences.

As used herein, the term "Fc region" or "Fc domain" refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region, such as an immunoglobulin heavy chain constant region other than the first constant region (CH 1). The Fc region as used herein includes native sequence Fc regions and/or Fc region variants, and may be part of an anti-EGFR antibody of the invention. It is understood in the art that the boundaries of the Fc region may vary, however, a human IgG heavy chain Fc region is generally defined as comprising a cysteine residue at position 226 or a proline residue at position 230 at its carboxy-terminus, according to the EU numbering system/scheme as seen in Kabat et al (1991, NIH publication 91-3242,National Technical Information Service [ national technical information service ], spin Philin, va.).

Herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population of antibodies are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, as compared to conventional (polyclonal) antibody preparations, which typically comprise different antibodies directed against different determinants (epitopes). The modifier "monoclonal" refers to the characteristics of the antibody as obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in accordance with the present technology may be made by the hybridoma method described for the first time by Kohler et al, nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "monoclonal antibodies" can also be isolated from phage antibody libraries using techniques such as those described in Clackson et al, nature 352:624-628 (1991), marks et al, journal of molecular biology 222:581-597 (1991).

In this context, the term "recombinant" when used in reference to, for example, a cell or nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein or modification of the native nucleic acid or protein, or that the material originates from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that otherwise perform abnormally, poorly, or not at all.

Herein, the term "chimeric antibody" refers to an antibody that contains sequences derived from two different antibodies (e.g., U.S. Pat. No. 4,816,567), which antibodies are typically derived from different species. For example, chimeric antibodies comprise human and rodent antibody fragments, typically human constant regions and mouse variable regions. Methods for producing chimeric antibodies include conventional recombinant DNA and gene transfection techniques known to those of ordinary skill in the art (e.g., morrison, S.L. et al, proc. Natl. Acad. Sci. USA) 81 (1984) 6851-6855;US 5,202,238 and US 5,204,244)

The anti-EGFR antibodies or antigen-binding fragments thereof of the invention may be selected from any one, or two, or more of the forms, including chimeric, non-human, humanized or fully human forms, provided that the forms are capable of specifically binding to human EGFR and inhibit human EGFR-mediated biological function.

In this context, the term "specific binding" or "binding specificity" or "specific for …" or "binding" refers to a binding reaction that determines the presence of a target molecule (or protein or antigen) in a heterogeneous population of proteins and other biological agents (e.g., in a biological sample (e.g., blood, serum, plasma, or tissue sample)), that is, the binding is selective for the target molecule and distinguishes between those undesired or non-specific interactions. For example, an antibody that specifically binds to a target molecule (which may be an antigen) is one that has greater affinity, binding activity, ease, and/or duration of binding to the target molecule than the antibody binds to other target molecules. In certain embodiments, the anti-EGFR antibodies or antigen-binding fragments thereof of the invention exhibit slightly less binding activity than cetuximab as measured by ELISA.

"Does not cross react", i.e., does not bind to molecules/proteins other than the specific target molecule/protein. In certain embodiments, the anti-EGFR antibodies or antigen-binding fragments thereof of the invention are found to not bind to other human ErbB receptor tyrosine kinases of the same family (e.g., HER2, HER3, and/or HER 4) as detected by ELISA.

As used herein, the term "epitope" means a protein-determining region capable of specifically binding to an antibody. Epitopes are generally composed of chemically active surface groups of molecules such as amino acids or sugar side chains and generally have specific three-dimensional structural features, as well as specific charge characteristics. Conformational epitopes differ from non-conformational epitopes in that binding to the former is lost but binding to the latter is not lost in the presence of denaturing solvents.

As used herein, the term "epitope binding domain" or "antigen binding region" is used interchangeably to refer to a portion of a binding molecule (e.g., an antibody or epitope-binding fragment or derivative thereof) that specifically interacts (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatially distributing) with a binding site on a target epitope. The term "antigen binding region" in the present invention also refers to one, or two, or more fragments of an antibody that retains specific interactions (e.g., by binding, steric hindrance, stabilization/destabilization, spatial distribution) with an EGFR epitope.

In this context, amino acid substitutions include conservative amino acid substitutions, which involve substitution with another amino acid in the same class (e.g., chemically or functionally similar), and non-conservative amino acid substitutions, which involve substitution of an amino acid in one of the classes with an amino acid in the other class. One of ordinary skill in the art can make conservative amino acid substitutions based on the similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, (I) nonpolar (hydrophobic) amino acids include alanine (Ala, a), leucine (Leu, L), isoleucine (Ile, I), valine (Val, V), proline (Pro, P), phenylalanine (Phe, F), tryptophan (Trp, W), and methionine (Met, M); (ii) Polar neutral amino acids include glycine (Gly, G), serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), tyrosine (Tyr, Y), asparagine (Asn, N), and glutamine (Gln, Q); (iii) Positively charged (basic) amino acids include arginine (Arg, R), lysine (Lys, K) and histidine (His, H); (iv) Negatively charged (acidic) amino acids include aspartic acid (Asp, D) and glutamic acid (Glu, E). Non-conservative amino acid substitutions may vary greatly in nature or function of the parent macromolecule. Substitutions that are generally expected to result in a maximum change in the properties of the polypeptide include, but are not limited to: (a) Hydrophilic residues such as serine or threonine, and hydrophobic residues such as leucine, isoleucine, phenylalanine, valine or alanine; (b) Cysteine or proline in place of (or in place of) any other amino acid residue; (c) A residue having an electropositive side chain such as lysyl, arginyl, or histidyl is substituted (or substituted) with an electronegative residue such as glutamyl or aspartyl; or (d) a residue having a bulky side chain such as phenylalanine is substituted (or substituted) with a residue having no side chain such as glycine. As known in the art, conservative substitutions typically do not result in a significant change in the conformational structure of the protein, and thus may preserve the biological activity of the protein. Although the site or region at which the mutation in the amino acid sequence is introduced may be predetermined, the alteration in the nature or function of the potential biomacromolecule by non-conservative substitutions is not anticipated.

In this context, the terms "identical" or "identity" or "percent sequence identity" are used interchangeably in two or more nucleic acid or polypeptide sequences to refer to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to a reference sequence after an amino acid sequence (or nucleic acid sequence) alignment is performed and, if necessary, a gap is introduced to maximize the number of identical amino acids (or nucleic acids). Conservative substitutions of the amino acid residues may or may not be considered the same residue. The sequences may be aligned to determine the percent sequence identity of amino acid (or nucleic acid) sequences by means disclosed in the art, such as BLASTN, BLASTP (national center for Biotechnology information website (NCBI), also see, altschul S.F. et al, J.mol.biol.,215:403-410 (1990), stephen F. Et al, nucleic acids Res.,25:3389-3402 (1997)), clustalW2 (European institute of biological information website, see, higgins D.G. et al, methods in Enzymology,266:383-402 (1996), larkin M.A. et al, bioinformation (Oxford, england), 23 (21): 2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software. The person skilled in the art can use the default parameters of the tool or adjust the parameters appropriately according to the needs of the alignment, for example by choosing an appropriate algorithm. )

As used herein, the term "isolated" when referring to a nucleic acid (or polynucleotide) or protein means that the nucleic acid or protein is substantially free of other cellular components to which it is associated in its native state, preferably in a homogeneous state, e.g., the isolated nucleic acid or protein may be removed from its natural or natural environment. The isolated nucleic acid or protein may be lyophilized or in aqueous solution. Typically, its purity and homogeneity can be determined using analytical chemistry techniques (e.g., polyacrylamide gel electrophoresis, isoelectric focusing, capillary electrophoresis, or chromatography, such as high performance liquid chromatography). As a main ingredient in the formulation. The protein is essentially obtained by purification. The term "purified" means that the nucleic acid or protein substantially produces a band in the electrophoresis gel. In particular, this means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. For the purposes of the present invention, recombinant proteins expressed in host cells are considered isolated in some embodiments, as well as natural or recombinant proteins that are separated, fractionated (fractionate), or partially or substantially purified by any technique well known to those of skill in the art. In some embodiments, an "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds human EGFR is substantially free of antibodies that specifically bind antigens other than human EGFR). However, isolated antibodies that specifically bind human EGFR may have cross-reactivity with other antigens (e.g., EGFR molecules from other species, such as mouse EGFR). Furthermore, the isolated antibodies may be substantially free of other cellular material and/or chemicals. In some embodiments, recombinant polynucleotides encoding a polypeptide or protein of the invention (e.g., an anti-EGFR antibody) contained in a vector are considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides contained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. An isolated polynucleotide includes a polynucleotide molecule contained in a cell that generally contains the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a location other than its natural chromosomal location. Isolated polynucleotides or nucleic acids of the invention also include synthetically produced such molecules. In addition, the polynucleotide or nucleic acid may be or include regulatory elements such as promoters, ribosome binding sites or transcription terminators.

As used herein, the term "polypeptide" refers to a polymer of amino acids and equivalents thereof, and not to a specific length of the product; thus, "peptide" and "protein" are included within the definition of polypeptide. Also included within the definition of polypeptide are "antibodies" as defined herein.

Unless the context indicates otherwise, a "derivative" is a polypeptide or fragment thereof having one, or two, or more non-conservative or conservative amino acid substitutions relative to a second polypeptide (also referred to as a "variant"); or by covalent attachment of a second molecule, e.g., by attachment of a heterologous polypeptide, or by glycosylation, acetylation, phosphorylation, etc. of a modified polypeptide or fragment thereof. The definition of "derivative" may also include, for example, polypeptides that contain analogs of one or more amino acids (e.g., unnatural amino acids, etc.), polypeptides with unsubstituted linkages, and other modifications known in the art (both natural and non-naturally occurring).

As used herein, the term "expression vector" or "vector" refers to a vehicle into which a polynucleotide or nucleic acid encoding a protein can be operably inserted and expressed. Vectors may be used to transform, transduce or transfect host cells such that elements of genetic material carried thereby are expressed within the host cells.

As used herein, the term "host cell" refers to a cell that is an exogenous polynucleotide or nucleic acid and/or vector. Host cells include "transformants" and "transformed cells" which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may comprise the mutation. Mutant progeny having the same function or biological activity, selected or selected in the initially transformed cells, are encompassed by the present application.

The term "EC ₅₀", also referred to herein as half maximal effective concentration, refers to the concentration of antibody that is capable of achieving 50% of maximum (i.e., 50% between baseline and maximum) biological effects (e.g., binding or inhibition, etc.) after a particular exposure time. EC50 values may be measured by methods known in the art, for example, sandwich methods such as ELISA, immunoblotting, flow cytometry, and other binding assays.

The term "IC ₅₀", also referred to herein as half maximal inhibitory concentration, refers to the concentration of an antibody corresponding to 50% inhibition of a particular biological or biochemical function relative to the absence of the antibody.

The terms "subject," "patient," or "individual" are used interchangeably herein, and include, but are not limited to: mammals, including, for example, humans, non-human primates (e.g., monkeys), mice, pigs, cows, goats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep, or other non-human mammals; non-mammalian, including, for example, non-mammalian vertebrates, such as birds (e.g., chickens or ducks) or fish; a non-mammalian invertebrate. In some embodiments, the subject and pharmaceutical compositions to which the use or method of the invention relates are for (prophylactically and/or therapeutically) treating a non-human animal.

In this context, "treating" or "treatment" of a disease or condition refers to alleviating a disease or condition, reducing the rate at which a disease or condition is raised or developed, reducing the risk of developing a disease or condition, or delaying the development of a condition associated with a disease or condition, reducing or terminating a condition associated with a disease or condition, producing a complete or partial reversal of a disease or condition, curing a disease or condition, or a combination thereof.

Herein, "preventing" includes inhibition of the occurrence or progression of a disease or disorder or a symptom of a particular disease or disorder. In some embodiments, the subject with a family history is a candidate for a prophylactic regimen. In general, the term "prevention" refers to administration of a drug prior to the occurrence of a sign or symptom, particularly in a subject at risk.

As used herein, an "EGFR-related" disease or condition refers to any disease or condition caused, exacerbated, or otherwise associated with increased or decreased expression or activity of EGFR. In certain embodiments, the EGFR-related disorder is cancer.

The term "therapeutically effective amount" or "effective dose" refers to a dose or concentration effective to achieve prevention or amelioration of symptoms associated with a disease or disorder and/or lessening the severity of a disease or disorder at a desired dose for a desired period of time. The therapeutically effective amount of the formulations, antibodies, or antigen binding fragments thereof, or compositions of the invention may vary depending on a variety of factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount may also be considered to be any toxic or detrimental effect of the formulation, antibody or antigen-binding fragment thereof or composition that is less than a therapeutically beneficial effect.

As used herein, the term "pharmaceutically acceptable" or "pharmaceutically acceptable" refers to carriers, vehicles, diluents, excipients and/or salts as indicated, which are generally compatible chemically and/or physically with the other ingredients in the formulation, and physiologically compatible with the subject.

The term "about" when used in conjunction with a numerical value is intended to encompass numerical values within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, or in one embodiment a lower limit of 10% less and an upper limit of 10% greater, or in another embodiment a lower limit of 15% less and an upper limit of 15% greater, or in another embodiment a lower limit of 20% less and an upper limit of 20% greater.

The term "and/or" is understood to mean any one of the selectable items or a combination of any two or more of the selectable items.

As used herein, the terms "comprises" or "comprising" or "includes" or "having" are used interchangeably to mean including the recited elements, integers or steps, but not excluding any other elements, integers or steps. In this document, the terms "comprises" or "comprising" or "includes" or "having" when used herein, unless otherwise indicated, also encompasses the circumstance that the recited elements, integers or steps consist of them.

Various aspects of the invention will be described in more detail in the following sections.

1. The anti-EGFR antibodies or antigen binding fragments thereof of the present invention

In one aspect, the invention provides an anti-EGFR antibody, or antigen-binding fragment thereof, that is capable of specifically binding to the extracellular domain of human EGFR.

The anti-EGFR antibodies or antigen binding fragments thereof of the present invention specifically bind to the extracellular domain of human EGFR with an EC50 value of no more than 1nM, preferably no more than 0.5nM, as measured by ELISA. The anti-EGFR antibodies or antigen-binding fragments thereof of the invention have slightly lower human EGFR binding activity than cetuximab.

The anti-EGFR antibodies or antigen binding fragments thereof of the present invention can also bind to the extracellular domain of human EGFR with high affinity, and the binding constant and dissociation constant between the anti-EGFR antibody or antigen binding fragment thereof and human EGFR of the present invention are detected by antigen-antibody binding kinetics and calculated by fitting, resulting in an affinity constant (KD) value of no more than 5X10 ^-8 M, preferably a KD value of about 10 ^-8 M.

The anti-EGFR antibodies or antigen binding fragments thereof of the invention are not cross-reactive (or do not bind) with other human ErbB receptor tyrosine kinases, e.g., HER2 (also known as ErbB 2), HER3 (also known as ErbB 3), and/or HER4 (also known as ErbB 4). The extent of binding of the anti-EGFR antibody or antigen-binding fragment thereof to a non-target protein (e.g., her2/ERBb2, her3/ERBb3, or Her4/ERBb 4) is more than about 10% less than the binding of the antibody or antigen-binding fragment thereof to EGFR, as determined by methods known in the art, such as ELISA, fluorescence Activated Cell Sorting (FACS) analysis, or Radioimmunoassay (RIA).

Thus, in some embodiments, "specifically binds" as used herein refers to a molecule (e.g., an anti-EGFR antibody of the invention or antigen-binding fragment thereof) that binds a particular polypeptide (e.g., human EGFR) or an epitope of a particular polypeptide, but does not substantially bind any other polypeptide or polypeptide epitope.

The anti-EGFR antibodies or antigen binding fragments thereof of the present invention are capable of inhibiting proliferation of EGFR-expressing tumor cells. In certain embodiments, the anti-EGFR antibody or antigen-binding fragment thereof has an IC50 value for tumor cells of no more than 1nM, preferably no more than 0.1nM, as detected by ELISA.

Antibodies of the invention may also optionally include F (ab) 2, F (ab ') 2, fab', scFv, single domain antibodies, and the like. The antibodies of the invention may be mouse antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, and antibody fragments so long as the antibodies specifically recognize human EGFR, and in particular the extracellular domain of human EGFR, and are capable of inhibiting a biological function mediated by human EGFR in tumor cells expressing EGFR, such as the growth or proliferation of tumor cells.

In another aspect, the anti-EGFR antibodies or antigen binding fragments thereof of the invention comprise CDRs (complementarity determining regions) comprising one or more of heavy chain CDR1 (HCDR 1), heavy chain CDR2 (HCDR 2), and heavy chain CDR3 (HCDR 3), or variants thereof, and/or one or more of light chain CDR1 (LCDR 1), light chain CDR2 (LCDR 2), and light chain CDR3 (LCDR 3), or variants thereof, including humanized antibodies or any other variants described herein, that retain specific binding activity to human EGFR.

In some embodiments, the antibody or antigen binding fragment thereof comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1, HCDR2 and HCDR3 defined by the IMGT numbering system comprise an amino acid sequence that is at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identical to HCDR1 shown in SEQ ID No. 1, HCDR2 shown in SEQ ID No. 2, and HCDR3 shown in SEQ ID No. 3, respectively. The HCDR1, HCDR2, and HCDR3 defined by the Kabat numbering system comprise amino acid sequences that are at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identical to the HCDR1 shown in SEQ ID NO 9, the HCDR2 shown in SEQ ID NO 10, and the HCDR3 shown in SEQ ID NO 11, respectively. The HCDR1, HCDR2, and HCDR3 defined by the Chothia numbering system comprise amino acid sequences that are at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identical to the HCDR1 shown in SEQ ID NO. 14, the HCDR2 shown in SEQ ID NO. 15, and the HCDR3 shown in SEQ ID NO. 11, respectively. The HCDR1, HCDR2 and HCDR3 defined by the Contact numbering system comprise amino acid sequences that are at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identical to the HCDR1 shown in SEQ ID NO. 16, the HCDR2 shown in SEQ ID NO. 17 and the HCDR3 shown in SEQ ID NO. 18, respectively.

In some embodiments, the antibody or antigen binding fragment thereof comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 defined by the IMGT numbering system comprise amino acid sequences that are at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%, or 100% identical to LCDR1, LCDR2, respectively, shown in SEQ ID No. 4, LCDR2, shown in SEQ ID No. 5, LCDR3, shown in SEQ ID No. 6. LCDR1, LCDR2 and LCDR3 defined by the Kabat or Chothia numbering system comprise amino acid sequences that are at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identical to LCDR1 shown in SEQ ID No. 12, LCDR2 shown in SEQ ID No. 13, LCDR3 shown in SEQ ID No. 6, respectively. LCDR1, LCDR2 and LCDR3 defined by the Contact numbering system comprise amino acid sequences at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identical to LCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:20, LCDR3 shown in SEQ ID NO:21, respectively.

In some embodiments, the anti-EGFR antibody or antigen binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, wherein the heavy chain variable region comprises any one, two, or three of HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, and HCDR3 as shown in SEQ ID NO:3 defined by the IMGT numbering system, or HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:11 defined by the Kabat numbering system, or HCDR1 as shown in SEQ ID NO:14, HCDR2 as shown in SEQ ID NO:15, and HCDR3 as shown in SEQ ID NO:11 defined by the Contact numbering system, or HCDR1 as shown in SEQ ID NO:16, HCDR2 as shown in SEQ ID NO:17, and HCDR3 as shown in SEQ ID NO: 18; the light chain variable region comprises LCDR1 as shown in SEQ ID NO. 4, LCDR2 as shown in SEQ ID NO. 5 and LCDR3 as shown in SEQ ID NO. 6 defined by the IMGT numbering system, or LCDR1 as shown in SEQ ID NO. 12, LCDR2 as shown in SEQ ID NO. 13 and LCDR3 as shown in SEQ ID NO. 6 defined by the Kabat or Chothia numbering system, or any one, two or three of LCDR1 as shown in SEQ ID NO. 19, LCDR2 as shown in SEQ ID NO. 20 and LCDR3 as shown in SEQ ID NO. 21 defined by the Contact numbering system.

In a specific embodiment, the anti-EGFR antibody, or antigen-binding fragment thereof, comprises (1) HCDR1 as set forth in SEQ ID NO:1, SEQ ID NO:2, and HCDR2 as set forth in SEQ ID NO:3, and the amino acid sequences of LCDR1 shown in SEQ ID NO. 4, LCDR2 shown in SEQ ID NO. 5, and LCDR3 shown in SEQ ID NO. 6; or (2) HCDR1 as defined by Kabat numbering system as shown in SEQ ID NO:9, SEQ ID NO:10, and HCDR2 as set forth in SEQ ID NO:11, and the amino acid sequences of LCDR1 shown in SEQ ID NO. 12, LCDR2 shown in SEQ ID NO. 13, and LCDR3 shown in SEQ ID NO. 6; or (3) HCDR1 as defined by the Chothia numbering system as shown in SEQ ID NO:14, SEQ ID NO:15, and HCDR2 as set forth in SEQ ID NO:11, and the amino acid sequences of LCDR1 shown in SEQ ID NO. 12, LCDR2 shown in SEQ ID NO. 13, and LCDR3 shown in SEQ ID NO. 6; or (4) HCDR1 as defined by the Contact numbering system as shown in SEQ ID NO:16, SEQ ID NO:17, and HCDR2 as set forth in SEQ ID NO:18, and the LCDR1 shown in SEQ ID NO. 19, the LCDR2 shown in SEQ ID NO. 20, and the LCDR3 amino acid sequence shown in SEQ ID NO. 21.

In some embodiments, the anti-EGFR antibody or antigen binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 7, or has an amino acid sequence at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identical to the heavy chain variable region and retains a specific binding activity to human EGFR.

In some embodiments, the anti-EGFR antibody, or antigen binding fragment thereof, comprises the heavy chain variable region as set forth in SEQ ID No. 7, and/or the light chain variable region as set forth in SEQ ID No. 8, or has at least 85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99% or 100% identical amino acid sequence to the heavy chain variable region and the light chain variable region, respectively, and retains specific binding activity to human EGFR.

The amino acid sequences of the CDR regions of the antibodies of the present invention, as determined by the exemplary numbering system/definition method used in the present invention, are summarized in table 2, and may be defined by any other existing method for numbering amino acid residues of antibodies and defining CDRs.

TABLE 2 amino acid sequences of antibody CDR regions defined by different numbering systems and ID numbers thereof

In addition, the heavy chain variable region and/or light chain variable region sequences (or CDR sequences) of other anti-EGFR antibodies that bind to human EGFR may also be "mixed and matched" with the heavy chain variable region and/or light chain variable region sequences (or CDR sequences) of the anti-EGFR antibodies of the invention. Preferably, when the heavy chain variable region sequences and the light chain variable region sequences (or CDRs within these chains) are mixed and matched, VH sequences from a particular VH/VL pair are replaced with structurally similar VH sequences. Likewise, preferably, the VL sequences from a particular VH/VL pair are replaced with structurally similar VL sequences, thereby producing more anti-EGFR antibodies.

Thus, in one embodiment, an antibody or antigen binding fragment thereof of the invention comprises:

(a) A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO. 7; and

(B) A light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 8, or the VL of another anti-EGFR antibody, wherein the antibody specifically binds human EGFR.

Or an antibody or antigen-binding fragment thereof of the invention comprises:

(a) A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 7, or the VL of another anti-EGFR antibody, wherein the antibody specifically binds human EGFR; and

(B) A light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 8.

In some embodiments, the anti-EGFR antibody or antigen-binding fragment thereof further comprises a Framework Region (FR) comprising a heavy chain Framework Region and/or a light chain Framework Region. In some embodiments, the heavy chain framework region is selected from a mouse or human antibody heavy chain framework region, and/or the light chain framework region is selected from a mouse or human antibody light chain framework region. In specific embodiments, the heavy chain framework region is selected from a mouse heavy chain framework region, and the light chain framework region is selected from a mouse light chain framework region.

In another embodiment, the antibodies of the invention comprise heavy and/or light chain variable region sequences having CDR1, CDR2, and CDR3 sequences that differ from the amino acid sequences of the anti-EGFR antibodies of the invention by one or more amino acid residue conservative modifications. It is understood in the art that modifications of certain conserved sequences do not cause the antibody to lose or reduce antigen binding capacity, e.g., can result in conservative modifications that do not substantially reduce human EGFR binding activity. See, e.g., brummel et al, (1993) Biochem 32:1180-8; de Wildt et al, (1997) Prot.Eng.10:835-41; komissarov et al, (1997) J.biol. Chem.272:26864-26870; hall et al, (1992) J.Immunol.149:1605-12; kelley and O' Connell, (1993) biochem.32:6862-35; adib-Conquy et al, (1998) int. Immunol.10:341-6; and bees et al, (2000) clin.can.res.6:2835-43.

Thus, in one embodiment, an antibody comprises a heavy chain variable region comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence, and/or a light chain variable region comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence, wherein:

(a) The heavy chain variable region CDR1 sequence comprises the sequence shown in SEQ ID NO. 1, 9, 14 or 16 and/or conservative modifications thereof; and/or

(B) The heavy chain variable region CDR2 sequence comprises the sequence shown in SEQ ID NO.2, 10, 15 or 17 and/or conservative modifications thereof; and/or

(C) The heavy chain variable region CDR3 sequence comprises the sequence shown in SEQ ID NO 3, 11 or 18 and conservative modifications thereof; and/or

(D) The light chain variable region CDR1 sequence comprises the sequence shown in SEQ ID NO. 4, 12 or 19 and conservative modifications thereof; and/or

(E) The light chain variable region CDR2 sequence comprises the sequence shown in SEQ ID NO. 5, 13 or 20 and conservative modifications thereof; and/or

(F) The light chain variable region CDR3 sequence comprises the sequence shown in SEQ ID NO. 6 or 21 and conservative modifications thereof; and

(G) The antibodies specifically bind human EGFR.

The antibodies of the invention described above have one or more of the following functional properties, e.g., high specific binding to human EGFR, and inhibition of EGFR expressing tumor cell proliferation.

One or more amino acid residues within the CDR regions of the antibodies of the invention may be conservatively substituted with other amino acid residues having the same side chain family, and the function retained by the altered antibody may be detected using the function assay methods described herein.

In various embodiments, the antibody may be a mouse, human, humanized or chimeric antibody.

In another aspect, one or more V _H/V_L sequence antibodies with an anti-EGFR antibody of the invention may be used as starting materials for engineering modifications to produce the antibodies of the invention. Antibodies may be engineered by modification of one or more amino acid residues within one or both variable regions (i.e., V _H and/or V _L), e.g., modification of one or more amino acid residues within one or more CDR regions and/or one or more framework regions.

In some embodiments, the variable regions of the anti-IL-2 antibodies of the invention are engineered by CDR grafting. Antibodies interact with target antigens primarily through the amino acid residues of the six CDRs of the heavy and light chains. Thus, the amino acid sequences within the CDR regions between individual antibodies are more diverse than sequences outside the CDR regions (e.g., FR). Because the amino acid sequences of the CDR regions are responsible for the majority of interactions of antibodies with antigens, recombinant antibodies can be expressed to mimic the properties of a particular naturally occurring antibody by constructing expression vectors comprising the CDR sequences from a particular naturally occurring antibody grafted to FR sequences of another antibody having different properties (see, e.g., riechmann et al, (1998) Nature 332:323-327; jones et al, (1986) Nature 321:522-525; queen et al, (1989) Proc. Natl. Acad. U.S.A.86:10029-10033. See also U.S. Pat. Nos. US5,225,539;US 5,530,101;US 5,585,089;US5,693,762 and US6,180,370).

In some embodiments, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising the amino acid sequences of HCDR1, HCDR2, and HCDR3 regions of the invention and/or a light chain variable region comprising the amino acid sequences of LCDR1, LCDR2, and LCDR3 regions of the invention. Although the antibodies comprise CDR region sequences of V _H and V _L of the monoclonal antibodies of the invention, they may also comprise different framework region sequences.

Such framework region sequences may be obtained from DNA public databases or published references relating to germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes are found in the "VBase" human germline sequence database (available on the Internet from www.mrc-cpe.cam.ac.uk/VBase); and, kabat et al, (1991), supra; tomlinson et al, (1992) j.mol. Biol.227:776-798; and Cox et al, (1994) Eur.J.Immunol.24:827-836; the contents of each of which are expressly incorporated herein by reference.

The comparison of antibody amino acid sequences can be performed using one of the sequence similarity search methods known to those skilled in the art as Gapped BLAST, based on a compiled protein sequence database (Altschul et al, (1997), supra).

Preferred framework sequences for use in the antibodies of the invention are acceptor framework regions that are structurally similar (or highly homologous) to the framework sequences used in the antibodies of the invention. In some embodiments, the CDR1, CDR2, and CDR3 region sequences of VH or VL, respectively, may be grafted to acceptor framework regions having the same or highest sequence homology to the germline immunoglobulin gene in which they are located; alternatively, the CDR sequences may be grafted onto a framework region comprising one or more mutations compared to the germline sequence. For example, it has been found that in some instances amino acid residues within the framework regions are mutated to maintain or enhance the antigen binding capacity of the antibody (see, e.g., U.S. Pat. Nos. 5,530,101;5,585,089;5,693,762 and 6,180,370).

In other embodiments, the variable region modification is a mutation of amino acid residues within the V _H and/or V _L CDR1 region, CDR2 region, and/or CDR3 region, thereby improving one or more binding properties (e.g., binding activity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutations and the effect of the mutations on antibody binding capacity or other functional properties can be assessed in vitro or in vivo detection methods known in the art. For example, the modification introduced may be a non-conservative modification (as known in the art). The mutation may be an amino acid substitution, addition or deletion, preferably an amino acid substitution. In particular, no more than 1,2, 3, 4 or 5 amino acid residues within the CDR regions are mutated.

In one embodiment, the invention provides an isolated anti-EGFR antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region comprising: (a) A V _H CDR1 region comprising a sequence of the invention or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions; and/or (b) a V _H CDR2 region comprising a sequence of the invention or having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions; and/or (c) a V _H CDR3 region comprising a sequence of the invention or having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions; the light chain variable region comprises: (d) A V _L CDR1 region comprising a sequence of the invention or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions; and/or (e) a V _L CDR2 region comprising a sequence of the invention or having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions; and/or (f) a V _L CDR3 region comprising a sequence of the invention or having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions.

The engineered antibodies of the invention may also include antibodies that have been modified (e.g., to improve the properties of the antibodies) with respect to amino acid residues of the V _H and/or V _L framework regions. In particular, such framework amino acid residue modifications may reduce the immunogenicity of the antibody. For example, one way is to "back-mutate" one or more framework amino acid residues to the corresponding germline sequence. More specifically, antibodies produced by somatic mutation may comprise a framework region that is different from the germline sequence from which the antibody is derived.

Another type of framework region amino acid residue modification comprises one or more amino acid residue mutations within the framework region, or within one or more CDR regions, to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This strategy is also known as "deimmunization" and is described in further detail in U.S. patent publication No. 20030153043.

In addition, in addition to modifications to the framework or CDR region amino acid residues, the Fc region of the antibodies of the invention may be engineered to alter one or more functional properties of the antibodies, such as serum half-life, complement fixation, fc receptor binding, and/or antigen-dependent cytotoxicity. Furthermore, the antibodies of the invention may also be chemically modified (e.g., one or more chemical groups may be attached to the antibody), or modified to alter its glycosylation, again altering one or more functional properties of the antibody.

In one embodiment, the hinge region of C _H1 is modified such that the number of cysteine residues in the hinge region is altered (e.g., increased or decreased). This method is further described in U.S. Pat. No. 5,677,425. For example, the number of cysteine residues in the C _H1 hinge region is altered to facilitate assembly of the light and heavy chains or to improve antibody stability.

Furthermore, each antibody has a characteristic isoelectric point (pI), typically between pH 6 and 9.5. The pI of IgG1 antibodies is typically pH 7-9.5, while the pI of IgG4 antibodies is typically pH 6-8. Antibodies with pI values outside the normal range may have partial unfolding and instability in vivo. Thus, it is preferred to obtain anti-EGFR antibodies having a pI value within the normal range, or to achieve the antibodies pI value within the normal range by mutating charged amino acid residues.

In another aspect, the anti-EGFR antibodies of the invention, or antigen-binding fragments thereof, may further comprise constant regions, including antibody heavy chain constant regions and light chain constant regions. The heavy chain constant region comprises a CH1, hinge, and/or CH2-CH3 region. The heavy chain constant regions of the invention include natural and/or mutant protein forms of the Fc region of human IgG heavy chain constant regions, as well as truncated forms of polypeptides comprising a hinge region that promotes dimer formation. In certain embodiments, the Fc region comprises the CH2 and CH3 domains of the antibody. Fusion proteins (and oligomers formed therefrom) comprising an Fc moiety offer the advantage of easy purification by affinity chromatography on a protein a or protein G column, as well as prolonged serum half-life. Preferred Fc regions are derived from human IgG, including IgG1, igG2, igG3, and IgG4.

One function of the Fc region of an antibody is to produce "effector functions" with the immune system when the antibody binds to its target, including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC). ADCC and ADCP are mediated by Fc binding to Fc receptors (fcrs) on the surface of immune cells; CDC is mediated by Fc binding to proteins of the complement system, e.g., C1 q. Effector function may be assessed by various methods, such as Fc receptor binding assays, C1q binding assays, and cell lysis assays.

2. Method for obtaining anti-EGFR antibodies or antigen binding fragments thereof of the present invention by screening

The anti-EGFR antibodies or antigen binding fragments thereof of the present invention can be obtained by screening using a variety of techniques, such as hybridoma techniques, phage display techniques, single lymphocyte gene cloning techniques, and the like. In some embodiments, the invention screens antibody libraries for antibodies that recognize an antigen of interest by constructing a phage display library and panning (panning) the antigen of interest. Various phage display methods known in the art can be used to generate antibodies of the invention, for example, U.S. Pat. nos. 5,223,409, 5,622,699 and 6,068,829 disclose methods for preparing phage libraries. The method can also be used according to "anti-body PHAGE DISPLAY: methods and Protocols (Philippa M.O' Brien, robert Aitken edit) ", to construct phage display libraries. In some embodiments, nucleic acid encoding the antibody variable region may be inserted into a phage coat protein gene such that the phage displays the antibody variable region on its surface, while nucleic acid encoding the antibody variable region is contained within the phage, thereby effecting a link between the antibody variable region phenotype and genotype.

In connection with screening of antibodies (or single chain antibodies), in phage display, a larger library of VH and/or VL regions of the antibodies (or single chain antibodies) can be expressed on the surface of the filamentous phage particle, thereby pairing them to form a binding domain. Phage may be screened from the library based on recognition and binding to the antigen of interest and the binding domain displayed thereby.

In some embodiments, the antibody variable region genes can be obtained by immunizing a mouse with an antigen of interest and amplifying from mature B cells of the mouse, displaying the mouse antibody variable region on the phage surface by phage display techniques, constructing a phage display library, panning through a specific target molecule (e.g., an antigen of interest for immunizing the mouse), detecting interaction of the antibody variable region displayed on the phage surface with the target molecule, screening and amplifying the antibody variable region library by an in vitro selection method, which is similar to the natural selection.

In some embodiments, the panning may be achieved by phage infection of host bacteria and propagation amplification in the host. The host bacteria may secrete phage with the antibody variable regions displayed on their surfaces without being lysed by the phage. Amplified phage may be re-exposed to target molecules as needed and collected for additional rounds of panning. Multiple rounds of panning may also be performed until a sub-population is obtained that selectively or specifically binds to the target molecule. The amino acid sequence of the antibody variable region can be determined by sequencing the genes corresponding to the antibody variable region in the phage genome (Arap et al, 1998, smith et al, 1985).

In a specific embodiment, the invention provides a technique for obtaining the anti-EGFR antibody or antigen binding fragment thereof according to the invention by constructing and panning a phage library, specifically, obtaining antibody variable region genes from spleen tissues of immunized mice, connecting the antibody variable region genes to phage surface structural protein genes III, and using a co-expression mode to make the antibody variable region genes participate in phage assembly and display on phage surfaces to form a phage display library. Using phage display libraries, phage that specifically bind to a target molecule (e.g., antigen for immunization of mice) can be enriched by repeated rounds of adsorption-elution-amplification, followed by gene sequencing techniques to obtain corresponding DNA sequence information, and thus infer the amino acid sequence of the antibody variable region.

3. Polynucleotides, vectors and host cells

In one aspect, the invention provides a nucleic acid encoding an anti-EGFR antibody or antigen binding fragment thereof. The invention also includes polynucleotide variants encoding the amino acid sequences described herein. The nucleic acid may be present in whole cells, or in cell lysates, or in partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially pure" when purified from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) by standard techniques. In a preferred embodiment, the nucleic acid is a cDNA molecule.

In some embodiments, the nucleic acids of the invention may be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas, the cDNA encoding the light and heavy chains of the antibody produced by the hybridoma can be obtained by standard Polymerase Chain Reaction (PCR) amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acids encoding such antibodies can be recovered from the gene library.

In other embodiments, nucleotide sequences corresponding to the amino acid sequences described herein, probes or primers for use in nucleic acid isolation, or databases providing interrogation may be obtained by amino acid sequence backtranslation. PCR procedures can be used to isolate and amplify DNA sequences encoding the anti-EGFR antibodies of the invention or antigen-binding fragments thereof. Oligonucleotides defining the desired ends of the combination of DNA fragments are used as 5 'and 3' primers. The oligonucleotides may additionally contain recognition sites for restriction endonucleases to facilitate insertion of amplified DNA fragment combinations into expression vectors. PCR techniques are described in Saiki et al, science 239:487 (1988); recombinant DNA Methodology, wu et al, editions ACADEMIC PRESS, in c., sa n Diego (1989), pages 189-196; PCR Pro to cols A Guide to Methods and Applications, innis et al, ACADEMIC PRESS, inc. (1990).

Nucleic acid molecules of the invention include single and double stranded forms of DNA and RNA, as well as corresponding complementary sequences, including isolated nucleic acid molecules, preferably derived from DNA or RNA that has been isolated at least once in a substantially pure form and whose constituent nucleotide sequences can be identified, manipulated and recovered in amounts or concentrations by standard biochemical methods (e.g., the methods described in Sambrook et al, molecular Cloning: ALaboratory Manual, 2nd edition, cold Spring Harbor Laboratory, cold Spring Harbor, N.Y. (1989)). Preferably, such sequences include those provided and/or constructed in the form of an open reading frame of internal untranslated sequences or intron breaks that are not commonly found in eukaryotic genes. The sequence of the non-translated DNA may be present 5 'or 3' to the open reading frame, wherein the sequence does not interfere with manipulation or expression of the coding region.

Preferred nucleic acid molecules of the invention include nucleic acid molecules encoding the sequences or CDR regions of anti-EGFR antibodies or antigen binding fragments thereof V _H and V _L. Once the DNA fragments encoding the V _H and V _L regions are obtained, these DNA fragments can be further recombined by standard recombinant DNA techniques, for example, converting the variable region genes into full length antibody chain genes, fab fragment genes or scFv genes. In these recombinations, this DNA fragment encoding V _L or V _H is ligated to a DNA fragment encoding another protein (e.g., an antibody constant region or flexible linker). As used herein, the term "operably linked" is intended to join two DNA fragments such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the V _H region can be converted to a gene encoding a full length heavy chain by operably linking the DNA encoding V _H to a DNA molecule encoding a heavy chain constant region (including C _H1、C_H2 and C _H3). The sequences of human heavy chain constant region genes are known in the art and DNA fragments comprising these regions can be obtained by standard PCR amplification techniques. The heavy chain constant region may be an IgG1, igG2, igG3, igG4, igA, igE, igM, or IgD constant region, but is most preferably an IgG1 or IgG4 constant region. For Fab fragment heavy chain genes, the DNA encoding V _H may be operably linked to a DNA molecule encoding only the heavy chain C _H1 constant region.

The isolated DNA encoding the V _L region can be converted to a gene encoding a full length light chain or Fab light chain gene by operably linking the DNA encoding the V _L region to another DNA molecule encoding the light chain constant region C _L. The sequences of human light chain constant region genes are known in the art and DNA fragments comprising these regions can be obtained by standard PCR amplification techniques. In preferred embodiments, the light chain constant region may be a kappa or lambda constant region.

To generate scFv genes, the DNA fragments encoding V _H and V _L are operably linked to another DNA fragment encoding a flexible linker (e.g., encoding the amino acid sequence (Gly 4-Ser) 3), such that the V _H and V _L sequences may be expressed as a continuous single chain protein, i.e., the V _L and V _H regions are linked by a flexible linker (see, e.g., bird et al, (1988) Science 242:423-426; huston et al, (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; mcCafferty et al, (1990) Nature 348:552-554).

The anti-EGFR antibodies or antigen-binding fragments thereof of the invention are prepared by the steps of: the nucleotides in the DNA encoding the anti-EGFR antibody or antigen-binding fragment thereof are subjected to site-specific mutagenesis using cassettes or PCR mutagenesis or other techniques known to one of ordinary skill in the art to produce DNA encoding the variant, after which the recombinant DNA is expressed in cell culture as outlined herein. However, anti-EGFR antibodies or antigen-binding fragments thereof may be prepared by in vitro synthesis using established techniques.

As known to those skilled in the art, due to the degeneracy of the genetic code, the anti-EGFR antibodies or antigen binding fragments thereof of the present invention are encoded by an extremely large number of nucleic acids, each of which is within the scope of the invention and can be made using standard techniques. Thus, identifying a particular amino acid sequence, one skilled in the art can prepare many different nucleic acids by simply modifying one or more codons of their respective coding sequences in a manner that does not alter the amino acid sequence of the anti-EGFR antibodies or antigen binding fragments thereof of the present invention. Specifically, degenerate codon substitutions may be achieved by substituting a mixed base and/or deoxyribose residue for the sequence resulting from the third position of one or more selected (or all) codons (see Batzer et al, nucleic Acid Res.19:5081 (1991); ohtsuka et al, J.biol. Chem.260:2605-2608 (1985); rossolini et al, mol. Cell. Probes 8:91-98 (1994)).

In another aspect, the invention also provides expression vectors encoding the nucleic acids of the anti-EGFR antibodies or antigen binding fragments thereof of the present invention.

Nucleic acids encoding the anti-EGFR antibodies or antigen binding fragments thereof of the present invention may be constructed in suitable vectors for introduction into host cells for expression of the protein of interest. The carrier component generally includes, but is not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. The nucleic acid encoding the protein of interest in the vector is operably linked to a promoter, i.e., a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcription regulator binding sites) and another nucleic acid sequence, and thus the control sequence controls transcription and/or translation of the other nucleic acid sequence.

Suitable vectors include plasmids, phagemids, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages such as lambda or M13 phages, animal viruses and the like. Animal virus species used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papilloma-virus vacuolated viruses (e.g., SV 40), lambda and M13 phages, plasmids pcDNA3.3、pMD18-T、pOptivec、pCMV、pEGFP、pIRES、pQD-Hyg-GSeu、pALTER、pBAD、pcDNA、pCal、pL、pET、pGEMEX、pGEX、pCI、pEGFT、pSV2、pFUSE、pVITRO、pVIVO、pMAL、pMONO、pSELECT、pUNO、pDUO、Psg5L、pBABE、pWPXL、pBI、p15TV-L、pPro18、pTD、pRS10、pLexA、pACT2.2、pCMV-SCRIPT.RTM.、pCDM8、pCDNA.1/amp、pcDNA3.1、pRc/RSV、PCR 2.1、pEF-1、pFB、pSG5、pXT1、pCDEF3、pSVSPORT、pEF-Bos, and the like. The vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin. The vector may also include components that assist it in entering the cell, including but not limited to viral particles, plasmids, liposomes, or protein shells.

In another aspect, the invention also provides a host cell comprising a nucleic acid or expression vector encoding an anti-EGFR antibody, or antigen-binding fragment thereof, of the invention.

The cell may be a eukaryotic cell, for example, a mammalian host cell, including, but not limited to, the SV40 transformed monkey kidney cell CV1 line (COS-7, ATCC CRL 1651), a human embryonic kidney cell line (293 or suspension cultured 293 cell subclone, graham et al, j.gen virol.36:59 (1977)), baby rat kidney cells (BHK, ATCC CCL 10), chinese hamster ovary cells/-DHFR (CHO, urlaub et al, proc.Natl. Acad.Sci.USA 77:4216 (1980)), mouse testis support cells (TM 4, mather, biol.Reprod.23:243-251 (1980)), monkey kidney cells (CV 1ATCC CCL 70), african green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (HepG 2, HB 8065), mouse mammary tumors (T060, ATCC CCL 51), TRI cells (Mather et al, annals N.Y.Acad.383.44:MR2 (1982), human liver cancer cells (MRC line 4, MRC 5).

The host cells are transformed with the above-described expression or cloning vectors to produce an anti-EGFR antibody or antigen binding fragment thereof, and cultured in conventional nutrient media suitably modified for the purpose of inducing promoters, selecting transformants or amplifying the genes encoding the desired sequences. In other embodiments, the antibodies may be produced by homologous recombination as known in the art.

4. Method for preparing anti-EGFR antibody or antigen binding fragment thereof

The invention provides methods of using the host cells to make the anti-EGFR antibodies or antigen binding fragments thereof of the present invention.

The method comprises transfecting a nucleic acid or expression vector encoding the anti-EGFR antibody or antigen binding fragment thereof of the present invention into a host cell, and culturing the host cell in a medium for a period of time to express the anti-EGFR antibody or antigen binding fragment thereof of the present invention. Without limitation, a commercially available medium may be used as the medium.

Preferably, the expressed anti-EGFR antibody or antigen-binding fragment thereof may be secreted into the medium in which the host cells are grown. Recovering the antibody from the culture medium using standard protein purification methods, such as removal of impurities by centrifugation or ultrafiltration, or purification of the resultant by affinity chromatography; other purification techniques may also be used, such as gel electrophoresis, dialysis, ammonium sulfate precipitation, salting out, anion or cation exchange chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography.

5. Bispecific/multispecific molecules

The invention also provides a bispecific molecule comprising an anti-EGFR antibody or antigen-binding fragment thereof of the invention linked to another functional molecule (e.g. another polypeptide or protein such as a Fab' fragment) to form a bispecific or multispecific molecule that binds to multiple binding sites or binding (target) epitopes. For example, an anti-EGFR antibody or antigen-binding fragment thereof of the invention may be functionally linked to one or more other binding molecules, such as another antibody, antigen-binding fragment, polypeptide, or binding mimetic, including chemical coupling, gene fusion, non-covalent association, or other means).

In one embodiment, the other functional molecule may be an Fc receptor (FcR), such as human fcγriii (CD 16), fcδri (CD 64) or fcαr (CD 89), and thus the bispecific or multispecific molecule of the present invention may be targeted to both fcγ R, fc αr or fcεr expressing effector cells (e.g., monocytes, macrophages or polymorphonuclear cells (PMNs)) and EGFR expressing tumor cells.

In one embodiment, the further functional molecule is selected from an antibody or antigen binding fragment thereof specific for a cancer-associated antigen (TAA) or an immune checkpoint protein antigen, which may be selected from HER2, HER3, VEGF, VEGFR, c-met, TFR (transferrin receptor), IGF-1R (human insulin-like growth factor I receptor), etc.; the immune checkpoint protein antigen may be selected from PD-1, PD-L1, CTLA-4, CD137, CD47, and the like.

In one embodiment, the bi-or multi-specific molecules of the invention may further comprise a third binding specificity in addition to the binding specificity and anti-EGFR binding specificity of the functional molecule described above. The third specificity may be an anti-Enhancement Factor (EF) moiety, for example, the molecule may bind to a surface protein involved in cytotoxic activity and thereby increase the immune response to the target cell. The anti-enhancer moiety may be an antibody (including scFv), and the anti-enhancer moiety may bind to FcR or target cell antigen. In addition, the target cells to which the anti-enhancer moiety binds may be different from the target cells to which the first and second binding specificity molecules bind, e.g., the anti-enhancer moiety may bind to cytotoxic T cells (e.g., via CD2, CD3, CD8, CD28, CD4, or ICAM-1) or other immune cells that produce an enhanced immune response to the target cells.

The term "target cell" as used herein refers to a cell targeted by a molecule of the invention (e.g., an anti-EGFR antibody or antigen-binding fragment thereof, a bispecific molecule, or a multispecific molecule) to any disorder in the subject. In some embodiments, the target cell is an EGFR-overexpressing cell, which generally includes tumor cells (e.g., bladder cancer, breast cancer, colon cancer, renal cancer, ovarian cancer, prostate cancer, renal cell carcinoma, squamous cell carcinoma, non-small cell lung cancer, head and neck cancer, etc.). Other EGFR-overexpressing cells may also include synovial fibroblasts and keratinocytes as target cells in the treatment of arthritis and psoriasis, respectively.

Bispecific molecules of the present invention can take many different forms. For example, a bispecific molecule retains the traditional antibody format, except that it does not have two binding arms of the same specificity, but two binding arms of different specificity, each of which may consist of two single chain antibody fragments (scFv) linked by a peptide chain, to construct a bispecific molecule, the so-called Bs (scFv) 2 structure. Bispecific molecules may also include two different F (ab) fragments linked by a peptide-based linker. These and other forms of bispecific molecules can be prepared by genetic engineering, somatic hybridization, or chemical synthesis methods. See, e.g., kufer, et al, supra; cao and Suresh, bioconjugate Chemistry,9 (6), 635-644 (1998); and VAN SPRIEL et al, immunology Today,21 (8), 391-397 (2000), and references cited therein.

6. Immunoconjugates, chimeric antigen receptors, engineered T cell receptors, or oncolytic viruses

In one aspect, the invention provides an immunoconjugate comprising an anti-EGFR antibody, or antigen-binding fragment thereof, of the invention coupled to a therapeutic agent or a detectable label (e.g., a radioisotope, luminescent label, fluorescent label, or enzyme substrate label).

In some embodiments, the anti-EGFR antibodies, or antigen-binding fragments thereof, of the invention can be conjugated to a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include cytotoxins (including paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, ipecine, mitomycin, etoposide, tenoposide, vincristine, MMAE, MMAF, DM, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, milpramine, actinomycin D, 1-dehydroproteinsterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol), alkylating agents (e.g., chloroform, chloro Ding Liuniao, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfoimide, dibromonitrobenzene, streptozotocin, mitomycin C and cisplatin (II) (DDP) cisplatin), puromycin and analogs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil norhydrazine), DNA minor groove binders, DNA intercalators, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, tubulin binding agents, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics (such as actinomycin, bleomycin, milbemycin, and Anthracycline (AMC)), and antimitotics (such as vincristine, vinblastine). Such as U.S. patent nos. 7,087,600, 6,989,452, and 7,129,261; PCT publications WO02/096910, WO 07/038,658, WO 07/051,081, WO 07/059,404, WO 08/083,312 and WO 08/103,693; ADCs are prepared as described in U.S. patent publications 20060024317, 20060004081, and 20060247295, the disclosures of which are incorporated herein by reference.

In some embodiments, the anti-EGFR antibodies of the invention, or antigen-binding fragments thereof, can be conjugated to a radioisotope (e.g., ,¹²³I、¹²⁴I、¹²⁵I、¹³¹I、³⁵S、³H、¹¹¹In、¹¹²In、¹⁴C、⁶⁴Cu、⁶⁷Cu、⁸⁶Y、⁸⁸Y、⁹ ⁰Y、¹⁷⁷Lu、²¹¹At、¹⁸⁶Re、¹⁸⁸Re、¹⁵³Sm、² ¹²Bi and ³² P, other lanthanides) to form a cytotoxic radiopharmaceutical that treats an EGFR-related disease or disorder.

In some embodiments, an anti-EGFR antibody or antigen-binding fragment thereof of the invention can be conjugated to a fluorescent label (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or texas red), or an enzyme substrate label (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, glycooxidase, or β -D-galactosidase), or a luminescent label (e.g., digoxin, biotin/avidin), to detect the level of EGFR expression in a test sample.

In another aspect, the invention also provides chimeric antigen receptors, engineered T cell receptors, or oncolytic viruses comprising the anti-EGFR antibodies or antigen-binding fragments thereof of the invention.

7. Pharmaceutical composition

The invention provides pharmaceutical compositions comprising an anti-EGFR antibody, or antigen-binding fragment thereof, of the invention (or an immunoconjugate, or a bispecific/multispecific molecule, or a chimeric antigen receptor, or an engineered T cell receptor, or an oncolytic virus), and a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable" means that the compositions containing the active ingredient do not produce adverse, allergic or other untoward reactions when properly administered to an animal or human. As used herein, a "pharmaceutically acceptable carrier" should be compatible with, i.e., capable of being admixed with, the molecules of the invention (e.g., monoclonal antibodies, bispecific molecules/multispecific molecules, immunoconjugates) without substantially reducing the efficacy of the composition in general.

The pharmaceutical composition may comprise an optional pharmaceutically acceptable excipient or stabilizer. Excipients that may be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersing or suspending aids, solubilizers, colorants, flavorants, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonic agents or combinations thereof. The selection and use of suitable excipients is taught below, gennaro et al, remington: THE SCIENCE AND PRACTICE of pharmacy, 20 th edition (Lippincott Williams & wilkins 2003), the disclosure of which is incorporated herein by reference.

The pharmaceutical composition of the present invention can be formulated into various dosage forms as needed, and the dosage beneficial to the patient can be determined by the physician according to the type, age, weight, general disease condition, administration mode, etc. of the patient. The administration may be, for example, injection or other therapeutic means. Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). In view of the different routes of administration, the active ingredient may be encapsulated in a material to protect it from acids and other natural conditions that may inactivate it. As used herein, the term "parenteral administration" is a non-enteral and topical mode of administration and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, the antibodies of the invention may be administered by a non-parenteral route (e.g., topical, epidermal, or mucosal route of administration), e.g., intranasal, oral, vaginal, rectal, sublingual, or topical.

The pharmaceutical composition may be a sterile aqueous solution or dispersion. The pharmaceutical compositions can also be formulated as solutions, microemulsions, liposomes, or other combinations of ordered structures suitable for high drug concentrations.

The amount of active ingredient that can be combined with the carrier material to form a single dosage form is determined according to the subject and the particular mode of administration and is also generally the amount of the composition that is capable of producing a therapeutic effect. Generally, the compositions formed with pharmaceutically acceptable carriers comprise from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% of the active ingredient.

The dosage regimen is adjusted to provide the best desired response (e.g., therapeutic response). For example, the dosage may be administered in a single dose at a time, multiple divided doses may be administered over time, or the dosage may be proportionally reduced or increased depending on the degree of urgency of the treatment. For ease of administration and to achieve dosage uniformity, it is highly advantageous to formulate compositions for parenteral administration in dosage unit form. As used herein, dosage unit form refers to a physiologically differentiated unit suitable as a unit dose for treating a subject; each unit dose contains a predetermined amount of the active ingredient, which is calculated to produce the desired therapeutic effect by administration of the active ingredient with the required pharmaceutical carrier. In addition, the anti-EGFR antibodies or antigen binding fragments thereof of the present invention may also be administered as a slow release formulation, which may reduce the frequency of administration.

The anti-EGFR antibodies, or antigen binding fragments thereof, or compositions comprising the anti-EGFR antibodies, or antigen binding fragments thereof, of the present invention may be administered in a dosage ranging from about 0.0001mg/kg to 100mg/kg body weight, typically 0.01mg/kg to 50mg/kg body weight.

The "therapeutically effective dose" of the anti-EGFR antibodies or antigen binding fragments thereof, or bispecific/multispecific molecules, or immunoconjugates, or chimeric antigen receptors, or engineered T cell receptors, or oncolytic viruses of the present invention preferably results in a decrease in severity of disease symptoms, an increase in the frequency and duration of disease symptoms in the absence of progression, or prevention of damage or disability to the body caused by disease affliction. For example, a "therapeutically effective dose" of a tumor-bearing subject preferably inhibits tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to an untreated subject. A therapeutically effective amount of a therapeutic antibody may reduce the size of a tumor, or ameliorate symptoms in a subject, typically a human or other mammal. The "therapeutically effective dose" may also be determined based on a variety of factors including, but not limited to, the method of formulation, the method of administration, the age, body weight, sex, physical or pathological condition of the patient, diet, time of administration, interval of administration, route of administration, rate of excretion, sensitivity of reaction, history of past diseases, and the like. One conventional method in the art involves determining the therapeutically effective amount based on factors such as the size of the target lesion, the severity of the subject's symptoms, and the particular composition or route of administration selected.

The pharmaceutical compositions may be selected from controlled release formulations including implants, transdermal patches and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid may be used. See, for example, sustained and Controlled Release Drug DELIVERY SYSTEMS, J.R.ROBINSON, editions, MARCEL DEKKER, inc., new york, 1978.

The therapeutic pharmaceutical composition may be delivered by a medical device selected from the group consisting of: (1) Needleless hypodermic injection devices (e.g., U.S. Pat. Nos. 5,399,163;5,383,851;5,312,335;5,064,413;4,941,880;4,790,824 and 4,596,556); (2) Micro infusion pumps, for example, U.S. patent 4,487,603 discloses implantable micro-infusion pumps that dispense drugs at a controlled rate; (3) transdermal devices (U.S. patent number 4,486,194); (4) Infusion devices, for example, U.S. Pat. No. 4,447,233 discloses drug infusion pumps that deliver drugs at a precise infusion rate, and U.S. Pat. No. 4,447,224 discloses implantable infusion pumps that are variable flow and capable of continuously delivering drugs; and (5) osmotic engine, such as U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having a multi-compartment container; U.S. patent 4,475,196 discloses a yihong osmotic drug delivery system, the disclosure of which is incorporated herein by reference.

In certain embodiments, the anti-EGFR antibodies, or antigen-binding fragments thereof, of the invention may be formulated to ensure proper biodistribution in vivo. For example, to ensure that the therapeutic antibodies of the invention are able to cross the Blood Brain Barrier (BBB) (if desired), they can be formulated as liposomes that can include one or more targeting moieties that selectively deliver to specific cells or organ tissues to enhance the selective delivery of targeted drugs. See, for example, U.S. Pat. nos. 4,522,811, 5,374,548, 5,416,016 and 5,399,331; v. ranade (1989) j. Clin. Pharmacol.29:685, a step of preparing a liquid; umezawa et al, (1988) biochem. Biophys. Res. Commun.153:1038; bloeman et al, (1995) FEBS Lett.357:140; m. Owais et al, (1995) Antimicrob. Agents Chemother.39:180; briscoe et al, (1995) am.J.Physiol.1233:134; schreier et al, (1994) J.biol. Chem.269:9090; keinanen and Laukkanen, (1994) FEBS Lett.346:123, a step of; and Killion and Fidler, (1994) Immunomethods 4:273.

8. Kit/combination product and kit

In one aspect, the invention provides a kit comprising a pharmaceutical composition of an anti-EGFR antibody or antigen-binding fragment of the invention, and one or more other therapeutic agents.

In some embodiments, the therapeutic agent includes chemotherapeutic agents including, but not limited to, vinca alkaloids, microtubule-forming disrupting agents (e.g., colchicine and derivatives thereof), anti-angiogenic agents, therapeutic antibodies, EGFR targeting agents, tyrosine kinase targeting agents (e.g., tyrosine kinase inhibitors), transition metal complexes, proteasome inhibitors, antimetabolites (e.g., nucleoside analogs), alkylating agents, platinum-based agents, anthracyclines, topoisomerase inhibitors, macrolides, retinoids (e.g., all-trans retinoic acid or derivatives thereof), geldanamycins or derivatives thereof (e.g., 17-AAG), and other standard chemotherapeutic agents recognized in the art. In some embodiments, the chemotherapeutic agent comprises one or more of, for example, doxorubicin, colchicine, cyclophosphamide, actinomycin, bleomycin, daunorubicin, epirubicin, mitomycin, methotrexate, mitoxantrone, fluorouracil, carboplatin, carmustine (BCNU), methyl-CCNU, cisplatin, etoposide, interferon, camptothecin and derivatives thereof, cholesterol-to-chlorambucil, taxane and derivatives thereof (e.g., paclitaxel and derivatives thereof, docetaxel (taxotere) and derivatives thereof, etc.), topotecan, vinblastine, vincristine, tamoxifen, piposulfan, nab-5404, nab-5800, nab-5801, irinotecan, HKP, ostatin, oxaliplatin, hypo,Vinorelbine,/>Capecitabine,/>Lapatinib, sorafenib, erlotinib, chemotherapeutic agents known in the art, and the like. In some embodiments, the chemotherapeutic agent is a composition comprising nanoparticles comprising a thiocolchicine derivative and a carrier protein (e.g., albumin).

In some embodiments, the chemotherapeutic agent is an anti-cancer agent including, but not limited to, a microtubule disrupting agent, an antimetabolite, a topoisomerase inhibitor, a DNA intercalating agent, an alkylating agent, a hormonal therapy agent, a kinase inhibitor (e.g., tyrosine kinase inhibitors including, but not limited to, EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, IGFR inhibitors, and Met inhibitors), a receptor antagonist, an activator of tumor cell apoptosis (including, but not limited to, IAP inhibitors, bcl2 inhibitors, MC11 inhibitors, TRAIL inhibitors, CHK inhibitors), or any anti-angiogenic drug;

in some embodiments, the therapeutic agent includes an immunomodulatory agent, including, but not limited to, PD-1, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or a TGFR inhibitor.

In another aspect, the invention relates to a kit, e.g., useful for isolating or detecting EGFR levels in a test sample, comprising an anti-EGFR antibody, or antigen-binding fragment thereof, of the invention, or comprising a pharmaceutical composition of the invention, or an immunoconjugate of the antibody, or antigen-binding fragment thereof, of the invention, with a detectable label.

As used herein, the term "sample to be tested" encompasses a variety of sample types, including blood and other bodily fluid samples of biological origin, solid tissue samples such as biopsy tissue samples or tissue cultures, or cells derived therefrom or their progeny. The term also includes samples that have been treated by any means after they have been obtained, for example by treatment with reagents, solubilization, or enrichment for certain components such as proteins or polynucleotides.

For convenience in detection, the kit may contain, in addition to the immunoconjugate comprising the anti-EGFR antibody or antigen-binding fragment thereof of the present invention and a detection label, other detection reagents or auxiliary reagents such as those conventionally used in ELISA or western blot kits, the characteristics of which and the methods of formulating them are well known to those skilled in the art, such as color-developing agents, labels, secondary antibodies, anti-antibodies, sensitizers, etc. It will be appreciated by those skilled in the art that various variations of the detection kit are included in the present invention as long as it utilizes the anti-EGFR antibody or antigen binding fragment thereof of the present invention as an agent that recognizes or binds EGFR.

A container may also be included in the kit, as well as a label or instructions on or associated with the container. For example, the container containing the contents comprising at least one anti-EGFR antibody of the invention, or antigen-binding fragment thereof, may comprise additional containers containing, for example, diluents and buffers, control antibodies. The label or instructions for use may provide instructions regarding the description of the composition and the intended in vitro or diagnostic use, for example, the label may indicate the intended use of the kit contents, may be provided on or with the kit outer packaging, or may be any written, marketing or recording material that is otherwise provided with the kit; the instructions for use may provide, for example, a diagnosis of a disease or disorder related to EGFR upregulation and/or EGFR.

After the anti-EGFR antibody or antigen binding fragment and/or kit provided by the invention are obtained, various immunological correlation methods can be used for detecting EGFR or the content thereof in the sample to be detected, so that whether the donor of the sample to be detected has related diseases or symptoms of EGFR over-expression or not can be known.

9. Therapeutic uses and methods

In one aspect, the invention relates to the use of an anti-EGFR antibody, or antigen-binding fragment thereof, of the invention for the preparation of a pharmaceutical composition or formulation for the treatment and/or prevention of EGFR-related diseases or disorders, including cancer or other EGFR-related diseases or disorders, for the preparation of a diagnostic reagent for the diagnosis of EGFR-related diseases or disorders.

In some embodiments, the cancer includes, but is not limited to, melanoma, lung cancer (non-small cell lung cancer or small cell lung cancer), colorectal cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, renal cancer or cell carcinoma, liver cancer, esophageal cancer, gall bladder cancer, pancreatic cancer, gastric cancer, thyroid cancer, bladder cancer, head and neck cancer, glioblastoma, skin cancer, and other solid and/or metastatic cancers.

In some embodiments, other EGFR-related diseases include, but are not limited to, autoimmune diseases, psoriasis, or inflammatory arthritis (e.g., rheumatoid arthritis, systemic lupus erythematosus-related arthritis, psoriatic arthritis).

In some embodiments, the other EGFR-related disease further comprises a cell proliferative disease comprising, in addition to the cancer: adrenal cortical hyperplasia (cushing's disease), congenital adrenal cortical hyperplasia, endometrial hyperplasia, benign prostatic hyperplasia, breast hyperplasia, intimal hyperplasia, focal epithelial hyperplasia (Heck's disease), sebaceous gland hyperplasia, compensatory liver hyperplasia, and any other cell proliferative disorder.

In another aspect, the invention provides a method of treating or preventing an EGFR-related disease or disorder, comprising administering to a subject a therapeutically effective amount of an anti-EGFR antibody, or antigen-binding fragment thereof, pharmaceutical composition, or kit of the invention, including cancer or other EGFR-related disease.

In some embodiments, the other EGFR-related disease comprises a cell proliferative disease comprising, in addition to the cancer: adrenal cortical hyperplasia (cushing's disease), congenital adrenal cortical hyperplasia, endometrial hyperplasia, benign prostatic hyperplasia, breast hyperplasia, intimal hyperplasia, focal epithelial hyperplasia (Heck's disease), sebaceous gland hyperplasia, compensatory liver hyperplasia, and any other cell proliferative disorder.

In some embodiments, the anti-EGFR antibodies, or antigen-binding fragments thereof, pharmaceutical compositions, or kits of the invention may be administered alone, or in combination with one or more other therapeutic agents or means of treatment.

In some embodiments, the other therapeutic agents include chemotherapeutic agents, anticancer agents, immunomodulatory agents, or therapeutic agents known in the art disclosed herein.

In some embodiments, an anti-EGFR antibody, or antigen-binding fragment thereof, pharmaceutical composition, or kit of the invention may be administered simultaneously with one or more other therapeutic agents, or may be administered separately from another therapeutic agent, e.g., before or after another therapeutic agent, which may have the same route of administration as the anti-EGFR antibody, or antigen-binding fragment thereof, pharmaceutical composition, or kit of the invention, or may be a different route of administration.

In some embodiments, the anti-EGFR antibodies, or antigen-binding fragments thereof, pharmaceutical compositions, or kits of the invention may be administered in combination with other therapeutic means including, but not limited to, surgery, chemotherapy, cytotoxic agents, photodynamic therapy, immunomodulation, or radiation therapy.

In another aspect, the invention provides a method of modulating EGFR activity in an EGFR-overexpressing cell, the method comprising eliciting one or more of the following biological activities in vivo or in vitro, e.g., inhibiting EGF-or TGF- α -induced autophosphorylation in the EGFR-expressing cell, using an anti-EGFR antibody, or antigen-binding fragment thereof, or a pharmaceutical composition, or a kit of the invention; inhibiting activation of EGFR cells expressed by induction of autocrine EGF or TGF-alpha; or inhibit the growth or proliferation of EGFR-expressing cells. In some embodiments, the method is inhibiting the growth or proliferation of an EGFR-overexpressing cell comprising treating the cell with an anti-EGFR antibody, or antigen-binding fragment thereof, or a pharmaceutical composition, or a kit of parts of the invention, including but not limited to a tumor cell of the invention.

In another aspect, the invention provides a method for detecting the level of EGFR in a test sample, comprising: (1) Detecting the content or expression level (quantitative or qualitative) of EGFR in a test sample using the kit of the present invention; or contacting the test sample with the anti-EGFR antibody or antigen binding fragment thereof of the present invention, and determining the EGFR content or expression level (quantitative or qualitative) in the test sample; (2) Comparing the level of EGFR in the sample with the level of EGFR in a control sample (e.g., a cell that is derived from the same tissue as the test sample, or equivalent to the level of EGFR expression in the normal cell), wherein a higher level of EGFR expression in the test sample as compared to the control sample can be determined to be present in the disease or disorder associated with EGFR.

In another aspect, the invention provides a method of detecting, diagnosing or monitoring an EGFR-related disease or disorder in a subject, the method comprising: (1) Detecting the level of EGFR in a test sample obtained from a subject (quantitative or qualitative) using a kit of the invention, or contacting a test sample obtained from a subject with an anti-EGFR antibody or antigen-binding fragment thereof of the invention; (2) Comparing the level of EGFR in the test sample with the level of EGFR in a control sample (e.g., a cell that is derived from the same tissue as the test sample or equivalent to the level of EGFR expression in the normal cell), wherein a higher level of EGFR expression in the test sample compared to the control sample can be indicative of the subject having a disease or disorder associated with EGFR, or a condition associated with EGFR.

10. Technical effects

The anti-EGFR antibodies or antigen-binding fragments thereof of the invention can specifically bind to human EGFR and do not cross-react with other human ErbB receptor tyrosine kinases (e.g., HER2, HER3, HER 4). The anti-EGFR antibody or antigen binding fragment thereof provided by the invention has an inhibition effect on the proliferation activity of tumor cells expressing EGFR, which indicates that the anti-EGFR antibody or antigen binding fragment thereof provided by the invention can block EGFR-mediated signaling pathways, thereby showing the activity of inhibiting the growth of tumor cells, but the anti-EGFR antibody or antigen binding fragment thereof provided by the invention has lower capability of binding human EGFR and/or inhibiting the proliferation of tumor cells expressing EGFR than cetuximab, which indicates that when the anti-EGFR antibody or antigen binding fragment thereof provided by the invention is applied to human body, the skin toxicity of the anti-EGFR antibody or antigen binding fragment thereof can be reduced, and the treatment window can be enlarged.

In summary, the anti-EGFR antibody or antigen binding fragment thereof provided by the present invention can be used as a candidate for therapeutic antibodies, and is also suitable for developing into bispecific or multispecific molecules, which can reduce its skin toxicity and expand the therapeutic window.

The application is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, patents and patent applications cited throughout this application are expressly incorporated herein by reference.

Examples

EXAMPLE 1 screening and preparation of mouse anti-EGFR monoclonal antibodies Using phage display technology

1. Immunized mice

Three 6-8 week old BALB/C female healthy mice (purchased from Shanghai Ji Hui laboratory animal feeding limited) were selected for immunization using the full length of the home-made human EGFR ectodomain protein with his tag at the C-terminus (i.e. hEGFR/ECD-his) as immunogens. Mice were immunized according to the immunization protocol shown in table 3, with the following specific immunization protocol:

primary immunization: balb/c mice were bled 4 days prior to immunization. On day 0, balb/c mice were injected subcutaneously in multiple spots, each with an amount of about 20. Mu.g of antigen, after thoroughly mixing and emulsifying the immunogen with complete Freund's adjuvant.

Secondary immunization: on day 14, after thoroughly mixing the immunogen with complete Freund's adjuvant for emulsification, balb/c mice were injected subcutaneously in multiple spots, each with an amount of about 20. Mu.g of antigen.

Third immunization: on day 28, after thoroughly mixing the immunogen with complete Freund's adjuvant for emulsification, balb/c mice were injected subcutaneously in multiple spots, each with an amount of about 20. Mu.g of antigen. And on day 35 mice were bled for tail vein and coated antigen was tested for serum antibody titer by conventional indirect ELISA.

The procedure of the indirect ELISA method was as follows:

(1) Coating: the self-made antigen hEGFR/ECD-his is diluted to 1 mug/ml by coating solution PBS to coat 96-well ELISA plates (100 mug/well), and incubated overnight at 4 ℃;

(2) Closing: the coating was discarded, the plate was washed 1 time with PBST (containing Tween 20 at a concentration of 0.05%), blocking solution 1% BSA (150. Mu.l/well) was added, and incubated at 37℃for 1 hour;

(3) Washing the plate: discarding the sealing solution and washing the PBST plate;

(4) Incubation of serum samples: the serum of immunized mice was diluted with blocking solution at a volume ratio of 1:10000,1:20000,1:40000,1:80000,1:160000,1:320000,1:640000,1:1280000 and added to 96-well plates at 100 μl/well and incubated for 1 hour at 37 ℃;

(5) And (3) incubation of enzyme-labeled antibodies: the supernatant was discarded, the plate was washed with PBST, goat anti-mouse IgG-HRP (Jackson) diluted with 1% BSA was added, 100. Mu.l/well, and incubated for 1 hour at room temperature;

(6) Color development: discarding the supernatant, washing the plate with PBST, adding TMB as a substrate, and adding 2M hydrochloric acid to stop after light shielding at room temperature;

(7) Colorimetric measurement: antigen-antibody binding OD values were detected at 450nm using a microplate reader assay.

Based on serum titers, boosting was performed on day 40, and 20 μg of immunogen was injected intraperitoneally into each Balb/c mouse.

After the mice were immunized as described above, the tail veins of the mice were bled, the serum of the immunized mice was diluted in the proportions of 1:10000,1:20000,1:40000,1:80000,1:160000,1:320000,1:640000,1:1280000, and serum antibody titer was detected by conventional indirect ELISA using antigen proteins, and mice with serum titers of 1:320000 or better were selected, and prepared for the subsequent experiments.

Preparation of spleen cells: on day 4 after the last immunization, immunized mice were collected under sterile conditions according to standard animal protocols, crushed until there was no tissue clumping and cells were uniform to obtain a single cell suspension, and counted for use.

TABLE 3 Balb immunization protocol for c mice

Flow name	Time of	Immunization dose/route
			Blood collection before immunization	T= -4 days
First immunization	T=0 days	20 Μg/each s.c.
			Second immunization	T=14 days	20 Μg/each s.c.
Third immunization	T=28 days	20 Μg/each s.c.
			Blood sampling test	T=35 days
Enhancing immunity	T=40±2 days	20 Μg/each, i.p.
			Spleen is taken	T=final-day +4 days

2. Construction of phage antibody display library against EGFR

RNA is extracted from spleen cells of immunized mice, light and heavy chain variable region cDNA of the whole set of antibodies is obtained by reverse transcription PCR (RT-PCR), VH and VL are amplified by using a heavy chain variable region (VH) primer and a light chain variable region (VL) primer respectively according to specific primers related to conserved sequences at two ends of the light and heavy chain variable regions of the antibodies, and a primer linker (linker) sequence (amino acid sequence of the linker is GGGGSGGGGSGGS) is introduced into a downstream primer sequence of the VH and an upstream primer sequence of the VL respectively when the primers are designed, and the VH and the VL are spliced and amplified by an overlap extension SOE-PCR technology to obtain single chain antibody (scFv) gene fragments.

The scFv gene fragment is cut by Sfi I enzyme and purified, the gene fragment is connected to pSEX81 phagemid vector, and is transformed into host escherichia coli TG1 competent cells by an electrotransformation instrument, then the host escherichia coli is subjected to secondary phage super-dyeing by M13KO7, so that phage containing the scFv is produced by the host escherichia coli, and the supernatant is subjected to centrifugal precipitation and filtering to obtain a phage display scFv library.

3. Panning phage antibodies that specifically bind to EGFR

Adding the obtained scFv library into an immune tube coated by an antigen hEGFR/ECD-his, standing and incubating at 4 ℃ for overnight, blocking, cleaning, eluting, neutralizing with 1M Tris (pH8.0), infecting TG1 escherichia coli cells in logarithmic growth phase, culturing, collecting bacterial cells, adding M13KO7 helper phage for infection, repeating 3-5 rounds of 'adsorption-elution-amplification' panning, enriching to obtain an anti-human EGFR phage display antibody library, picking single colony for amplification culture, carrying out bacterial liquid PCR identification and monoclonal phase-ELISA identification, and carrying out gene sequencing to obtain scFv capable of specifically binding with the antigen, wherein the amino acid sequence of the VH is shown as SEQ ID NO:7, and the amino acid sequence of the VL is shown as SEQ ID NO: 8.

4. Methods of making anti-EGFR antibodies

VH and VL of scFv obtained in the above steps were cloned into a human IgG1 heavy chain constant region (shown in SEQ ID NO: 22) and a human kappa light chain constant region (shown in SEQ ID NO: 23), respectively, i.e., a nucleotide sequence containing a sequence encoding VH and human IgG1 heavy chain constant region and a nucleotide sequence containing a sequence encoding VL and human kappa light chain constant region were constructed as a heavy chain expression vector and a light chain expression vector, respectively, and the heavy chain expression vector and the light chain expression vector were mixed in a ratio of 1:1, transiently transfected into Expi-CHOS cells in a logarithmic growth phase, and cultured at 32 ℃ for 10 days. Cell supernatant was collected, cells were pelleted by centrifugation, filtered, and antibodies were purified by Protein A affinity chromatography and designated mouse anibody158,158 (abbreviated mAb 158)

EXAMPLE 2ELISA detection of binding Activity of anti-EGFR antibodies to the antigen hEGFR/ECD-his

The ELISA detection method is as follows:

(1) Coating: hEGFR/EDC-his antigen was diluted to 1. Mu.g/ml with 1 XPBS buffer and added to 96-well ELISA plates at 100. Mu.l/well and incubated overnight at 4 ℃;

(2) Closing: the coating was discarded, the plate was washed with PBST buffer (containing 0.1% Tween 20), 150. Mu.l of blocking solution (PBST containing 1% BSA (Sigma)) was added to each well and incubated for 1 hour at 37 ℃;

(4) Incubating primary antibody: 100 μl of samples to be tested, containing different concentrations after gradient dilution (i.e. 3-fold gradient dilution with PBST buffer containing 1% BSA, diluted to a minimum concentration of about 0.0004 μg/mL with 1 μg/mL as starting concentration), were added to each well, and the samples were incubated at 37℃for 1 hour with the mouse anti-EGFR antibody and the control antibody cetuximab prepared in example 1;

(5) Washing: washing the plate with PBST buffer solution;

(6) Incubating the secondary antibody: 100 μl of anti-hIgG Fc-HRP (Jackson) diluted 1:10000 in blocking solution was added to each well and incubated for 1 hour at room temperature;

(7) Color development: the supernatant is discarded, the plate is washed by PBST buffer solution, 100 mu L of TMB color development solution is added into each hole, and the plate is incubated for 3 to 10 minutes at room temperature and in a dark place;

(8) And (3) terminating: adding 50 mu L of 2M hydrochloric acid stop solution into each hole;

(9) Reading measurement: the 96-well microplate was placed in a microplate reader for detection and OD was read at 450 nm.

The results of the assay are shown in FIG. 1 and Table 4, where the anti-EGFR antibody mAb158 has an EC50 value of about 0.2122 μg/mL for binding to the antigen hEGFR, slightly lower than that of cetuximab.

TABLE 4 ELISA detection of binding Activity of anti-EGFR antibodies to the antigen hEGFR/ECD-his

EXAMPLE 3ELISA detection of the Cross-reactivity of anti-EGFR antibodies with other human ErbB receptor tyrosine kinases of the same family

Cross-reactivity of anti-EGFR antibody mAb158 with other human ErbB receptor family members of the same family (including HER2, HER3, and HER 4) was separately tested as described in example 2, with initial concentrations of anti-EGFR antibody and control antibody (cetuximab) of 2 μg/mL, and 3-fold gradient concentration dilutions were made.

As shown in fig. 2, none of the anti-EGFR antibodies mAb158 cross-reacted (or did not bind) to HER2, HER3, and HER 4.

EXAMPLE 4 detection of biological Activity of anti-EGFR antibodies

The biological activity of the anti-EGFR antibodies was tested using the a431 cell line (ATCC) as follows:

(1) Digesting with pancreatin, fully blowing and collecting A431 cells in logarithmic phase, centrifuging to remove supernatant;

(2) The cell pellet was resuspended to 1.25X10 ⁵/mL in DMEM medium containing 1% FBS and the cell suspension was seeded into 96 well cell culture plates at 80. Mu.L per well;

(3) Samples to be tested, including anti-EGFR antibody mAb158 and control samples cetuximab and mAb1127 (self-made) were diluted to 500 μg/mL in DMEM medium containing 1% FBS and subjected to 1:3 gradient dilution;

(4) Adding the diluted sample to be tested into a 96-hole cell culture plate containing cells in 20 mu L of each hole, and carrying out light beating and uniform mixing, wherein the concentration of the sample to be tested is 100 mu g/mL all the time;

(5) Placing the cell culture plate in a incubator with 5% CO ₂ at 37 ℃ for standing culture for 4 days;

(6) After the cell culture is finished, adding into each hole 2.0 (Nanjinouzan, cat. DD 1101-02) cell viability assay reagent for quantifying viable cells by measuring the amount of ATP to detect cell proliferation activity;

(7) The cell culture plate was placed in an enzyme-labeled instrument and the chemiluminescent values of each well were read.

As shown in fig. 3 and table 5, the anti-EGFR antibody mAb158 was able to inhibit proliferation of a431 cells, but its IC50 value was slightly higher than that of the control antibody cetuximab, indicating that the inhibition of proliferation of a431 cells by anti-EGFR antibody mAb158 was slightly lower than that of cetuximab.

TABLE 5 detection of inhibition of A431 cell proliferation by anti-EGFR antibodies

Example 5 detection of binding kinetics of anti-EGFR antibodies to the antigen hEGFR/ECD-his

The binding kinetics of the anti-EGFR antibodies screened in example 1 to the antigen hEGFR/EGFR-his were examined by a molecular interaction device (forteBio, model Qke) as follows:

1) Diluting the antibody to be detected to 3 mug/mL by using 1 XPBS solution, and adding 200mL of the antibody to a 96-well blackboard;

2) Human EGFR protein was diluted to 20. Mu.g/mL with 1 XPBS solution and was added to the same 96 well blackboard at 200mL per well after 1:2 gradient dilution;

3) Placing a 96-hole blackboard containing a sample to be tested and a Protein A detection probe in a machine;

4) Setting a program to enable the Protein A probe to bind the antibody to be detected to a signal of 1.5nm, and then carrying out binding and dissociation detection with human EGFR for 300s and 300s respectively;

5) After each cycle, immersing the probe in 10mM glycine buffer (pH 1.5) to regenerate the probe, and starting the next cycle detection;

6) After the detection is finished, the binding constant and the dissociation constant between the corresponding antibody and the antigen are subjected to fitting calculation by using analysis software, and affinity constant values are obtained.

The results of the assay are shown in Table 6, where anti-EGFR antibody mAb158 binds to hEGFR ectodomain structural protein with high affinity, with a binding affinity KD of about 1.19X10 ^-8 M.

TABLE 6 results of kinetic analysis of anti-EGFR antibodies against antigen binding

(Full R2 represents the similarity between the fitted curve and the measured curve)

While the invention has been described in terms of one or more embodiments, it will be understood that the invention is not limited to those embodiments, but is intended to cover all alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All references cited herein are incorporated herein by reference in their entirety.

Claims

1. An anti-human Epidermal Growth Factor (EGFR) antibody or antigen binding fragment thereof, comprising a heavy chain variable region having a CDR1 region, a CDR2 region and a CDR3 region and a light chain variable region having a CDR1 region, a CDR2 region and a CDR3 region,

When defined by IMGT numbering system, the CDR1, CDR2 and CDR3 amino acid sequences of the heavy chain variable region are set forth in SEQ ID NOs: 1.2 and 3, wherein the amino acid sequences of the CDR1 region, the CDR2 region and the CDR3 region of the light chain variable region are shown as SEQ ID NOs 4, 5 and 6 respectively; or (b)

When defined by the Kabat numbering system, the CDR1, CDR2, and CDR3 amino acid sequences of the heavy chain variable region are set forth in SEQ ID NOs: 9. 10 and 11, and the amino acid sequences of the CDR1 region, CDR2 region and CDR3 region of the light chain variable region are shown in SEQ ID NOs: 12. 13 and 6; or (b)

When defined by the Chothia numbering system, the CDR1, CDR2 and CDR3 amino acid sequences of the heavy chain variable region are set forth in SEQ ID NOs: 14. 15 and 11, and the amino acid sequences of the CDR1 region, the CDR2 region and the CDR3 region of the light chain variable region are shown in SEQ ID NOs 12, 13 and 6 respectively; or (b)

When defined by the Contact numbering system, the CDR1, CDR2 and CDR3 amino acid sequences of the heavy chain variable region are set forth in SEQ ID NOs: 16. 17 and 18, and the amino acid sequences of the CDR1 region, CDR2 region and CDR3 region of the light chain variable region are shown in SEQ ID NOs: 19, 20 and 21, respectively.

2. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region and a light chain variable region having the amino acid sequences set forth in SEQ ID NOs 7 and 8, respectively.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, further comprising an immunoglobulin constant region that is a constant region of human IgG.

4. The antibody or antigen binding fragment thereof of any one of claim 1 or 2, having one or more of the following characteristics,

(1) Specifically binds to human EGFR extracellular domain ECD, and its EC50 value is no more than 1nM as detected by ELISA;

(2) Does not have cross-reactivity with other human ErbB receptor tyrosine kinases including HER2, HER3 and/or HER4;

(3) Has an inhibitory effect on the proliferation activity of EGFR-expressing tumor cells, and the anti-EGFR antibody or antigen binding fragment thereof has an IC50 value of not more than 1nM for the tumor cells as detected by ELISA;

(4) The binding affinity constant KD value for the human EGFR extracellular domain ECD is not more than 5X 10 ^-8 M.

5. The antibody or antigen-binding fragment thereof of any one of claims 1 or 2, which is a mouse, chimeric or humanized antibody.

6. A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-5.

7. An expression vector for expressing the nucleic acid of claim 6.

8. A host cell comprising the nucleic acid of claim 6 or the expression vector of claim 7.

9. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-5 using the host cell of claim 8, comprising the steps of (1) expressing the antibody or antigen-binding fragment thereof in the host cell, and (2) isolating the antibody or antigen-binding fragment thereof from the host cell or cell culture.

10. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-5 and a detectable label comprising a radioisotope, luminescent label, fluorescent label, or enzyme substrate label.

11. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-5, and a pharmaceutically acceptable carrier.

12. A kit comprising the pharmaceutical composition of claim 11, together with one or more other therapeutic agents selected from chemotherapeutic agents, anticancer agents, immunomodulators.

13. A kit comprising the antibody or antigen-binding fragment of any one of claims 1-5, or the immunoconjugate of claim 10, or the pharmaceutical composition of claim 11.

14. Use of an antibody or antigen-binding fragment thereof of any one of claims 1-5 in the manufacture of a pharmaceutical composition or formulation for treating an EGFR-related disease or disorder, which disease or disorder is cancer that is melanoma, lung cancer, colorectal cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, renal cancer or renal cell cancer, liver cancer, esophageal cancer, gall bladder cancer, pancreatic cancer, gastric cancer, thyroid cancer, bladder cancer, head and neck cancer, glioblastoma or skin cancer.

15. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-5 in the manufacture of a kit for detecting EGFR levels or expression levels in a test sample.

16. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-5 in the manufacture of a diagnostic reagent for diagnosing an EGFR-related disease or disorder, wherein the EGFR-related disease or disorder is a solid cancer that is melanoma, lung cancer, colorectal cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, renal cancer or renal cell cancer, liver cancer, esophageal cancer, gall bladder cancer, pancreatic cancer, gastric cancer, thyroid cancer, bladder cancer, head and neck cancer, glioblastoma or skin cancer.