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WO2025237392A1 - Nouvel anticorps anti-egfr, conjugué de médicament et son utilisation - Google Patents

Nouvel anticorps anti-egfr, conjugué de médicament et son utilisation

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Publication number
WO2025237392A1
WO2025237392A1 PCT/CN2025/095291 CN2025095291W WO2025237392A1 WO 2025237392 A1 WO2025237392 A1 WO 2025237392A1 CN 2025095291 W CN2025095291 W CN 2025095291W WO 2025237392 A1 WO2025237392 A1 WO 2025237392A1
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Prior art keywords
amino acid
egfr
acid sequence
seq
sequence shown
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Chinese (zh)
Inventor
李约翰
李生伟
周明
徐阳
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Latticon Suzhou Biopharmaceuticals Co Ltd
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Latticon Suzhou Biopharmaceuticals Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • This invention relates to anti-epidermal growth factor receptor (EGFR) antibodies or antigen-binding fragments thereof and anti-EGFR antibody-drug conjugates constructed therefrom, as well as methods for preparing the antibodies and antibody-drug conjugates thereof and methods for treating diseases associated with abnormal EGFR expression.
  • EGFR epidermal growth factor receptor
  • Epidermal growth factor receptor also known as ErbB-1 or HER1
  • EGFR Epidermal growth factor receptor
  • HER1 is a member of the ErbB/HER family of receptor tyrosine kinases. Its molecular structure mainly includes an extracellular domain (ECD), a transmembrane domain, and an intracellular tyrosine kinase domain involved in ligand binding.
  • ECD extracellular domain
  • Ligand binding induces conformational changes in the receptor protein, promotes the formation of homodimers or heterodimers of EGFR, and activates downstream signal transduction pathways, thereby participating in the regulation of cell proliferation, survival, differentiation, migration, and adhesion (Arteaga, J Clin Oncol 2001, 19:32s-40s; Lemmon and Schlessinger, Cell 2010, 141:1117-1134).
  • EGFR gene mutations and/or gene amplification may lead to EGFR overexpression.
  • Reports show that EGFR is overexpressed in various epithelial cell-derived tumors, such as lung cancer, colorectal cancer, glioma, head and neck cancer, pancreatic cancer, breast cancer, prostate cancer, esophageal cancer, gastric cancer, ovarian cancer, bladder cancer, and kidney cancer (Yarden, Eur J Cancer 2001, 37:S3-S8; Mendelsohn et al., Oncogene 2000, 19:6550-6565); furthermore, EGFR overexpression is associated with tumor invasiveness, poor prognosis, and drug resistance (Baselga, Oncologist 2002, 7:2-8).
  • EGFR-targeting therapeutics mainly fall into two categories: anti-EGFR antibodies (e.g., Cetuximab, Panitumumab, Nimotuzumab, and Necitumumab) and tyrosine kinase inhibitors (e.g., Afatinib, Erlotinib, Gefitinib, and Osimertinib).
  • anti-EGFR antibodies e.g., Cetuximab, Panitumumab, Nimotuzumab, and Necitumumab
  • tyrosine kinase inhibitors e.g., Afatinib, Erlotinib, Gefitinib, and Osimertinib.
  • EGFR-targeted therapies are often accompanied by on-target off-tumor toxicities, such as skin toxicity, hypomagnesemia, stomatitis, and diarrhea (Shah et al., Drug Saf 2019, 42:181-198); among these, skin toxicity is the most common dose-limiting toxicity of EGFR-targeted therapies.
  • This invention provides an antibody targeting EGFR and an antibody-drug conjugate (ADC) constructed therefrom.
  • the antibody and ADC can specifically bind to the epitope of amino acid residues 447-491 of the extracellular domain of EGFR (its sequence is shown in SEQ ID NO: 107).
  • the inventors' research found that the accessibility of this antigen-binding epitope is related to the expression abundance of EGFR on the cell surface, that is, it can only be bound when EGFR is overexpressed on the cell surface. Therefore, the antibody and ADC can specifically target cells that overexpress EGFR (e.g., tumor cells) and not bind to normal tissues or cells with relatively low EGFR expression abundance (e.g., skin tissue).
  • the ADC can produce targeted killing effect on the cell and subsequent bystander killing effect. Therefore, the ADC of the present invention is expected to be used to treat cancers that have developed resistance to existing EGFR-targeted therapies.
  • the antibody and ADC do not bind or weakly bind to skin tissue and other normal tissues or cells, and do not affect the downstream signal transduction pathway mediated by EGFR. Therefore, the ADC does not kill normal tissues and cells expressing EGFR, nor does it interfere with the normal biological function of EGFR, thereby avoiding non-tumor-targeting toxic effects (e.g., skin toxicity).
  • the present invention provides an isolated polypeptide containing amino acid residues 447-491 of the EGFR extracellular domain, and an antibody or antigen-binding fragment thereof that specifically binds to said polypeptide.
  • the sequence of amino acid residues 447-491 of the EGFR extracellular domain contained in the polypeptide is YANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGC (SEQ ID NO: 107).
  • the present invention provides the use of a polypeptide comprising amino acid residues 447-491 of the extracellular domain of EGFR in the preparation of isolated anti-EGFR antibodies or antigen-binding fragments thereof that specifically bind to EGFR-overexpressing cells but do not bind or weakly bind to normal tissues or cells with relatively low EGFR expression levels.
  • the present invention provides an isolated anti-EGFR antibody or its antigen-binding fragment thereof, which specifically binds to the epitope of amino acid residues 447-491 of the EGFR extracellular domain. Since the epitope of amino acid residues 447-491 of the EGFR extracellular domain is only exposed in a state of EGFR overexpression, the binding activity of the antibody or its antigen-binding fragment to cells depends on the abundance of EGFR expression on the cell surface. This means it specifically binds to EGFR-overexpressing tumor cells, while not binding or weakly binding to normal tissues or cells expressing EGFR, and it does not inhibit or activate the regulation of EGFR and its downstream signal transduction pathways.
  • the binding affinity constant (K ⁇ sub> D ⁇ /sub> ) of the antibody or its antigen-binding fragment to the polypeptide containing amino acid residues 447-491 of the EGFR extracellular domain is ⁇ 5 ⁇ 10 ⁇ sup>-7 ⁇ /sup> M, ⁇ 1 ⁇ 10 ⁇ sup>-7 ⁇ /sup> M, ⁇ 5 ⁇ 10 ⁇ sup>-8 ⁇ /sup> M, or ⁇ 1 ⁇ 10 ⁇ sup> -8 ⁇ /sup> M.
  • the antigen-binding epitopes of existing therapeutic anti-EGFR antibodies differ from those recognized by the antibodies or antigen-binding fragments of this invention.
  • the antigen-binding epitope of Cetuximab is located in subdomain D3 of the extracellular domain of EGFR, and it exhibits strong binding activity with cells exhibiting both high and low EGFR expression levels. Therefore, Cetuximab can bind to normal tissues or cells expressing EGFR and can inhibit the activation of EGFR and its downstream signal transduction pathways.
  • the anti-EGFR antibody or its antigen-binding fragment of the present invention upon binding to cells overexpressing EGFR, can be internalized by the cells, and the internalization rate is comparable to that of Cetuximab.
  • the anti-EGFR antibody includes fully human anti-human EGFR antibody, mouse anti-human EGFR antibody or its derived chimeric antibody and/or humanized antibody and its optimized antibody (e.g., affinity-matured antibody).
  • the anti-EGFR antibody or its antigen-binding fragment of the present invention does not cross-bind with other members of the ErbB/HER family (including HER2, HER3 and HER4).
  • the present invention provides an ADC that couples a small molecule toxin compound to the anti-EGFR antibody or its antigen-binding fragment via a linker.
  • the ADC can specifically target EGFR-overexpressing tumor cells and exert a killing effect on them, as well as a subsequent bystander killing effect; furthermore, the ADC has no killing activity against normal tissues or cells, thereby solving the non-tumor-targeting toxicity problem of existing EGFR-targeted therapies.
  • the ADC can be represented by equation (I):
  • Ab represents the anti-EGFR antibody or its antigen-binding fragment of the present invention
  • D indicates a small molecule toxic compound (Drug);
  • L represents a cleavable linker that couples D to Ab
  • p represents the number of (L-D) copies coupled to Ab, ranging from 2 to 8.
  • the small molecule toxin compound includes cytotoxins, chemotherapeutic agents, and radioisotopes.
  • the small molecule toxin compound is a cytotoxin, including tubulin inhibitors and DNA damaging agents; preferably, the tubulin inhibitors include eribulin, auristatins derivatives (e.g., MMAE, MMAF, MMAD), tubulysins, cryptomycins, and maytansinoids derivatives (e.g., DM1, DM2, DM3, DM4), and the DNA damaging agents include topoisomerase inhibitors (e.g., camptothecin derivatives SN-38, exatecan, and DXd [Exatecan derivative for ADC]), pyrrolobenzodiazepines (PBD), calic acid and its derivatives (e.g., N-acetylcalic acid [CMC]), and duocarmycin.
  • the small molecule toxin compound is e
  • the cleavable linker can be any linker containing a cleavable portion comprising any cleavable chemical bond.
  • the cleavable linker comprises a cleavable peptide portion capable of being hydrolyzed by intracellular peptidases or proteases, the cleavable peptide portion comprising an amino acid unit comprising a dipeptide, tripeptide, or tetrapeptide.
  • the cleavable linker may comprise at least one spacer conjugated to the anti-EGFR antibody or its antigen-binding fragment (Ab) of the present invention.
  • the spacer conjugated to the antibody or its antigen-binding fragment is hydrophilic, and exemplary spacers comprise polyethylene glycol (PEG).
  • the spacer is via a maleic butylene diimide group (Mal, ) or 4-(5-thio-1,3,4-oxadiazol-2-yl)benzamide
  • Mal maleic butylene diimide group
  • the cleavable linker includes a Mal-spacer region.
  • the Mal-spacer region or It can be linked to the thiol group of cysteine residues at specific positions in the constant and/or variable regions of the antibody.
  • the cleavable linker is connected to the anti-EGFR antibody or its antigen-binding fragment via a thioether bond or a dithioether bond.
  • the cleavable portion of the cleavable adapter (e.g., a cleavable peptide) can be directly conjugated to the small molecule toxin compound portion of the ADC, wherein the small molecule toxin compound is eribulin or a derivative thereof.
  • p is 2 to 8, for example 4 to 8.
  • p can be an integer greater than 0 or a non-integer.
  • the present invention relates to isolated nucleic acid molecules (also referred to as “polynucleotides”) encoding the anti-EGFR antibody or its antigen-binding fragment described herein, as well as expression vectors comprising said nucleic acid molecules and host cells comprising said nucleic acid molecules or expression vectors.
  • the invention also relates to methods for preparing the polypeptides described herein and/or the anti-EGFR antibody or its antigen-binding fragment using said host cells, said methods comprising culturing said host cells and recovering said polypeptides and/or said antibodies or their antigen-binding fragments from the culture medium.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-EGFR antibody or an antigen-binding fragment thereof as described herein, an anti-EGFR antibody-drug conjugate, and a pharmaceutically acceptable carrier.
  • the present invention relates to a kit containing an effective amount of the anti-EGFR antibody-drug conjugate or pharmaceutical composition of the present invention, and optionally at least one other tumor therapeutic agent.
  • the present invention relates to a method for treating EGFR-overexpressing tumors in a subject, the method comprising administering the anti-EGFR antibody-drug conjugate, or pharmaceutical composition, or kit described herein to a subject in need.
  • the present invention relates to the use of the anti-EGFR antibody-drug conjugate, or pharmaceutical composition, or kit described herein in the preparation of a medicament for treating EGFR-overexpressing tumors in a subject.
  • the present invention relates to the anti-EGFR antibody-drug conjugate, or pharmaceutical composition, or kit described herein for treating EGFR-overexpressing tumors in a subject.
  • the present invention relates to the use of the anti-EGFR antibody-drug conjugate, or pharmaceutical composition, or kit described herein for treating EGFR-overexpressing tumors in a subject.
  • the tumor includes tumors that overexpress EGFR (e.g., EGFR gene amplification) regardless of whether their EGFR intracellular tyrosine kinase domains are mutated (e.g., primary EGFR mutations and acquired EGFR mutations caused by tyrosine kinase inhibitors, including but not limited to L688P, Q701H, E709V, L718Q, Q724S, K745N, L747P, D761Y, M766Q, C781R, exon 19 deletion, exon 21 insertion, L858R, T790M, C797S).
  • EGFR e.g., EGFR gene amplification
  • tyrosine kinase inhibitors including but not limited to L688P
  • the anti-EGFR antibody-drug conjugate or its pharmaceutical composition has cytotoxic activity against EGFR-overexpressing tumor cells. In some embodiments, the anti-EGFR antibody-drug conjugate or its pharmaceutical composition has a bystander killing effect on EGFR-overexpressing tumor cells in addition to direct killing. In some embodiments, the subject is a mammal.
  • the tumor includes lung cancer, head and neck cancer, glioblastoma, colorectal cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, kidney cancer or renal cell carcinoma, liver cancer, esophageal cancer, gallbladder cancer, pancreatic cancer, stomach cancer, thyroid cancer, bladder cancer, skin cancer, nasopharyngeal carcinoma, melanoma, and other solid tumors that are EGFR overexpressing.
  • the present invention relates to a method for detecting and/or measuring EGFR in a sample, or a method for screening cancer patients who respond to anti-EGFR ADC therapy of the present invention, comprising incubating the anti-EGFR antibody of the present invention or an antigen-binding fragment thereof with the sample or a biological sample isolated from the patient, and detecting whether the antibody or the antigen-binding fragment thereof binds to the sample or the biological sample.
  • Figure 1 Schematic diagram of the structure of EGFR on the cell membrane, including the ligand-unbound EGFR monomer (Figure 1A) and dimer (Figure 1B) in the resting state, and the ligand-bound activated EGFR dimer ( Figure 1C).
  • Figure 1A Schematic diagram of the structure of EGFR on the cell membrane, including the ligand-unbound EGFR monomer (Figure 1A) and dimer (Figure 1B) in the resting state, and the ligand-bound activated EGFR dimer ( Figure 1C).
  • the epitopes of amino acid residues 447-491 of the EGFR extracellular domain are located at the junction of subdomains 3 and 4 of the EGFR extracellular region and are shown as dashed circles in Figure 1.
  • Figure 2 Flow cytometry detection of the binding activity of anti-EGFR chimeric antibody mAb1135 and fully human antibody hAb173 with 293T engineered cells (293T-EGFR#2, 293T-EGFR#16-2D6) expressing different EGFR abundances, with Cetuximab as the control antibody.
  • Figure 3 Flow cytometry detection of the competitive binding activity of anti-EGFR chimeric antibody mAb1135 and fully human antibody hAb173 to A431 cells, wherein the biotin-labeled antibody is hAb173 (Biotin-hAb173).
  • Figure 4 Schematic diagram of the structure of human EGFR/HER4 chimeric recombinant protein.
  • FIG. 5 ELISA assay to detect the binding activity of anti-EGFR chimeric antibody mAb1135 and fully human antibody hAb173 to various EGFR/HER4 chimeric recombinant proteins, EGFR, and HER4.
  • the control antibody was Cetuximab.
  • Figure 6 Effects of anti-EGFR antibodies mAb1135 and hAb173 on the in vitro proliferation of A431 cells, with Cetuximab as the control antibody.
  • Figure 7 Flow cytometry detection of the binding activity of various mutants of the anti-EGFR humanized antibody Hu1135-H1L1 to A431 cells, with control antibodies including the maternal antibody Hu1135-H1L1 and Cetuximab.
  • Figure 8 Flow cytometry detection of the binding activity of anti-EGFR humanized antibodies containing single or multiple mutations and the maternal antibody Hu1135-H1L1-BM02 to A431 cells and MCF-10A cells, respectively.
  • Figure 9 Flow cytometry analysis of the binding activity of anti-EGFR humanized antibodies containing combined mutations (including BM02-H301-L202, BM02-H302-L202 and BM02-H303-L202) to A431 cells and MCF-10A cells, respectively.
  • the control antibodies included chimeric antibodies mAb1135 and Cetuximab.
  • Figure 10 Flow cytometry analysis of the binding activity of anti-EGFR humanized antibodies BM02-H302-L202 and BM02-H303-L202 to 293T engineered cells expressing different amounts of EGFR and the efficiency of their endocytosis in the cells, wherein the control antibody was Cetuximab.
  • FIG. Reporter gene assay for ADCC activity of anti-EGFR humanized antibody L202-H302TM, with Cetuximab as the control antibody.
  • Figure 12 Flow cytometry detection of the binding activity of the anti-EGFR humanized antibody L202-H302TM to cell lines expressing different amounts of EGFR, with the control antibody being Cetuximab.
  • FIG. 13 Immunohistochemistry (IHC) detection of the binding activity of anti-EGFR humanized antibody L202-H302TM to skin tissue of healthy individuals and lung cancer tissue of tumor patients.
  • the positive control antibody was Cetuximab
  • the negative control antibody was an IgG isotype antibody.
  • Figure 14 ELISA assay to detect the binding activity of anti-EGFR antibodies L202-H302TM and hAb173 to recombinant EGFR extracellular domain proteins containing different amino acid mutations at the junction of extracellular subdomains D3 and D4, with Cetuximab as the control antibody.
  • Figure 15 ELISA assay to detect the binding activity of anti-EGFR antibodies E45-mab304 and E45-mab309 to recombinant EGFR extracellular domain proteins containing different amino acid mutations at the junction of extracellular subdomains D3 and D4, with Cetuximab as the control antibody.
  • FIG. 16 Flow cytometry analysis of the binding activity of antibodies (including E45-mab304, E45-mab309, and L202-H302TM) that specifically recognize the EGFR extracellular domain epitopes 447-491 with EGFR expressed at different abundances in 293T engineered cell lines.
  • the control antibody was Cetuximab.
  • Figure 17 Effects of antibodies that specifically bind to the epitopes of amino acid residues 447-491 of the extracellular domain of EGFR (including L202-H302TM, E45-mab304 and E45-mab309) on the in vitro proliferation of A431 cells, with Cetuximab as the control antibody.
  • Figure 18 Effects of antibodies that specifically bind to the EGFR extracellular domain epitopes 447-491 (including L202-H302TM, E45-mab304, and E45-mab309) on EGF-induced AKT phosphorylation in A431 cells, with Cetuximab as the control antibody.
  • FIG. 19 ELISA method for detecting the binding activity or binding specificity of anti-EGFR ADC (L202-H302TM-ADC) with ErbB/HER family members (including EGFR, HER2, HER3 and HER4), wherein the control antibody includes Cetuximab and the corresponding naked anti-L202-H302TM of the ADC.
  • Figure 20 Flow cytometry detection of the binding activity of anti-EGFR ADC (L202-H302TM-ADC) to 293T engineered cell lines expressing different abundances of EGFR, wherein the control antibody included Cetuximab and the corresponding naked anti-L202-H302TM of the ADC.
  • Figure 21 In vitro cell killing assay to detect the in vitro killing activity of anti-EGFR ADC (L202-H302TM-ADC) and control small molecule toxin compound Eribulin in cell lines expressing different abundances of EGFR.
  • Figure 22 Bystander effect of anti-EGFR ADC (L202-H302TM-ADC) detected by flow cytometry by culturing MDA-MB-468 cells overexpressing EGFR and Jurkat cells not expressing EGFR, either alone or in co-culture.
  • the percentage values shown in the figure represent the ratio of the number of surviving MDA-MB-468 cells and Jurkat cells after treatment with L202-H302TM-ADC (1 nM) to the number of surviving cells in blank medium (Medium).
  • Figure 23 Detection of in vivo tumor-suppressive activity of anti-EGFR ADC (L202-H302TM-ADC) in mouse subcutaneous xenograft tumor models constructed based on tumor cell lines A431 ( Figure 23A) and JIMT-1 ( Figure 23B).
  • the control group included an antibody-small molecule drug mixture group (ADMix) and a solvent group (Vehicle). All tumor-bearing mice were administered via tail vein injection. The arrows in the figure indicate the time points of administration.
  • EGFR epidermal growth factor receptor
  • the protein is also referred to as ErbB-1 or HER1.
  • EGFR refers to any naturally occurring form of human EGFR, which may have the amino acid sequence shown in SEQ ID NO:105 and/or the full-length EGFR amino acid sequence containing the signal peptide as shown in Swiss-Prot registry number P00533 (where amino acid residues 1-24 are the signal peptide and amino acid residues 25-645 are the extracellular domain), and may be naturally expressed by cells (including tumor cells) or expressed by cells transfected with the EGFR gene or cDNA.
  • wild-type EGFR or EGFR extracellular domain as described herein refers to the mature form of wild-type EGFR (the amino acid sequence of which is shown in SEQ ID NO:106) or the mature form of EGFR extracellular domain (the sequence of amino acid residues 1-621 shown in SEQ ID NO:106).
  • the extracellular domain of EGFR consists of four subdomains: subdomain 1 (D1, approximately amino acid residues 1-165), subdomain 2 (D2, approximately amino acid residues 166-312), subdomain 3 (D3, approximately amino acid residues 313-481), and subdomain 4 (D4, approximately amino acid residues 482-621).
  • the amino acid residue numbers do not include the signal peptide.
  • D1 and D3 are involved in ligand binding, while D2 and D4 are cysteine-rich domains responsible for receptor dimerization (Cochran et al., J Immunol Methods 2004, 287:147-158; Ward et al., Proteins 1995, 22:141-153).
  • cells expressing EGFR can be naturally occurring cells or cell lines (e.g., tumor cells) or cells generated by recombinantly introducing nucleic acids encoding EGFR into host cells.
  • antigen-binding domain As used interchangeably to refer to a specific region on an antibody or antigen-binding fragment or its derivative that is directly involved in the specific interaction with the target antigen. This interaction occurs, for example, through binding, steric hindrance, stabilization/instability, or spatial distribution, to achieve a dynamic equilibrium with the target antigen.
  • antigen-binding domain also refers to a specific region on the antibody or antigen-binding fragment or its derivative that interacts with a specific epitope on EGFR, achieving a dynamic equilibrium through binding, steric hindrance, stabilization/instability, or spatial distribution.
  • An antibody is a polypeptide or protein that is generally encoded by one or more immunoglobulin genes or fragments thereof and is capable of specifically recognizing and binding to an antigen. Recognized immunoglobulin genes include constant regions of ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , as well as numerous variable regions. Light chains are classified as ⁇ or ⁇ . Heavy chains are classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , which respectively define the immunoglobulin class/isotype IgG, IgM, IgA, IgD, and IgE.
  • the typical structural unit of an immunoglobulin (e.g., an antibody) is a tetramer.
  • Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa).
  • the N-terminal domain of each chain defines a variable region (V) consisting of approximately 100 to 110 or more amino acids, primarily responsible for antigen recognition.
  • the antibody heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH), where the heavy chain constant region typically includes three domains: CH1, CH2, and CH3.
  • the light chain consists of a light chain variable region (VL) and a light chain constant region (CL), where the light chain constant region typically contains one domain, CL.
  • VH and VL together forms a single antigen-binding site.
  • Endogenous VL is encoded by gene segments V (variable) and J (conjugating), and endogenous VH is encoded by V, D (diversity), and J.
  • Both VL and VH include a region of hypervariability, also known as a complementarity-determining region (CDR), and a framework region (FR).
  • CDR complementarity-determining region
  • FR framework region
  • variable region or “V region” are used interchangeably and refer to a heavy chain variable region or light chain variable region arranged in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the amino terminus to the carboxyl terminus.
  • J region refers to a subsequence encoding a variable region containing the C-terminal portions of CDR3 and FR4. V or J regions can be naturally occurring, recombinant, or synthetic.
  • antibody light chain variable regions and/or antibody heavy chain variable regions are sometimes collectively referred to as “antibody variable regions,” and antibody light chains and/or antibody heavy chains are collectively referred to as “antibody chains.”
  • the FR of the antibody or its antigen-binding fragment provided herein may be identical to a human germline sequence or may be natural or artificially modified.
  • CDR and FR can be determined using a variety of definitions well known in the art, such as Kabat, Chothia, IMGT, and Contact (see: Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, 1991, NIH Publication No.
  • antigen binding sites are also described in the following literature: Ruiz et al., Nucleic Acids Res 2000, 28:219-221; Lefranc, Nucleic Acids Res 2001, 29:207-209; Lefranc, The Immunologist 1999, 7:132-136; Lefranc et al., Dev Comp Immunol 2003, 27:55-77; MacCallum et al., J Mol Biol 1 996,262:732-745; Martin et al., Proc Natl Acad Sci USA 1989,86:9268-9272; Martin et al., Methods Enzymol 1991,203:121-153; Sternberg (ed.), Protein Structure Prediction: A Practical Approach, Oxford University Press 1996,141-172.
  • This document contains any definitional method for determining the CDR in the anti-EGFR antibody or its antigen-binding fragment of the present invention.
  • Table 1 shows the positional numbers of the amino acid sequences of the antibody CDR determined using different definitional methods. The exact number of amino acid residues covering a particular CDR varies with the CDR sequence. Given the amino acid sequence of the antibody variable region, those skilled in the art can determine the CDR of the antibody using conventional methods, including but not limited to the definitions described herein.
  • Kabat et al. defined a numbering system for the variable region sequence, which can be applied to any antibody. Those skilled in the art can readily apply this "Kabat numbering" system to the variable region sequence of any antibody without relying on any experimental data other than the antibody sequence itself to determine the variable region sequence. Unless otherwise stated, the numbering of specific amino acid residue positions in the variable region of the antigen-binding domain of the anti-EGFR antibody or ADC of the present invention is determined according to the Kabat numbering system.
  • Antibodies exist either as intact immunoglobulins or as numerous fragments produced by digestion with various peptidases. While various antibody fragments are defined according to the enzymatic digestion products of intact antibodies, those skilled in the art will understand that such antibody fragments can also be generated by chemical cleavage or synthesized de novo using recombinant DNA methods.
  • the term "antigen-binding fragment” refers to an antibody moiety containing one or more CDRs or any other antibody fragment capable of binding to an antigen (e.g., EGFR or its extracellular domain) but not possessing an intact antibody structure. Antigen-binding fragments can have the same specific antigen-binding activity as intact antibodies.
  • the antigen-binding fragments also retain the ability to internalize into cells expressing the target antigen.
  • the antigen-binding fragment may contain one or more CDRs from a particular human antibody, transposed to a frame region from one or more different human antibodies.
  • Antigen-binding fragments include, but are not limited to: (i) “Fab” fragments, monovalent antibody fragments composed of VH, VL, CL, and CH1 domains; (ii) “F(ab')2” fragments, bivalent fragments containing two Fab fragments linked by disulfide bonds in the hinge region; (iii) “Fv” fragments, composed of the VL and VH domains of the antibody arm, which are the smallest antibody fragments containing complete antigen-binding sites; (iv) “Fd” fragments, composed of VH and CH1 domains; (v) “Single-chain Fv antibody (scFv)” or “single-chain antibody”, which refers to engineered antibodies composed of light chain variable regions directly linked to heavy chain variable regions or linked by a single peptide chain (Huston et al., Proc Natl Acad Sci USA 1988, 85:5879-5883; Bird et al., Science 19 88,242:423-4
  • Single-domain antibodies are independent immunoglobulin domains;
  • "Diabody” is a bivalent bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to pair the two domains on the same chain, thus forcing these two domains to pair with the complementary domain of another chain to form two antigen-binding sites (Holliger et al., Proc Natl Acad Sci USA 1993, 90: 6444-6448; Poljak et al., Structure 1994, 2: 1121-1123; EP404097; WO93/11161).
  • Fc region refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region, such as the immunoglobulin heavy chain constant region other than the first constant region (CH1).
  • the Fc region may comprise immunoglobulin domains CH2 and CH3, as well as a hinge region between CH1 and CH2.
  • the Fc region as used herein includes native sequence Fc regions and/or Fc region variants, and may be part of the anti-EGFR antibody of the present invention or its ADC.
  • the boundaries of the Fc region can vary; however, the human IgG heavy chain Fc region is generally defined as containing a cysteine residue at position 226 or a proline residue at position 230 at its amino terminus, according to the EU numbering system/scheme, as seen in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, 1991, NIH Publication No. 91-3242.
  • anti-EGFR antibody or “antibody that specifically binds to EGFR” refers to any form of antibody or fragment thereof that specifically binds to EGFR, and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, as long as the fragment can specifically bind to EGFR.
  • binding refers to a binding reaction that determines the presence of a target molecule (e.g., an antigen) in a heterogeneous population of proteins and other biological products (e.g., biological samples such as blood, serum, plasma, or tissue samples).
  • a target molecule e.g., an antigen
  • biological products e.g., biological samples such as blood, serum, plasma, or tissue samples.
  • this binding is selective for the target molecule and distinguishes between undesirable or nonspecific interactions.
  • an antibody that specifically binds to a target molecule exhibits higher affinity, stronger binding activity, easier binding, and/or longer binding duration when binding to the target molecule compared to binding to other non-target molecules.
  • Various immunoassays can be used to screen for antibodies that specifically react with a particular protein, such as solid-phase ELISA assays.
  • the specific or selective binding reaction of an antibody or conjugate to an antigen produces a signal at least twice the background signal, more generally at least 10-100 times the background signal, and substantially does not bind to other antigens present in the sample in significant quantities.
  • the equilibrium dissociation constant (K ⁇ sub> D ⁇ /sub>) for the specific binding of the antibody to the target antigen is ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, or ⁇ 0.1 nM.
  • the term "monoclonal antibody” refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that the individual antibodies constituting the group are identical except for the possibility of small amounts of naturally occurring mutations. Monoclonal antibodies exhibit high binding specificity and affinity for specific epitopes. Monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., Nature 1975, 256:495, or by recombinant DNA methods (see US4816567), or isolated from phage antibody libraries, for example, using techniques described in Clackson et al., Nature 1991, 352:624-628; Marks et al., J Mol Biol 1991, 222:581-597.
  • chimeric antibody refers to an antibody containing sequences derived from two different antibodies (e.g., US4816567), which are typically derived from different species.
  • a chimeric antibody comprises human and rodent antibody fragments, typically a human constant region and a mouse variable region.
  • Methods for generating chimeric antibodies include conventional recombinant DNA and gene transfection techniques known to those skilled in the art (e.g., Morrison et al., Proc Natl Acad Sci USA 1984, 81:6851-6855; US5202238; US5204244).
  • humanized antibody or antigen-binding fragment refers to an antibody or antigen-binding fragment comprising a CDR derived from a non-human animal, a FR region derived from a human, and a constant region derived from a human.
  • the humanized antibody optionally also comprises at least a portion of the constant region of a human immunoglobulin. Because humanized antibodies or antigen-binding fragments have reduced immunogenicity, they can be used as therapeutic agents administered to humans.
  • the non-human animal is a mammal such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.
  • the humanized antibody or antigen-binding fragment is substantially composed entirely of human sequences, except that the CDR sequence is non-human.
  • the humanized antibody can be further modified, improved, and optimized for specificity, affinity, and/or activity by replacing corresponding residues in the FR framework region of human immunoglobulins with residues from antibodies from non-human species.
  • the human-derived FR region may comprise the same amino acid sequence as the human antibody from which it originates, or may comprise some amino acid changes, for example, no more than five amino acid changes.
  • the amino acid change may exist only in the heavy chain FR region, only in the light chain FR region, or in both chains.
  • corresponding germline sequence refers to an antibody variable region amino acid sequence or subsequence that, when aligned with all other known germline immunoglobulin variable region amino acid sequences, exhibits the highest amino acid sequence identity with a reference germline immunoglobulin variable region amino acid sequence.
  • the corresponding germline sequence can be a single frame region, a single complementarity-determining region, a frame region and a complementarity-determining region, a variable region, or other combinations containing a variable region sequence or subsequence. Sequence identity can be determined by aligning two sequences using the methods described herein, for example, BLAST, ALIGN, or other alignment algorithms known in the art.
  • the corresponding germline amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a reference germline immunoglobulin variable region amino acid sequence.
  • the antibody portion of the anti-EGFR antibody or ADC of the present invention may be selected from any one or more of the following forms, including chimeric, non-human, humanized, or fully human forms, as long as the form can specifically bind to EGFR.
  • the term "antigen" refers to a molecule that can be bound by a binding agent, such as an antibody or an antigen-binding fragment thereof.
  • the antigen can be used to immunize an animal and generate antibodies in the animal that can bind to the antigen.
  • the antigen may have one or more epitopes that interact with the antibody.
  • the antigen in this invention can be EGFR expressed on the surface of naturally occurring cells or cell lines (e.g., tumor cells), or it can be a "chimeric antigen polypeptide" (also called a "recombinant antigen polypeptide” or “chimeric polypeptide") composed of any fragment of the EGFR amino acid sequence replacing corresponding amino acid residues of other members of the ErbB/HER family (e.g., HER4).
  • the chimeric antigen polypeptide can be produced by introducing the nucleic acid encoding this polypeptide into a host cell for expression.
  • the "chimeric antigen polypeptide" can be constructed by replacing a subdomain sequence of the EGFR extracellular domain with a corresponding subdomain sequence of HER4, or by replacing a subdomain sequence of the HER4 extracellular domain with a corresponding subdomain sequence of EGFR, thereby constructing an EGFR/HER4 chimeric antigen polypeptide.
  • the terms "epipote,” “antigen-binding epitope,” or “binding epitope” refer to a protein determinant, an antigenic moiety that can be recognized and specifically bound by an antibody.
  • Epitopes typically consist of surface groups of a molecule, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural features and specific charge characteristics.
  • the portion of the antibody or its antigen-binding fragment that recognizes the epitope is called the paratope.
  • the anti-EGFR antibody of this invention can specifically bind to the epitope with the amino acid sequence shown in SEQ ID NO:107, corresponding to amino acid residues 447-491 of the EGFR extracellular domain.
  • affinity refers to the intrinsic binding strength of an interaction between a molecule (e.g., a receptor or antigen) and its pair (e.g., a ligand or antibody), i.e., the strength of the sum of all non-covalent interactions.
  • binding affinity as used herein is used to reflect the intrinsic binding strength of a one-to-one interaction between members of a binding pair (e.g., receptor and ligand or antigen and antibody).
  • the affinity of molecule X for its pair Y is typically expressed as an equilibrium dissociation constant (K ⁇ sub>dis ⁇ /sub> ), which is the ratio of the dissociation rate constant (k ⁇ sub>dis ⁇ /sub> or k ⁇ sub> off ⁇ /sub> ) to the binding rate constant (k ⁇ sub> a ⁇ /sub> or k ⁇ sub> on ⁇ /sub> ).
  • K ⁇ sub>dis ⁇ /sub> is the ratio of the dissociation rate constant (k ⁇ sub>dis ⁇ /sub> or k ⁇ sub> off ⁇ /sub> ) to the binding rate constant (k ⁇ sub> a ⁇ /sub> or k ⁇ sub> on ⁇ /sub> ).
  • Affinity can be measured by common methods known in the art, including those used in this invention.
  • internalization or “endocytosis” refers to the process by which an antibody or its antigen-binding fragment or ADC binds to a target antigen on the cell surface and then enters the cell's endosome through internalization (i.e., endocytosis) via the cell's lipid bilayer membrane.
  • antibody variant refers to an antibody polypeptide sequence containing at least one amino acid mutation in the variable region of a reference antibody. Variants may be substantially homologous to or substantially identical to unmodified antibodies. In some embodiments, amino acid mutations are present in one, two, three, four, five, and/or six CDRs of the antibody portion of the anti-EGFR antibody or ADC of the present invention to improve and optimize the performance of the antibody or antigen-binding moiety, including but not limited to improving humanization, enhancing binding specificity or affinity or binding activity to EGFR, increasing yield, and/or improving stability (e.g., reducing or eliminating the risk of asparagine deacylation).
  • amino acid mutations are present in one, two, three, and/or four FRs of the antibody portion of the anti-EGFR antibody or ADC of the present invention to improve and optimize the performance of the antibody or antigen-binding moiety, for example, enhancing binding affinity to EGFR.
  • one or more amino acid mutations are made within the CDR and FR of the antibody portion of the anti-EGFR antibody or ADC of the present invention to improve the humanization of the antibody or antigen-binding fragment, enhance its binding specificity or affinity or binding activity to EGFR, increase its expression level, and/or improve its stability (e.g., reduce or eliminate the risk of asparagine deacylation).
  • the amino acid mutations include amino acid substitution, removal, insertion, or any combination thereof.
  • Amino acid substitution includes conserved amino acid substitution and non-conserved amino acid substitution.
  • conserved amino acid substitution involves replacing the residue with another amino acid of the same class (with similar chemical properties or functions), while non-conserved substitution involves replacing the residue with an amino acid of a different class (with dissimilar chemical properties or functions).
  • Those skilled in the art can perform conserved or non-conserved amino acid substitution based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphiphilic properties of the residues involved.
  • nonpolar (hydrophobic) amino acids include alanine (Ala, A), leucine (Leu, L), isoleucine (Ile, I), valine (V, V), proline (Pro, P), phenylalanine (Phe, F), tryptophan (Trp, W), and methionine (Met, M);
  • 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);
  • positively charged (basic) amino acids include arginine (Arg, R), lysine (Lys, K), and histidine (His, H); and
  • negatively charged (acidic) amino acids include aspartic acid (Asp, D) and glutamic acid (Glu, E).
  • the terms “identical,” “identity,” “percentage of identity,” or “percentage sequence identity” are used interchangeably, referring to the percentage of amino acid residues in a candidate sequence that are identical to those in a reference sequence, relative to the total number of amino acid residues in the reference sequence, after introducing necessary intervals to maximize the number of identical amino acids during amino acid sequence alignment. Conservative substitutions of these amino acid residues may or may not be considered identical residues.
  • Sequence alignment can be performed using tools disclosed in the art, such as BLASTp, ClustalW2 (see also Higgins et al., Methods Enzymol 1996, 266:383-402; Larkin et al., Bioinformatics 2007, 23:2947-2948), and ALIGN or Megalign (DNASTAR) software to determine the percentage sequence identity of the amino acid sequences.
  • tools disclosed in the art such as BLASTp, ClustalW2 (see also Higgins et al., Methods Enzymol 1996, 266:383-402; Larkin et al., Bioinformatics 2007, 23:2947-2948), and ALIGN or Megalign (DNASTAR) software to determine the percentage sequence identity of the amino acid sequences.
  • BLASTp Altschul et al., Altschul et al., Altschul et al., Altschul et al., Altschul et al., Altschul et al., Altschul e
  • amino acid sequences referred to in this article as "reducing or eliminating the risk of deamidation” are those containing amino acid residues that are readily deamidated but are replaced by amino acid residues that are less readily or reluctantly deamidated.
  • Deamidation is a chemical reaction in which the amide functional group in the side chain of asparagine or glutamine is removed or converted into another functional group.
  • asparagine (Asn) can be converted into aspartic acid or isoaspartic acid
  • glutamine (Gln) can be converted into glutamic acid or pyroglutamic acid.
  • Amino acid sequences containing Asn-Gly, Asn-Ser, or Asn-Thr sites are prone to deamidation, and asparagine is more readily deamidated than glutamine.
  • Deamidation of asparagine and/or glutamine can alter the structure and stability and/or function of antibodies (e.g., antibody-antigen binding), therefore, it is necessary to reduce or eliminate the risk of deamidation.
  • the risk of deamidation at these sites can be reduced or eliminated by predicting amino acid residues in the antibody variable region that are prone to deamidation (Sydow et al., PLoS ONE 2014, 9:e100736) and by substituting them with amino acid residues that are less or not prone to deamidation.
  • the term "isolated” means that the protein is substantially free of other cellular components bound to it in its native state, preferably in a homogeneous state; for example, the isolated protein may be removed from its native or natural environment.
  • the isolated protein may be lyophilized or aqueous. Typically, its purity and homogeneity can be determined using analytical chemistry techniques (e.g., polyacrylamide gel electrophoresis or high-performance liquid chromatography).
  • the protein is substantially purified.
  • purified means that the protein shows substantially only one band in a non-reducing electrophoresis gel.
  • the protein has a purity of at least 85%, more preferably at least 95%, and most preferably at least 99%.
  • recombinant proteins expressed in host cells are considered isolated, and the same applies to native or recombinant proteins separated, fractionated, or partially or substantially purified by any technique well known to those skilled in the art.
  • isolated antibody refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to EGFR is substantially free of antibodies that specifically bind to antigens other than EGFR).
  • an isolated antibody that specifically binds to EGFR may have cross-binding reactivity with EGFR proteins from other species (e.g., monkey EGFR). Furthermore, the isolated antibody may be substantially free of other cellular material and/or chemicals.
  • a recombinant polynucleotide containing a vector encoding a polypeptide or protein described herein e.g., a polypeptide containing amino acid residues 447-491 of the EGFR extracellular domain, an anti-EGFR antibody
  • isolated polynucleotides include recombinant polynucleotides contained in heterologous host cells or purified (partially or substantially) polynucleotides in solution.
  • engineering includes any manipulation of the polypeptide or protein backbone, or post-translational modification of naturally occurring or recombinant proteins or polypeptides.
  • Engineering includes mutation of the amino acid sequence, glycosylation, or modification of the side chain groups of individual amino acids, as well as combinations of these methods.
  • polypeptide refers to a polymer of amino acids and their equivalents, rather than to the specific length of the product; therefore, “peptide” and “protein” are included within the definition of a polypeptide.
  • the definition of a polypeptide also includes “antibody” as defined in this invention.
  • expression vector refers to a delivery vehicle into which a polynucleotide or nucleic acid encoding a protein can be operatively inserted to enable the expression of that protein.
  • Vectors can be used to transform, transduce, or transfect host cells, enabling the expression of the genetic material they carry within the host cells.
  • host cell refers to a cell into which a foreign polynucleotide or nucleic acid and/or vector has been introduced.
  • Host cells include “transformers” and “transformed cells,” which include the initially transformed cell and its progeny, regardless of the number of passages.
  • Progeny cells may not be identical to parental cells in their nucleic acid content and may contain mutations. This invention includes screening or selecting mutant progeny cells with the same function or biological activity from the initially transformed cells.
  • the terms “subject,” “patient,” or “individual” are used interchangeably and include, but are not limited to: mammals, including, for example, humans, non-human primates (e.g., monkeys), mice, pigs, dogs, cats, cattle, goats, rabbits, rats, guinea pigs, hamsters, horses, sheep, or other non-human mammals; non-mammals, including, for example, non-mammalian vertebrates such as birds (e.g., chickens, emus, or ducks) or fish; and non-mammalian invertebrates.
  • the subject and pharmaceutical composition involved in the use or method of the present invention are used (preventively and/or therapeutically) to treat non-human animals.
  • treating means alleviating (or “to alleviate”) a disease or symptom, slowing the onset or development of a disease or symptom, reducing the risk of developing a disease or symptom, or delaying the development of symptoms associated with a disease or symptom, reducing or terminating symptoms associated with a disease or symptom, producing a complete or partial reversal of a disease or symptom, curing a disease or symptom, or a combination of the above.
  • therapeutic effective amount refers to the dose and duration required to achieve effective prevention or improvement of symptoms associated with a disease or condition and/or to reduce the severity of the disease or condition.
  • Therapeutic effective amounts of the formulations, antibodies, or antigen-binding fragments thereof, ADCs, or compositions of the present invention can vary depending on various factors such as disease state, individual age, sex, and weight, and the ability of the antibody or antibody fraction or ADC to elicit the desired response in the individual. Therapeutic effective amounts can also be considered as any toxic or harmful effects of the formulation, antibody, or antigen-binding fragment thereof, ADC, or composition being less than the therapeutically beneficial effects.
  • effective amount refers to the amount of an active ingredient or agent sufficient to provide a clinical benefit to a subject (including, but not limited to, improvement, relief, or reduction of the disease, condition, or related symptoms, delaying or halting disease progression).
  • EGFR expression levels are relatively highest in normal tissues, particularly in skin.
  • “EGFR overexpression” or “EGFR overexpression” refers to a statistically significant increase in EGFR expression abundance on the cell surface compared to normal skin tissue/cell surface. Overexpression can result from gene amplification or enhanced transcription or translation. Cell surface EGFR expression levels can be assessed using methods known in the art.
  • immunohistochemical staining can be performed on paraffin-embedded tissue sections from tumor biopsies using the EGFR pharmDx TM kit (DakoCytomation), and EGFR expression levels can be described based on the score of EGFR staining intensity on the cell membrane (Pirker et al., Lancet Oncol 2012, 13:33-42). EGFR expression levels can also be described by measuring EGFR fluorescence staining levels on the cell surface using flow cytometry (FACS) or by measuring the levels of nucleic acid molecules encoding EGFR in cells using in situ hybridization, Western blotting, or PCR.
  • FACS flow cytometry
  • the expression level of EGFR in the breast epithelial cell line MCF-10A is comparable to that in normal human skin tissue, both showing IHC 2+ (see EGFR pharmDx TM kit instructions; Subik et al., Breast Cancer: Basic and Clinical Research 2010, 4:35-41).
  • the expression level of EGFR on the cell surface was detected by FACS, and the results showed that the surface EGFR expression level of the engineered cell line 293T-EGFR#16-2D6 was close to or at the same order of magnitude as that of MCF-10A cells.
  • the EGFR expression level on the cell surface is statistically significantly higher than that of normal skin tissue or the MCF-10A cells and 293T-EGFR#16-2D6 cells in this study, it can be considered as EGFR overexpression, for example, an IHC result of IHC 3+, or an MFI value detected by FACS that is 10 times or higher than that of MCF-10A cells.
  • the "bystander effect” or “bystander killing effect” of ADCs is mediated by free cytotoxins (e.g., small molecule toxic compounds with cell membrane permeability) that are released into the tumor microenvironment after being endocytosed by tumor cells overexpressing EGFR and degraded in their lysosomes.
  • free cytotoxins e.g., small molecule toxic compounds with cell membrane permeability
  • These released free cytotoxins can enter and kill neighboring cells, including those that do not express EGFR or have relatively low levels of EGFR expression, through passive diffusion.
  • the terms “pharmaceutical acceptable” or “medicinally acceptable” mean that the carrier, solvent, diluent, excipient and/or salt is chemically and/or physically compatible with other ingredients in the formulation and physiologically compatible with the subject.
  • the terms “comprising,” “including,” “containing,” “having,” or “involving” are used interchangeably to mean including the stated elements, integers, or steps, but not excluding any other elements, integers, or steps.
  • the terms “comprising,” “including,” “containing,” “having,” or “involving” are used, unless otherwise specified, they also cover situations consisting of the stated elements, integers, or steps.
  • the term “optional” indicates whether the object it modifies is present or not; for example, “the kit contains at least one additional oncology treatment agent” means that the kit may or may not contain at least one additional oncology treatment agent.
  • the two EGFRs form a stable dimer through the interaction of D2 and the interaction near the C-terminus of the transmembrane domain.
  • the D4s of the two EGFR extracellular regions exhibit an antiparallel conformation and the contact between D4 and the cell membrane is somewhat loose (Figure 1B).
  • the inventors discovered that during the transformation of EGFR from monomer to dimer, the amino acid sequence at the junction of D3 and D4 in its extracellular region changes from incomplete exposure in the monomeric state to complete exposure on the molecular surface in the dimer state. Furthermore, in the monomeric state, because D4 is in close contact with the cell membrane, the amino acid residues at the junction of D3 and D4 in the extracellular region of EGFR are too close to the cell membrane and are thus inaccessible, preventing antibody binding.
  • the present invention provides an isolated anti-EGFR antibody or its antigen-binding fragment thereof, the antibody or its antigen-binding fragment being capable of specifically binding to a polypeptide containing amino acid residues 447-491 of the EGFR extracellular domain, wherein the polypeptide includes any fragment derived from EGFR or HER4, as long as the polypeptide contains or is intercalated with amino acid residues 447-491 of the EGFR extracellular domain.
  • amino acid residues 447-491 of the EGFR extracellular domain are located at the junction of D3 and D4 in the EGFR extracellular region, and their amino acid sequence is shown below:
  • the polypeptide containing amino acid residues 447-491 of the EGFR extracellular domain includes an EGFR extracellular domain polypeptide, or a full-length EGFR polypeptide with an amino acid sequence as shown in SEQ ID NO: 105 or 106; in some embodiments, the polypeptide is a chimeric polypeptide, including a chimeric polypeptide recombined by replacing the corresponding amino acid sequence of HER4 with amino acid residues 447-491 of the EGFR extracellular domain, with an amino acid sequence as shown in SEQ ID NO: 109.
  • the binding activity of the anti-EGFR antibody or its antigen-binding fragment that specifically binds to the polypeptide containing the amino acid sequence of the EGFR extracellular domain 447-491 described in this invention depends on the expression abundance of EGFR on the cell surface. That is, the antibody or its antigen-binding fragment only binds to cells that overexpress EGFR, and does not bind or binds weakly to normal tissues or cells.
  • the antibody or its antigen-binding fragment does not interfere with the binding of ligands to EGFR, that is, it does not affect the regulation of EGFR and its downstream signal transduction pathways.
  • the present invention provides an isolated anti-EGFR antibody or its antigen-binding fragment thereof, said antibody or its antigen-binding fragment being capable of specifically binding amino acid residues 447-491 of the extracellular domain of EGFR or polypeptides containing therein.
  • the anti-EGFR antibody provided by this invention can be a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.
  • the screening and/or preparation methods for the antibody include any method known in the art, such as hybridoma technology, phage display technology, single-lymphocyte gene cloning technology, etc.
  • the phage display technology includes constructing the antibody library of this invention using various phage display methods known in the art. For example, US5223409, US5622699, and US6068829 disclose methods for preparing phage libraries; phage display libraries can also be constructed according to the methods described in "Antibody Phage Display: Methods and Protocols (edited by O'Brien and Aitken)".
  • phage display libraries can be panned to identify phages expressing the target antibody (or single-chain antibody).
  • the panning can be achieved by infecting host bacteria with phages and allowing them to multiply and amplify within the host. Phages secreted by host bacteria with single-chain antibody fragments on their surface are collected and panned multiple times as needed until phages capable of selectively or specifically binding to the target antigen are obtained. Finally, the amino acid sequence of the antibody variable region is obtained by sequencing the antibody gene in the phage genome (Arap et al., Science 1998, 279:377-380; Smith et al., Science 1985, 228:1315-1317).
  • a polypeptide containing any fragment of EGFR (preferably the EGFR extracellular domain) or any fragment of HER4 (preferably the EGFR extracellular domain) can be selected as the antigen to immunize mice.
  • the antibody variable region gene is then amplified from mature mouse B cells, and the mouse antibody variable region is displayed on the phage surface using phage display technology to construct a mouse immune library.
  • a fully human antibody natural library is constructed using human PBMCs as the starting material.
  • the antibody libraries can be panned using specific target molecules (e.g., the aforementioned antigens), and the interaction between the antibody variable region displayed on the phage surface and the target molecule can be detected.
  • specific target antigen e.g., a polypeptide containing any fragment of HER4 containing amino acid sequences of EGFR extracellular domains 447-491, preferably a chimeric polypeptide with an amino acid sequence as shown in SEQ ID NO:109
  • pan the antibody library to screen for antibodies that recognize amino acid residues of EGFR extracellular domains 447-491.
  • this invention utilizes the EGFR extracellular domain as the target antigen to immunize mice.
  • Mouse antibody heavy chain variable region and light chain variable region gene fragments are obtained from the spleen tissue of immunized mice, or fully human antibody heavy chain variable region and light chain variable region gene fragments are obtained from human PBMCs. These mouse or fully human antibody variable region gene fragments are then linked to phage surface structural protein gene III and co-expressed to participate in phage assembly and be displayed on the phage surface, thereby constructing a mouse immune library or a fully human antibody natural library.
  • phages that specifically bind to the target antigen can be enriched through multiple rounds of adsorption-elution-amplification (panning).
  • the corresponding DNA sequence information is then obtained using gene sequencing technology, allowing the deduction of the amino acid sequence of the antibody variable region.
  • the anti-EGFR antibody of the present invention or its antigen-binding fragment specifically binds to an epitope located at the junction of the D3 and D4 extracellular domains of EGFR, the epitope comprising the amino acid sequence of positions 447-491 of the extracellular domain of EGFR as shown in SEQ ID NO:107.
  • One or more key amino acid sequences in the antigen that contact/bind with the anti-EGFR antibody or its antigen-binding fragment can be identified by substituting or deleting amino acid sequences in specific regions of wild-type EGFR and determining whether the anti-EGFR antibody or its antigen-binding fragment can bind to the mutated EGFR.
  • These key amino acid sequences contain antigen-binding epitopes of the anti-EGFR antibody or its antigen-binding fragment.
  • multiple specific EGFR mutants can be constructed by single-point or multi-point mutation of amino acid residues at specific positions in wild-type EGFR and determining whether these mutants can bind to the anti-EGFR antibody or its antigen-binding fragment. This identifies key amino acid residues that play a direct contact role in antibody-antigen binding, as well as amino acid residues that are spatially close enough to the key amino acid residues that affect the contact between the key amino acid residues and the antibody after mutation. All these directly contacting key amino acid residues and amino acid residues affecting the contact constitute the antigen-binding epitopes of the antibody.
  • any corresponding extracellular subdomain sequence of EGFR and HER4 can be substituted to construct various EGFR/HER4 chimeric peptides with original tertiary structures, i.e., the tertiary structure of the constructed EGFR/HER4 chimeric peptides is similar to that of wild-type EGFR or HER4.
  • any subdomain sequence in the extracellular region of wild-type HER4 is substituted to replace the corresponding subdomain sequence in the extracellular region of wild-type EGFR to construct various EGFR/HER4 chimeric peptides.
  • binding activity of these peptides with the anti-EGFR antibody or its antigen-binding fragment of the present invention is then detected by ELISA. If the binding activity is significantly lower than that of wild-type EGFR (including a decrease of at least 50% compared to wild-type EGFR, such as 50%, 60%, 70%, 80%, 90%, 99% or more, or any value between the listed values), it can be determined that the substituted EGFR extracellular subdomain sequence contains key amino acids that bind to the anti-EGFR antibody or its antigen-binding fragment and constitutes an antigen-binding epitope.
  • each HER4/EGFR chimeric polypeptide is constructed by replacing a corresponding subdomain sequence in wild-type HER4 with any subdomain sequence of the wild-type EGFR extracellular region.
  • the binding activity of these polypeptides with the anti-EGFR antibody or its antigen-binding fragment of the present invention is then detected by ELISA.
  • the binding activity is not less than 50% of the binding activity of wild-type EGFR (e.g., 50%, 60%, 70%, 80%, 90%, 99% or more, or any value between the listed values), it can be determined that the EGFR extracellular region subdomain sequence used to replace the sequence in HER4 contains a key amino acid that binds to the anti-EGFR antibody or its antigen-binding fragment and the antigen-binding epitope it constitutes.
  • the region containing the key amino acid that binds to the anti-EGFR antibody or its antigen-binding fragment and the antigen-binding epitope it constitutes includes EGFR extracellular regions D3 and D4 (e.g., the junction sequence of EGFR extracellular regions D3 and D4), which is different from the binding epitope of Cetuximab (located in EGFR extracellular region D3).
  • amino acid residues at the D3-D4 junction i.e., amino acid sequences 447-508 of the extracellular region of wild-type EGFR, as shown in SEQ ID NO:106, are mutated to amino acid residues corresponding to the same positions in wild-type HER4 (as shown in SEQ ID NO:108) to construct multiple EGFR mutants.
  • the binding activity of these mutants with the anti-EGFR antibody or its antigen-binding fragment of the present invention is then detected to determine the antigen-binding epitopes and key amino acids of the antibody.
  • Various methods known in the art can be used to determine the binding of the anti-EGFR antibody or its antigen-binding fragment to each EGFR mutant; exemplary methods are described in Example 3.
  • the binding activity of the anti-EGFR antibody or its antigen-binding fragment to the EGFR mutant is reduced by at least 50% compared to wild-type EGFR, and may even be reduced below the detection limit.
  • the binding activity of the anti-EGFR antibody or its antigen-binding fragment to EGFR mutants with mutations at one or more of the following positions in the sequence shown in SEQ ID NO:106 is significantly reduced (including a reduction of at least 50% compared to wild-type EGFR): 448, 449, 454, 455, 458, 460, 461, 463, 465, 467, 471, 472, 473, 474, 476, 478, 480, 488 and 489.
  • the binding activity of the anti-EGFR antibody or its antigen-binding fragment to an EGFR mutant with mutations at one or more of the following positions in the sequence shown in SEQ ID NO:106 is reduced by at least 50%: 448, 449, 454, 455, 458, 460, 461, 463, 465, 488, and 489.
  • the binding activity of the anti-EGFR antibody or its antigen-binding fragment to an EGFR mutant with mutations at one or more of the following positions in the sequence shown in SEQ ID NO:106 is reduced by at least 50%: 454, 455, 467, 471, 472, 473, 474, 476, 478, and 480.
  • the binding activity of the anti-EGFR antibody or its antigen-binding fragment to an EGFR mutant with mutations at one or more of the following positions in the sequence shown in SEQ ID NO:106 is reduced by at least 50%: 465, 471, 472, 473, 474 and 476.
  • the binding activity of the anti-EGFR antibody or its antigen-binding fragment to an EGFR mutant with mutations at one or more of the following positions in the sequence shown in SEQ ID NO:106 is reduced by at least 50%: 448, 449, 454, 455, 458, 460, 461, 465, 488, and 489.
  • EGFR mutants having one or more of the following amino acid mutations exhibit at least a 50% reduction in binding activity to the anti-EGFR antibody or its antigen-binding fragment: A448Y, N449H, K454T, K455T, G458S, S460I, G461N, K463R, K465V, I467R, G471K, E472A, N473E, S474N, K476T, T478E, Q480M, P488S, and E489D.
  • EGFR mutants having one or more of the following amino acid mutations exhibit at least a 50% decrease in binding activity to the anti-EGFR antibody or its antigen-binding fragment: A448Y, N449H, K454T, K455T, G458S, S460I, G461N, K463R, K465V, P488S, and E489D.
  • EGFR mutants having one or more of the following amino acid mutations exhibit at least a 50% decrease in binding activity to the anti-EGFR antibody or its antigen-binding fragment: K454T, K455T, I467R, G471K, E472A, N473E, S474N, K476T, T478E, and Q480M.
  • EGFR mutants having one or more of the following amino acid mutations exhibit at least a 50% decrease in binding activity to the anti-EGFR antibody or its antigen-binding fragment: K465V, G471K, E472A, N473E, S474N, and K476T.
  • EGFR mutants having one or more of the following amino acid mutations exhibit at least a 50% decrease in binding activity to the anti-EGFR antibody or its antigen-binding fragment: A448Y, N449H, K454T, K455T, G458S, S460I, G461N, K465V, P488S, and E489D.
  • the binding activity of the anti-EGFR antibody or its antigen-binding fragment to an EGFR mutant having the following amino acid mutations is reduced by at least 50%: A448Y and N449H, or K454T and K455T, or G458S, S460I and G461N, or K463R, or K465V, or I467R, or G471K, E472A, N473E, S474N and K476T, or K476T, T478E and Q480M, or P488S and E489D.
  • the binding activity of the anti-EGFR antibody or its antigen-binding fragment with EGFR mutants having the following amino acid mutations is reduced by at least 50%: A448Y and N449H, or K454T and K455T, or G458S, S460I and G461N, or K463R, or K465V, or P488S and E489D.
  • the binding activity of the anti-EGFR antibody or its antigen-binding fragment to EGFR mutants having the following amino acid mutations is reduced by at least 50%: K454T and K455T, or I467R, or G471K, E472A, N473E, S474N and K476T, or K476T, T478E and Q480M.
  • the binding activity of the anti-EGFR antibody or its antigen-binding fragment to an EGFR mutant having the following amino acid mutations is reduced by at least 50% compared to wild-type EGFR: K465V, or G471K, E472A, N473E, S474N, and K476T.
  • the binding activity of the anti-EGFR antibody or its antigen-binding fragment to an EGFR mutant having the following amino acid mutations is reduced by at least 50%: A448Y and N449H, or K454T and K455T, or G458S, S460I and G461N, or K465V, or P488S and E489D.
  • Cetuximab compared with wild-type EGFR, exhibits significantly reduced binding activity only with EGFR mutants having the S468D mutation compared to the sequence shown in SEQ ID NO:106 (i.e., a decrease of at least 50% compared to wild-type EGFR); conversely, the binding activity of the EGFR mutants containing the S468D mutation with the anti-EGFR antibody of the present invention or its antigen-binding fragment is not significantly reduced (i.e., a decrease of less than 50% compared to wild-type EGFR), indicating that the antigen-binding epitopes and/or key binding sites of the anti-EGFR antibody of the present invention or its antigen-binding fragment are completely different from those of Cetuximab.
  • the antibody or its antigen-binding fragment when the anti-EGFR antibody or its antigen-binding fragment binds to EGFR, the antibody or its antigen-binding fragment is in direct contact with or sufficiently close in spatial position to at least one of the following amino acid residues located at the junction of D3 and D4 in the extracellular region of EGFR: A448, N449, K454, K455, G458, S460, G461, K463, K465, I467, G471, E472, N473, S474, K476, T478, Q480, P488, and E489.
  • the antibody or its antigen-binding fragment when the anti-EGFR antibody or its antigen-binding fragment binds to EGFR, the antibody or its antigen-binding fragment is in direct contact with or spatially close to at least one of the following amino acid residues located at the junction of D3 and D4 in the extracellular region of EGFR: A448, N449, K454, K455, G458, S460, G461, K463, K465, P488, and E489.
  • the antibody or its antigen-binding fragment when the anti-EGFR antibody or its antigen-binding fragment binds to EGFR, the antibody or its antigen-binding fragment is in direct contact with or spatially close to at least one of the following amino acid residues located at the junction of D3 and D4 in the extracellular region of EGFR: K454, K455, I467, G471, E472, N473, S474, K476, T478, and Q480.
  • the antibody or its antigen-binding fragment when the anti-EGFR antibody or its antigen-binding fragment binds to EGFR, the antibody or its antigen-binding fragment is in direct contact with or spatially close to at least one of the following amino acid residues located at the junction of D3 and D4 in the extracellular region of EGFR: K465, G471, E472, N473, S474, and K476.
  • the antibody or its antigen-binding fragment when the anti-EGFR antibody or its antigen-binding fragment binds to EGFR, the antibody or its antigen-binding fragment is in direct contact with or sufficiently close in spatial position to at least one of the following amino acid residues located at the junction of D3 and D4 in the extracellular region of EGFR: A448, N449, K454, K455, G458, S460, G461, K465, P488, and E489.
  • the antigen-binding epitope of the anti-EGFR antibody or its antigen-binding fragment comprises amino acid residues at one or more of the following positions in the extracellular domain of EGFR: A448, N449, K454, K455, G458, S460, G461, K463, K465, I467, G471, E472, N473, S474, K476, T478, Q480, P488, and E489.
  • the antigen-binding epitope is located at the junction of D3 and D4 of the EGFR extracellular region.
  • the antigen-binding epitope is contained in a polypeptide sequence as shown in SEQ ID NO: 107.
  • the antigen-binding epitope of the anti-EGFR antibody or its antigen-binding fragment comprises amino acid residues at one or more of the following positions of the EGFR extracellular domain: A448, N449, K454, K455, G458, S460, G461, K463, K465, P488, and E489.
  • the antigen-binding epitope of the anti-EGFR antibody or its antigen-binding fragment comprises amino acid residues at one or more of the following positions of the EGFR extracellular domain: K454, K455, I467, G471, E472, N473, S474, K476, T478, and Q480.
  • the antigen-binding epitope of the anti-EGFR antibody or its antigen-binding fragment comprises amino acid residues at one or more of the following positions of the EGFR extracellular domain: K465, G471, E472, N473, S474, and K476.
  • the antigen-binding epitope of the anti-EGFR antibody or its antigen-binding fragment comprises amino acid residues at one or more of the following positions of the EGFR extracellular domain: A448, N449, K454, K455, G458, S460, G461, K465, P488, and E489.
  • the binding activity of the anti-EGFR antibody or its antigen-binding fragment of the present invention to cells depends on the expression abundance of EGFR on the cell surface. It is characterized by the ability to specifically bind to cells that overexpress EGFR (e.g., tumor cells), while not binding or weakly binding to normal tissues or cells with relatively low EGFR expression levels.
  • FACS can be used to detect EGFR expression levels on the cell surface, wherein the EGFR expression levels of MCF-10A and the engineered cell line 293T-EGFR#16-2D6 are comparable to those of normal skin tissue. Therefore, cells with significantly higher EGFR expression levels than MCF-10A and 293T-EGFR#16-2D6 are considered EGFR-overexpressing cells (e.g., whose MFI value as determined by FACS is 10 times or higher than that of MCF-10A).
  • the EGFR overexpression includes wild-type EGFR overexpression and various mutant EGFR overexpression, provided that the mutation does not affect the binding of the antibody or its antigen-binding fragment to the epitope of amino acid residues 447-491 of the EGFR extracellular domain.
  • the EGFR-overexpressing cells include EGFR-overexpressing tumor cells and EGFR-overexpressing engineered cell lines.
  • the tumor cells include human skin squamous cell carcinoma cells A431, human non-small cell lung cancer cells HCC827 lacking the E746-A750 intracellular tyrosine kinase domain of EGFR, human pharyngeal squamous cell carcinoma cells FaDu, and human breast cancer cells JIMT-1.
  • the EGFR-overexpressing engineered cell line includes 293T-EGFR#2.
  • the EGFR abundance on the cell surface of the above-mentioned EGFR-overexpressing cell lines is 10 times or higher than that of MCF-10A cells.
  • the expression of EGFR on the cell surface is detected using FACS, and the cell lines with the highest to lowest expression abundance are, in descending order: A431, HCC827, 293T-EGFR#2, FaDu, JIMT-1, NCI-N87, 293T-EGFR#16-2D6, and MCF-10A.
  • the binding activity of the antibody or its antigen-binding fragment that specifically binds to the epitopes of amino acid residues 447-491 of the extracellular domain of EGFR described in this invention with the above-mentioned cell lines is positively correlated with the expression abundance of EGFR on their surface.
  • the binding activity of the antibody or its antigen-binding fragment with A431, HCC827, and 293T-EGFR#2 cells is comparable to that of Cetuximab; it can significantly bind to FaDu and JIMT-1 cells, but its binding activity is lower than that of Cetuximab; it shows weak binding to NCI-N87 cells, whose EGFR expression level is higher than that of MCF-10A cells but less than 10 times (MFI value); and it basically does not bind to 293T-EGFR#16-2D6 and MCF-10A cells, whose EGFR expression level is comparable to that of normal skin tissue/cells.
  • Cetuximab can significantly bind to all of the above-mentioned cell lines, regardless of the level of EGFR expression on their surface.
  • the antibody or its antigen-binding fragment that specifically binds to the epitope of amino acid residues 447-491 of the extracellular domain of EGFR specifically binds to lung cancer tissue overexpressing EGFR, but does not bind to any normal tissue (including skin tissue); Cetuximab, on the other hand, binds strongly to both lung cancer tissue and normal epidermal tissue (including skin tissue).
  • the anti-EGFR antibody or its antigen-binding fragment shows weak positive staining in the cytoplasm and no staining on the cell membrane in normal epidermal tissue, while Cetuximab shows strong positive staining in both the cell membrane and cytoplasm in epidermal tissue (including skin tissue); both the anti-EGFR antibody or its antigen-binding fragment and Cetuximab show strong positive staining in both the cell membrane and cytoplasm in the epithelial cells of lung cancer tissue.
  • IHC immunohistochemical
  • the binding affinity constant (K ⁇ sub> D ⁇ /sub> ) of the anti-EGFR antibody or its antigen-binding fragment to a polypeptide containing amino acid residues 447-491 of the EGFR extracellular domain is ⁇ 5 ⁇ 10 ⁇ sup>-7 ⁇ /sup> M, ⁇ 1 ⁇ 10 ⁇ sup>-7 ⁇ /sup> M, ⁇ 5 ⁇ 10 ⁇ sup>-8 ⁇ /sup> M, or ⁇ 1 ⁇ 10 ⁇ sup> -8 ⁇ /sup> M.
  • the polypeptide containing amino acid residues 447-491 of the EGFR extracellular domain includes wild-type EGFR, or a polypeptide containing any fragment of HER4 containing amino acid residues 447-491 of the EGFR extracellular domain (preferably a chimeric polypeptide with the amino acid sequence shown in SEQ ID NO:109, where amino acid residues 447-491 of the EGFR extracellular domain are substituted for the corresponding amino acid sequences in HER4).
  • the antigen-binding epitopes of the anti-EGFR antibody or its antigen-binding fragment of the present invention are different from those of Cetuximab, and therefore do not compete with Cetuximab for binding to EGFR; the antibody or its antigen-binding fragment of the present invention also does not block the binding of the ligand to EGFR, and does not affect (e.g., does not inhibit or activate) the regulation of EGFR and its downstream signal transduction pathways.
  • the anti-EGFR antibody of the present invention or its antigen-binding fragment specifically binds to the epitope of amino acid residues 447-491 of the EGFR extracellular domain, wherein one or more amino acid residues in the EGFR extracellular domain are in direct contact with or sufficiently close in spatial position to the antibody of the present invention or its antigen-binding fragment: A448, N449, K454, K455, G458, S460, G461, K463, K465, P488, and E489; or K454, K455, I467, G471, E472, N473, S474, K476, T478, and Q480; or K465, G471, E472, N473, S474, and K476; or A448, N449, K454, K455, G458, S460, G461, K465, P488, and E489.
  • the antigen-binding epitope of Cetuximab is located in D3 of the extracellular domain of EGFR, as described in WO2006009694, and includes amino acids at the following positions in the extracellular domain of EGFR: Q384, Q408, S418, S440, K465, S468, and N469.
  • the key binding site of Cetuximab includes a serine residue at position 468 of the EGFR extracellular domain.
  • the anti-EGFR antibody or its antigen-binding fragment of the present invention can be internalized by cells that specifically bind to EGFR.
  • the efficiency of internalization of the anti-EGFR antibody or its antigen-binding fragment of the present invention into cells that specifically bind to EGFR is comparable to that of Cetuximab.
  • the anti-EGFR antibody or its antigen-binding fragment of the present invention has no effect on the in vitro proliferation of tumor cells expressing EGFR, while Cetuximab can significantly inhibit the in vitro proliferation of cells expressing EGFR.
  • the antibody or its antigen-binding fragment of the present invention does not induce, block, or inhibit phosphorylation and/or dephosphorylation of tyrosine residues in the intracellular domain of EGFR, or does not induce, block, or inhibit phosphorylation and/or dephosphorylation of signaling molecules (e.g., AKT, ERK) in downstream signal transduction pathways of EGFR.
  • the antibody or its antigen-binding fragment of the present invention does not inhibit phosphorylation and/or dephosphorylation of the Ser473 site of AKT in downstream signal transduction pathways of EGFR.
  • the anti-EGFR antibody of the present invention may optionally include F(ab’)2, Fab, Fab’, Fv, scFv, scFv-Fc, single-domain antibody (sdAb), or of IgG type.
  • the antibody of the present invention may be a murine antibody, chimeric antibody, humanized antibody, or fully human antibody; it may be a monoclonal antibody, polyclonal antibody, monospecific antibody, bispecific antibody, multispecific antibody, or antibody fragment, provided that the antibody specifically recognizes the epitope of amino acid residues 447-491 of the extracellular domain of EGFR, and its binding activity to cells depends on the abundance of EGFR expression on the cell surface, and does not affect EGFR expressed on the cell surface or its signal transduction pathway.
  • the anti-EGFR antibody is selected from mouse anti-human EGFR antibodies and their humanized antibodies and optimized antibodies; the anti-EGFR antibody may also be a fully human antibody.
  • the anti-EGFR antibody or its antigen-binding fragment binds specifically to EGFR only and has no cross-binding activity with other members of the ErbB/HER family.
  • the anti-EGFR antibody includes scFv, scFv-Fc, Fab fragment, and/or has an IgG type. In some embodiments, the anti-EGFR antibody is an IgG type.
  • the present invention provides anti-EGFR antibodies or antigen-binding fragments thereof comprising CDRs and/or variable regions of the antibodies shown in Table 2, which recognize the epitope of amino acid residues 447-491 of the extracellular domain of EGFR and are capable of specifically binding to cells overexpressing EGFR, while not binding or weakly binding to normal tissues or cells with relatively low EGFR expression levels (e.g., skin tissue), and do not induce, block, or inhibit phosphorylation and/or dephosphorylation of signaling molecules (e.g., AKT) in downstream signal transduction pathways of EGFR.
  • AKT phosphorylation and/or dephosphorylation of signaling molecules
  • CDRs heavy chain variable region
  • CDRs light chain variable region
  • CDRs can also be defined based on other heavy/light chain variable region sequences, such as Chothia and IMGT, AbM, or Contact numbering systems/methods.
  • CDRs defined by other numbering systems/methods, as well as those defined by Kabat as used herein, are all within the scope of protection of this invention.
  • the anti-EGFR antibody or its antigen-binding fragment of the present invention is capable of specifically binding to the epitope of amino acid residues 447-491 of the extracellular domain of EGFR, comprising: a VH containing the amino acid sequence shown in SEQ ID NO: 1, 3, 5, or 119 and/or a VL containing the amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 120.
  • the anti-EGFR antibody or its antigen-binding fragment of the present invention comprises: a VH containing the amino acid sequence shown in SEQ ID NO: 1 and/or a VL containing the amino acid sequence shown in SEQ ID NO: 2.
  • the anti-EGFR antibody or its antigen-binding fragment of the present invention comprises: a VH containing the amino acid sequence shown in SEQ ID NO: 3 and/or a VL containing the amino acid sequence shown in SEQ ID NO: 4. In one embodiment, the anti-EGFR antibody or its antigen-binding fragment of the present invention comprises: a VH containing the amino acid sequence shown in SEQ ID NO: 5 and/or a VL containing the amino acid sequence shown in SEQ ID NO: 6. In one embodiment, the anti-EGFR antibody or its antigen-binding fragment of the present invention comprises: VH containing the amino acid sequence shown in SEQ ID NO:119 and/or VL containing the amino acid sequence shown in SEQ ID NO:120.
  • the anti-EGFR antibody or its antigen-binding fragment of the present invention comprises: one or more CDRs of VH containing the amino acid sequence shown in SEQ ID NO:1 or a CDR containing the amino acid sequence shown in SEQ ID NOs:7, 9 and 17 or a variant thereof, said variant including the CDR of humanized antibody or any other variant of the present invention.
  • the anti-EGFR antibody or its antigen-binding fragment according to the present invention further comprises: one or more CDRs containing the amino acid sequence shown in SEQ ID NO:2 or a CDR containing the amino acid sequence shown in SEQ ID NOs:30, 37 and 47 or a variant thereof, said variant including the CDR of a humanized antibody or any other variant according to the present invention.
  • the anti-EGFR antibody or its antigen-binding fragment of the present invention comprises: one or more CDRs of VH containing the amino acid sequence shown in SEQ ID NO:3 or CDRs containing the amino acid sequences shown in SEQ ID NOs:8, 16 and 28 or variants thereof, said variants including CDRs of humanized antibodies or any other variants of the present invention.
  • the anti-EGFR antibody or its antigen-binding fragment according to the present invention further comprises: one or more CDRs containing the amino acid sequence shown in SEQ ID NO:4 or a CDR containing the amino acid sequence shown in SEQ ID NOs:35, 45 and 58 or a variant thereof, said variant including the CDR of a humanized antibody or any other variant according to the present invention.
  • the anti-EGFR antibody or its antigen-binding fragment of the present invention comprises: one or more CDRs of VH containing the amino acid sequence shown in SEQ ID NO:5 or a CDR containing the amino acid sequence shown in SEQ ID NOs:7, 9 and 29 or a variant thereof, said variant including the CDR of humanized antibody or any other variant of the present invention.
  • the anti-EGFR antibody or its antigen-binding fragment according to the present invention further comprises: one or more CDRs containing the amino acid sequence shown in SEQ ID NO:6 or a CDR containing the amino acid sequence shown in SEQ ID NOs:36, 46 and 59 or a variant thereof, said variant including the CDR of a humanized antibody or any other variant according to the present invention.
  • the anti-EGFR antibody or its antigen-binding fragment of the present invention comprises: one or more CDRs of VH containing the amino acid sequence shown in SEQ ID NO:119 or CDRs containing the amino acid sequences shown in SEQ ID NOs:113, 114 and 115 or variants thereof.
  • the anti-EGFR antibody or its antigen-binding fragment according to the present invention further comprises: one or more CDRs in a VL containing the amino acid sequence shown in SEQ ID NO: 120 or a CDR or a variant thereof containing the amino acid sequences shown in SEQ ID NOs: 116, 117 and 118.
  • the anti-EGFR antibody or its antigen-binding fragment comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO:7, HCDR2 having the amino acid sequence shown in SEQ ID NO:9, and HCDR3 having the amino acid sequence shown in SEQ ID NO:17; and LCDR1 having the amino acid sequence shown in SEQ ID NO:30, LCDR2 having the amino acid sequence shown in SEQ ID NO:37, and LCDR3 having the amino acid sequence shown in SEQ ID NO:47.
  • the anti-EGFR antibody or its antigen-binding fragment comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO:8, HCDR2 having the amino acid sequence shown in SEQ ID NO:16, and HCDR3 having the amino acid sequence shown in SEQ ID NO:28; and LCDR1 having the amino acid sequence shown in SEQ ID NO:35, LCDR2 having the amino acid sequence shown in SEQ ID NO:45, and LCDR3 having the amino acid sequence shown in SEQ ID NO:58.
  • the anti-EGFR antibody or its antigen-binding fragment comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO:7, HCDR2 having the amino acid sequence shown in SEQ ID NO:9, and HCDR3 having the amino acid sequence shown in SEQ ID NO:29; and LCDR1 having the amino acid sequence shown in SEQ ID NO:36, LCDR2 having the amino acid sequence shown in SEQ ID NO:46, and LCDR3 having the amino acid sequence shown in SEQ ID NO:59.
  • the anti-EGFR antibody or its antigen-binding fragment comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO:113, HCDR2 having the amino acid sequence shown in SEQ ID NO:114, and HCDR3 having the amino acid sequence shown in SEQ ID NO:115; and LCDR1 having the amino acid sequence shown in SEQ ID NO:116, LCDR2 having the amino acid sequence shown in SEQ ID NO:117, and LCDR3 having the amino acid sequence shown in SEQ ID NO:118.
  • the VH and/or VL of the anti-EGFR antibody of the present invention or its antigen-binding fragment can be used as starting materials for engineering modification to prepare an anti-EGFR antibody more suitable for human administration.
  • the antibody can be engineered by mutating one or more amino acid residues within one or both variable regions (i.e., VH and/or VL), for example, by mutating one or more CDR regions and/or one or more frame regions.
  • variable region of the anti-EGFR antibody of the present invention is engineered via CDR grafting.
  • the antibody primarily interacts with the target antigen through the amino acid residues of six CDRs in the heavy chain and light chain variable regions. Therefore, the amino acid sequence within the CDR regions of individual antibodies is more diverse than the sequence outside the CDR regions (e.g., FR). Because the amino acid sequence of the CDR region is responsible for most of the antibody-antigen interactions, recombinant antibodies can be expressed by constructing expression vectors to mimic the characteristics of specific naturally occurring antibodies.
  • These expression vectors comprise the CDR sequence from a specific naturally occurring antibody transplanted into the FR sequence of another antibody with different characteristics (see, for example, Riechmann et al., Nature 1998, 332:323-327; Jones et al., Nature 1986, 321:522-525; Queen et al., Proc Natl Acad Sci USA 1989, 86:10029-10033. See also US5225539, US5530101, US5585089, US5693762, and US6180370).
  • the anti-EGFR antibody of the present invention may also contain different framework region sequences.
  • framework region sequences can be obtained from public DNA databases or from publicly available references relating to germline antibody gene sequences.
  • germline DNA sequences of human heavy and light chain variable region genes are available in the “V Base” human germline sequence database (available at www.mrc-cpe.cam.ac.uk/vbase); and Kabat et al., Sequences of Proteins of Immunological Interest, 1991, 5th edition, NIH Publication No.
  • germline DNA sequences of human heavy and light chain variable region genes are available in the IMGT database.
  • the following heavy chain germline sequences found in human immunoglobulins can be obtained through the IMGT registry number: IGHV1-46*01 or IGHV3-15*06.
  • the following light chain germline sequence found in human immunoglobulins can be obtained through the IMGT registry number: IGKV1-33.
  • Antibody amino acid sequences can be compared using one of the sequence similarity search methods known to those skilled in the art, called Gapped BLAST, based on a compiled protein sequence database (Altschul et al., Nucleic Acids Res 1997, 25:3389-3402).
  • the preferred framework sequence in the anti-EGFR antibody used in this invention is a receptor framework region that is structurally similar (or highly homologous) to the framework sequence of the murine maternal antibody of this invention.
  • the CDR1, CDR2, and CDR3 regions of VH or VL can be transplanted into the receptor framework region, wherein the receptor framework region has a sequence that is identical or highly homologous to the amino acid sequence of the immunoglobulin of its germline.
  • this invention selects to transplant the CDR regions of VH shown in SEQ ID NO:1 and VL shown in SEQ ID NO:2 into the human IgG FR region to obtain a humanized antibody.
  • the humanized antibody can maintain similar antigen-binding activity, cell-binding activity, binding affinity, and biological activity characteristics to the maternal antibody containing the amino acid sequences of VH shown in SEQ ID NO:1 and VL shown in SEQ ID NO:2. For example, it does not inhibit the in vitro proliferation of cells expressing EGFR and/or does not affect the phosphorylation/dephosphorylation of signaling molecules (e.g., AKT) in downstream EGFR signal transduction pathways.
  • signaling molecules e.g., AKT
  • VH and VL sequences (or CDR sequences, or full-length heavy chain and full-length light chain sequences) of other anti-EGFR antibodies binding to EGFR can be "mixed and matched" with the VH and VL sequences (or CDR sequences, or full-length heavy chain and full-length light chain sequences) of the anti-EGFR antibody of the present invention.
  • VH and VL chains or the CDRs within these chains, or full-length heavy chain and full-length light chain sequences
  • the VH sequence from a specific VH/VL pair is replaced by a structurally similar VH sequence.
  • the VL sequence from a specific VH/VL pair is replaced by a structurally similar VL sequence.
  • the full-length heavy chain sequence from a specific full-length heavy chain/full-length light chain pair should be replaced with a structurally similar full-length heavy chain sequence.
  • the full-length light chain sequence from a specific full-length heavy chain/full-length light chain pair should be replaced with a structurally similar full-length light chain sequence.
  • the antibody or antigen-binding fragment of the present invention comprises: (a) a heavy chain variable region containing the amino acid sequence listed in Table 2; and (b) a light chain variable region containing the amino acid sequence listed in Table 2, or, a VL of another anti-EGFR antibody, wherein the antibody specifically binds to the amino acid sequence at the D3 and D4 junction of the EGFR extracellular domain (e.g., amino acid residues 447-491 of the EGFR extracellular domain).
  • the antibody or antigen-binding fragment of the present invention comprises: (a) a heavy chain variable region comprising the amino acid sequence listed in Table 2, or the VH of another anti-EGFR antibody, wherein the antibody specifically binds to the amino acid sequence at the D3-D4 junction of the EGFR extracellular domain (e.g., amino acid residues 447-491 of the EGFR extracellular domain); and (b) a light chain variable region comprising the amino acid sequence listed in Table 2.
  • the anti-EGFR humanized antibody or its antigen-binding fragment of the present invention comprises VH, said VH containing an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 60 or 61.
  • the anti-EGFR humanized antibody or its antigen-binding fragment of the present invention further comprises a VL containing an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:78.
  • the humanized antibody or its antigen-binding fragment of the present invention comprises VH and VL, wherein the VH contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 60 or 61, and the VL contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO: 78.
  • amino acids that differ from the amino acid sequences shown in SEQ ID NO:60, SEQ ID NO:61, or SEQ ID NO:78, having at least 85% identity are mainly or entirely located in the frame region (FR).
  • the anti-EGFR antibodies described herein, their antigen-binding fragments, or various variants thereof, specifically bind to amino acid residues 447-491 of the extracellular domain of EGFR.
  • the CDR sequence can be transplanted into a frame region containing one or more mutations compared to the germline sequence. For example, mutating amino acid residues within the frame region can maintain or enhance the antigen-binding ability of the antibody (see, for example, US5530101, US5585089, US5693762, and US6180370).
  • transplanting the CDR of the maternal antibody of the present invention into a receptor frame region having one or more mutations compared to the germline sequence can improve the humanization degree of the anti-EGFR antibody and/or improve its binding properties or affinity.
  • the FR of the anti-EGFR humanized antibody of the present invention has one or more amino acid mutations (e.g., reversion mutations) to improve the binding affinity and/or binding activity of the humanized antibody.
  • the FR of VH shown in SEQ ID NO:60 is mutated by one or more amino acid mutations.
  • amino acid mutations may be made in HFR3, for example, an amino acid mutation at position 32 of HFR3 (corresponding to position 94 of the amino acid sequence of VH shown in SEQ ID NO:60); and/or the FR of VL shown in SEQ ID NO:78 has one or more amino acid mutations relative to the receptor framework region (e.g., IGKV1-33), for example, an amino acid mutation at position 17 of LFR3 (corresponding to position 73 of the amino acid sequence of VL shown in SEQ ID NO:78) and/or position 27 (corresponding to position 83 of the amino acid sequence of VL shown in SEQ ID NO:78).
  • the receptor framework region e.g., IGKV1-33
  • the anti-EGFR humanized antibody of the present invention has an amino acid mutation R94N in FR relative to the VH amino acid sequence shown in SEQ ID NO:60, and/or has amino acid mutations F73L and I83V in the VL amino acid sequence shown in SEQ ID NO:78 relative to the receptor framework region IGKV1-33, which can significantly improve the binding affinity of the antibody ( KD value ⁇ 5 ⁇ 10-8 M) and maintain the binding properties of the antibody (e.g., specifically binding to cells overexpressing EGFR, while not binding or weakly binding to normal tissues or cells).
  • the present invention mutates the CDRs of the VH and/or VL of humanized antibodies to improve one or more properties of the target antibody, such as increasing humanization, increasing binding affinity, increasing expression levels, and enhancing stability (e.g., reducing the potential risk of asparagine deamidation).
  • Site-directed mutagenesis or PCR-induced mutagenesis can be performed, and the effect of said mutation on antibody binding capacity or other functional properties can be evaluated using in vitro or in vivo detection methods known in the art.
  • Mutagenesis can be amino acid substitution, addition, or deletion, preferably amino acid substitution. Specifically, mutations occur in no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues within the heavy chain CDR or light chain CDR.
  • the heavy chain CDR or light chain CDR of the anti-EGFR humanized antibody of the present invention has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations.
  • the CDR in VH shown in SEQ ID NO:62 is mutated by one or more amino acids. Further, one, two, three, four, five, six, or seven amino acid mutations can be performed on HCDR2 and/or HCDR3.
  • one or more amino acid mutations can be made at the asparagine residue at position 6 of HCDR2 (corresponding to position 54 of VH in SEQ ID NO:62), the glycine residue at position 7 (corresponding to position 55 of VH in SEQ ID NO:62), the glutamate residue at position 13 (corresponding to position 61 of VH in SEQ ID NO:62), and/or at the tyrosine residue at position 1 of HCDR3 (corresponding to position 95 of VH in SEQ ID NO:62), the lysine residue at position 4 (corresponding to position 98 of VH in SEQ ID NO:62), the glutamate residue at position 6 (corresponding to position 100a of VH in SEQ ID NO:62), and the methionine residue at position 10 (corresponding to position 100d of VH in SEQ ID NO:62).
  • one or more amino acid mutations are made in HCDR2 and/or HCDR3 in the VH shown in SEQ ID NO:62.
  • the 7th glycine residue (corresponding to position 55 of the VH shown in SEQ ID NO:62), the 13th glutamic acid residue (corresponding to position 61 of the VH shown in SEQ ID NO:62), and/or the 1st tyrosine residue (corresponding to position 95 of the VH shown in SEQ ID NO:62), the 4th lysine residue (corresponding to position 98 of the VH shown in SEQ ID NO:62), and the 10th methionine residue (corresponding to position 100d of the VH shown in SEQ ID NO:62) of HCDR3 are mutated.
  • one or more amino acid mutations are made in the CDR of the VL shown in SEQ ID NO:78.
  • one, two, or three amino acid mutations can be made in LCDR1, LCDR3, and/or LCDR3.
  • mutations can be made in the 5th aspartic acid residue (corresponding to position 28 of the VL shown in SEQ ID NO:78), the 8th asparagine residue (corresponding to position 31 of the VL shown in SEQ ID NO:78) of LCDR1, the 1st tyrosine residue (corresponding to position 50 of the VL shown in SEQ ID NO:78), the 2nd threonine residue (corresponding to position 51 of the VL shown in SEQ ID NO:78), the 4th arginine residue (corresponding to position 53 of the VL shown in SEQ ID NO:78), and the...
  • One or more amino acid mutations are made at the 5th serine residue (corresponding to position 54 of VL shown in SEQ ID NO:78), the 6th histidine residue (corresponding to position 55 of VL shown in SEQ ID NO:78), the 7th serine residue (corresponding to position 56 of VL shown in SEQ ID NO:78), and/or at the 4th lysine residue (corresponding to position 92 of VL shown in SEQ ID NO:78) and the 5th threonine residue (corresponding to position 93 of VL shown in SEQ ID NO:78).
  • one or more amino acid mutations are made in LCDR1 and/or LCDR3 in the VL shown in SEQ ID NO:78.
  • an amino acid mutation is made in the 5th aspartic acid residue of LCDR1 in the VL shown in SEQ ID NO:78 (corresponding to the 28th position of the VL shown in SEQ ID NO:78), and/or in the 4th lysine residue of LCDR3 (corresponding to the 92nd position of the VL shown in SEQ ID NO:78) and the 5th threonine residue (corresponding to the 93rd position of the VL shown in SEQ ID NO:78).
  • the present invention performs multiple amino acid mutations on the CDRs in the VH and VL of the above-mentioned anti-EGFR humanized antibodies to improve the degree of humanization of the antibodies, improve binding properties, increase expression levels, and/or improve stability (e.g., reduce the potential risk of asparagine deamidation).
  • amino acid mutations are performed on the 7th glycine residue (corresponding to position 55 of the VH shown in SEQ ID NO:62) and the 13th glutamic acid residue (corresponding to position 61 of the VH shown in SEQ ID NO:62) of HCDR2 in the VH shown in SEQ ID NO:62, and/or on the 1st tyrosine residue (corresponding to position 95 of the VH shown in SEQ ID NO:62), the 4th arginine residue (corresponding to position 98 of the VH shown in SEQ ID NO:62), and the 10th methionine residue of HCDR3.
  • Amino acid mutations are performed on one or more of the acid residues (corresponding to position 100d of VH shown in SEQ ID NO:62), and on the aspartic acid residue at position 5 of LCDR1 of VL shown in SEQ ID NO:78 (corresponding to position 28 of VL shown in SEQ ID NO:78), and/or on the lysine residue at position 4 of LCDR3 (corresponding to position 92 of VL shown in SEQ ID NO:78) and the threonine residue at position 5 of LCDR3 (corresponding to position 93 of VL shown in SEQ ID NO:78).
  • amino acid mutations are performed on the 7th glycine residue (corresponding to position 55 of VH in SEQ ID NO:62), the 13th glutamic acid residue (corresponding to position 61 of VH in SEQ ID NO:62), and/or the 4th arginine residue (corresponding to position 98 of VH in SEQ ID NO:62), the 10th methionine residue (corresponding to position 100d of VH in SEQ ID NO:62) of HCDR2 in VH, and on SEQ ID NO:7.
  • Amino acid mutations were made in the 5th aspartic acid residue of LCDR1 (corresponding to position 28 of VL shown in SEQ ID NO:78) and/or the 4th lysine residue of LCDR3 (corresponding to position 92 of VL shown in SEQ ID NO:78) and the 5th threonine residue (corresponding to position 93 of VL shown in SEQ ID NO:78) of LCDR3.
  • These mutations not only gave the antibody a higher degree of humanization, but also significantly enhanced its binding activity with EGFR-overexpressing cells, while reducing the potential risk of asparagine deamidation and increasing the expression level.
  • the antibody or its antigen-binding fragment can specifically bind to cells overexpressing EGFR, while not binding or weakly binding to normal tissues or cells, and has a high degree of humanization, high expression level, and eliminates the risk of asparagine deamidation.
  • the antibody or antigen-binding fragment thereof of the present invention comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO:7; HCDR2 having the amino acid sequence shown in SEQ ID NO:9, 10, 11, 12, 13, 14, or 15; and HCDR3 having the amino acid sequence shown in SEQ ID NO:17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27; LCDR1 having the amino acid sequence shown in SEQ ID NO:30, 31, 32, 33, or 34; LCDR2 having the amino acid sequence shown in SEQ ID NO:37, 38, 39, 40, 41, 42, 43, or 44; and LCDR3 having the amino acid sequence shown in SEQ ID NO:47, 48, 49, 50, 51, 52, 53, 54, 55, 56, or 57.
  • the anti-EGFR antibody or its antigen-binding fragment of the present invention comprises:
  • HCDR1 having the amino acid sequence shown in SEQ ID NO:7
  • HCDR2 having the amino acid sequence shown in SEQ ID NO:9, 10, 11, 12, 13, or 14,
  • HCDR3 having the amino acid sequence shown in SEQ ID NO:17
  • LCDR1 having the amino acid sequence shown in SEQ ID NO:30
  • LCDR2 having the amino acid sequence shown in SEQ ID NO:37
  • LCDR3 having the amino acid sequence shown in SEQ ID NO:47, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or
  • HCDR1 having the amino acid sequence shown in SEQ ID NO:7
  • HCDR2 having the amino acid sequence shown in SEQ ID NO:9
  • HCDR3 having the amino acid sequence shown in SEQ ID NO:18, 19, 20, 21, 22, 23, or 24
  • LCDR1 having the amino acid sequence shown in SEQ ID NO:30
  • LCDR2 having the amino acid sequence shown in SEQ ID NO:37
  • LCDR3 having the amino acid sequence shown in SEQ ID NO:47
  • HCDR1 having the amino acid sequence shown in SEQ ID NO:7
  • HCDR2 having the amino acid sequence shown in SEQ ID NO:9
  • HCDR3 having the amino acid sequence shown in SEQ ID NO:17
  • LCDR1 having the amino acid sequence shown in SEQ ID NO:30
  • LCDR2 having the amino acid sequence shown in SEQ ID NO:38, 39, 40, 41, 42, 43, or 44
  • LCDR3 having the amino acid sequence shown in SEQ ID NO:47, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or
  • HCDR1 having the amino acid sequence shown in SEQ ID NO:7
  • HCDR2 having the amino acid sequence shown in SEQ ID NO:9
  • HCDR3 having the amino acid sequence shown in SEQ ID NO:17
  • LCDR1 having the amino acid sequence shown in SEQ ID NO:30
  • LCDR2 having the amino acid sequence shown in SEQ ID NO:37
  • LCDR3 having the amino acid sequence shown in SEQ ID NO:48, 49, 50, 51, 52, 53, 54, 55, or 56, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or
  • HCDR1 having the amino acid sequence shown in SEQ ID NO:7
  • HCDR2 having the amino acid sequence shown in SEQ ID NO:15
  • HCDR3 having the amino acid sequence shown in SEQ ID NO:25, 26, or 27
  • LCDR1 having the amino acid sequence shown in SEQ ID NO:33
  • LCDR2 having the amino acid sequence shown in SEQ ID NO:37
  • LCDR3 having the amino acid sequence shown in SEQ ID NO:57
  • the anti-EGFR antibody or its antigen-binding fragment according to the present invention comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO:7; HCDR2 having the amino acid sequence shown in SEQ ID NO:9, 12, 14, or 15; HCDR3 having the amino acid sequence shown in SEQ ID NO:17, 18, 19, 20, 23, 25, 26, or 27; and LCDR1 having the amino acid sequence shown in SEQ ID NO:30 or 33; and having the amino acid sequence shown in SEQ ID NO:30 or 33.
  • LCDR2 having the amino acid sequence shown in SEQ ID NO:37
  • LCDR3 having the amino acid sequence shown in SEQ ID NO:47, 50, 53, or 57, or having 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 the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.
  • the antibody or its antigen-binding fragment comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO:7, HCDR2 having the amino acid sequence shown in SEQ ID NO:15, HCDR3 having the amino acid sequence shown in SEQ ID NO:26, LCDR1 having the amino acid sequence shown in SEQ ID NO:33, LCDR2 having the amino acid sequence shown in SEQ ID NO:37, and LCDR3 having the amino acid sequence shown in SEQ ID NO:57, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.
  • the antibody or its antigen-binding fragment can specifically bind to cells that overexpress EGFR, while not binding or weakly binding to normal tissues or cells.
  • HCDR1 having the amino acid sequence shown in SEQ ID NO:7
  • HCDR2 having the amino acid sequence shown in SEQ ID NO:9
  • HCDR3 having the amino acid sequences shown in SEQ ID NO:18, 19, 20, 23
  • LCDR1 having the amino acid sequence shown in SEQ ID NO:30
  • LCDR2 having the amino acid sequence shown in SEQ ID NO:37
  • LCDR3 having the amino acid sequence shown in SEQ ID NO:47;
  • HCDR1 having the amino acid sequence shown in SEQ ID NO:7
  • HCDR2 having the amino acid sequence shown in SEQ ID NO:9
  • HCDR3 having the amino acid sequence shown in SEQ ID NO:17
  • LCDR1 having the amino acid sequence shown in SEQ ID NO:33
  • LCDR2 having the amino acid sequence shown in SEQ ID NO:37
  • LCDR3 having the amino acid sequence shown in SEQ ID NO:47;
  • HCDR1 having the amino acid sequence shown in SEQ ID NO:7
  • HCDR2 having the amino acid sequence shown in SEQ ID NO:9
  • HCDR3 having the amino acid sequence shown in SEQ ID NO:17
  • LCDR1 having the amino acid sequence shown in SEQ ID NO:30
  • LCDR2 having the amino acid sequence shown in SEQ ID NO:37
  • LCDR3 having the amino acid sequence shown in SEQ ID NO:50 or 53;
  • HCDR1 having the amino acid sequence shown in SEQ ID NO:7
  • HCDR2 having the amino acid sequence shown in SEQ ID NO:15
  • HCDR3 having the amino acid sequence shown in SEQ ID NO:26 or 27
  • LCDR1 having the amino acid sequence shown in SEQ ID NO:33
  • LCDR2 having the amino acid sequence shown in SEQ ID NO:37
  • LCDR3 having the amino acid sequence shown in SEQ ID NO:57.
  • the anti-EGFR antibody or its antigen-binding fragment of the present invention comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO:7, HCDR2 having the amino acid sequence shown in SEQ ID NO:15, HCDR3 having the amino acid sequence shown in SEQ ID NO:26 or 27, LCDR1 having the amino acid sequence shown in SEQ ID NO:33, LCDR2 having the amino acid sequence shown in SEQ ID NO:37, and LCDR3 having the amino acid sequence shown in SEQ ID NO:57.
  • the anti-EGFR antibody or its antigen-binding fragment of the present invention further comprises a heavy chain variable region (VH) having said HCDR1, HCDR2 and HCDR3 and a light chain variable region (VL) having said LCDR1, LCDR2 and LCDR3.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or antigen-binding fragment of the present invention comprises VH and VL, wherein the VH and VL contain amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the VH amino acid sequence shown in any one of SEQ ID NOs:60-77 and the VL amino acid sequence shown in any one of SEQ ID NOs:78-99.
  • the amino acids differing from those shown in the amino acid sequences of at least 85% identity with SEQ ID NOs:60-77 and 78-99 are predominantly or entirely located in the FR region.
  • the antibody or antigen-binding fragment of the present invention comprises VH and VL, wherein the VH and VL contain: (1) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the VH amino acid sequence shown in any one of SEQ ID NO:60-74 and the VL amino acid sequence shown in SEQ ID NO:78, respectively; or (2) amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 99%, or 100% identity with the VH amino acid sequence shown in SEQ ID NO:62 and any one of SEQ ID NO:79-98, respectively.
  • the VL amino acid sequence shown in SEQ ID NO: 75-77 has 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 the VH amino acid sequence shown in SEQ ID NO: 75-77 and the VL amino acid sequence shown in SEQ ID NO: 99, respectively.
  • the antibody or its antigen-binding fragment of the present invention comprises VH and VL, wherein VH and VL contain:
  • amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the VH amino acid sequence shown in SEQ ID NO: 65, 67, 68, 69, 70 or 73 and the VL amino acid sequence shown in SEQ ID NO: 78, respectively;
  • Amino acid sequences that have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the VH amino acid sequence shown in SEQ ID NO:62 and the VL amino acid sequence shown in SEQ ID NO:81, 89, or 92, respectively;
  • the anti-EGFR antibody or its antigen-binding fragment of the present invention comprises: VH and VL containing amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the VH amino acid sequence shown in SEQ ID NO: 76 or 77 and the VL amino acid sequence shown in SEQ ID NO: 99, respectively.
  • the antibody or antigen-binding fragment of the present invention comprises: VH and VL containing amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 70, 73, 75, 76, or 77 VH amino acid sequences and VL amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences shown in SEQ ID NOs: 62, 65, 67, 68, 69, 70, 73, 75, 76, or 77 and the amino acid sequences shown in SEQ ID NOs: 78, 81, 89, 92, or 99, respectively.
  • the antibody or its antigen-binding fragment comprises: VH and VL containing amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the VH amino acid sequence shown in SEQ ID NO: 76 or 77 and the VL amino acid sequence shown in SEQ ID NO: 99, respectively.
  • the antibody or its antigen-binding fragment comprises: VH and VL containing amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the VH amino acid sequence shown in SEQ ID NO: 76 and the VL amino acid sequence shown in SEQ ID NO: 99, respectively.
  • the binding activity of the anti-EGFR humanized antibody or its antigen-binding fragment to cells in this invention depends on the expression abundance of EGFR on the cell surface. It can specifically bind to cells that overexpress EGFR, while it does not bind or binds weakly to normal tissues or cells with relatively low EGFR expression levels. Furthermore, it does not affect downstream signal transduction pathways mediated by EGFR and does not cross-bind with other members of the human ErbB/HER family (including HER2, HER3, and HER4).
  • the anti-EGFR antibody of the present invention may also comprise a constant region, which includes an antibody heavy chain constant region and a light chain constant region.
  • the heavy chain constant region comprises native and mutant protein forms of the Fc region of the human IgG heavy chain constant region, and also includes truncated polypeptide forms containing hinge regions that promote dimerization.
  • the Fc region comprises antibody CH2 and CH3 domains. Fusion proteins (and the resulting oligomers) comprising the Fc moiety offer the advantage of easy purification by affinity chromatography using protein A or protein G, and extended serum half-life.
  • Preferred Fc regions are derived from human IgG, including IgG1, IgG2, IgG3, and IgG4. In this document, the positions of specific amino acid residues in the Fc region are determined according to the EU numbering system.
  • the light chain constant region comprises the light chain constant region of the human ⁇ or ⁇ chain, or a mutant protein form thereof.
  • ADCC antibody-dependent cytotoxicity
  • ADCP antibody-dependent phagocytosis
  • CDC complement-dependent cytotoxicity
  • ADCC and ADCP are mediated by the binding of the antibody's Fc region to Fc receptors (FcRs) expressed on the surface of immune cells (Raghavan et al., Annu Rev Cell Dev Biol 1996, 12:181-220; Ghetie et al., Annu Rev Immunol 2000, 18:739-766; Ravetch et al., Annu Rev Immunol 2001, 19:275-290), including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, NK cells, and T cells.
  • CDC is mediated by Fc binding to complement system proteins such as C1q (Ward et al., Ther Immunol 1995, 2:77-94).
  • the anti-EGFR antibody of the present invention comprises an engineered IgG Fc region to reduce its mediated effector function.
  • Exemplary Fc molecules with reduced effector function include Fc molecules having the following amino acid substitution mutations:
  • the preferred engineered Fc region is human IgG1 Fc with L234F/L235E/P331S (EU numbering system) amino acid substitutions, which can reduce the binding of the Fc region to one or more Fc ⁇ R and C1q (Oganesyan et al., Acta Crystallogr D Biol Crystallogr 2008, 64:700-704; US5624821; US6194551).
  • the Fc ⁇ R protein family includes: Fc ⁇ RI (also known as CD64), including isoforms Fc ⁇ RIa, Fc ⁇ RIb, and Fc ⁇ RIc; Fc ⁇ RII (also known as CD32), including isoforms Fc ⁇ RIIa, Fc ⁇ RIIb, and Fc ⁇ RIIc; and Fc ⁇ RIII (also known as CD16), including isoforms Fc ⁇ RIIIa and Fc ⁇ RIIIb (Jefferis et al., Immunol Lett 2002, 82:57-65). Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIc, and Fc ⁇ RIIIa can induce ADCC, endocytosis, phagocytosis, and/or cytokine release.
  • Binding properties include, but are not limited to, binding specificity, binding affinity (K ⁇ sub>dis ⁇ /sub> ), and dissociation and binding rates (k ⁇ sub> dis ⁇ /sub> and k ⁇ sub>a ⁇ /sub>, respectively).
  • the heavy chain constant region and light chain constant region of the antibody of the present invention may be the heavy chain constant region (which may be natural or mutated) derived from human IgG1 or IgG4, and the human ⁇ chain and ⁇ chain constant regions.
  • the heavy chain constant region comprises an amino acid sequence as shown in SEQ ID NO:111 having the L234F/L235E/P331S mutation
  • the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO:112.
  • introducing the L234F/L235E/P331S mutation into the heavy chain constant region of the antibody can make the antibody substantially non-binding to Fc ⁇ RI, Fc ⁇ RIIa(H131), Fc ⁇ RIIb, Fc ⁇ RIIIa(V158), Fc ⁇ RIIIa(F158) and/or C1q, without affecting the binding affinity of the antibody to FcRn.
  • introducing the L234F/L235E/P331S mutation into the heavy chain constant region of the antibody can significantly reduce the ADCC activity of the anti-EGFR antibody.
  • the ADCC activity of the antibody is significantly reduced or eliminated compared to antibodies without amino acid mutations in the Fc region.
  • the antibody exhibits ADCC activity reduced to undetectable levels, and this reduction or elimination of ADCC activity may be due to a significant decrease in the binding affinity of the anti-EGFR antibody to Fc ⁇ R.
  • This invention further provides an anti-EGFR ADC comprising a small molecule toxin compound coupled to the anti-EGFR antibody of this invention via a cleavable linker.
  • the ADC specifically binds to the epitope of amino acid residues 447-491 of the EGFR extracellular domain.
  • the ADC specifically targets cells overexpressing EGFR (e.g., EGFR-overexpressing tumor cells), and after binding to EGFR on the cell surface, it can be endocytosed and transported into lysosomes for degradation, thereby releasing the small molecule toxin compound into the cytoplasm to kill the target cells.
  • the released small molecule toxin compound not only directly kills the target cells, but also, due to its cell membrane permeability, can exert a bystander effect in the tumor microenvironment, i.e., killing neighboring cells, including tumor cells that express low or no EGFR.
  • the ADC does not bind or weakly binds to normal tissues or cells, and the ADC does not affect EGFR-mediated downstream signal transduction pathways; therefore, the ADC does not kill normal tissues or cells or interfere with the biological function of EGFR in normal tissues and cells.
  • the ADC has better safety (i.e., does not cause non-tumor-targeted toxicity, such as skin toxicity) and broader-spectrum tumor cell killing activity (e.g., can kill tumor cells containing EGFR signaling pathway mutations that do not respond to existing EGFR-targeted therapies) compared to existing EGFR-targeted therapies.
  • the anti-EGFR ADC of the present invention has one or more of the following characteristics:
  • the anti-EGFR ADC of the present invention does not need to inhibit the EGFR signaling pathway to exert its anti-tumor effect. Instead, it targets and binds to EGFR-overexpressing tumor cells, is endocytosed and degraded by the cells, and releases a small molecule toxic compound into the cytoplasm to exert its killing effect.
  • the overexpressed EGFR can be wild-type EGFR or various mutant EGFR, as long as the mutation does not affect the binding of the ADC to the epitope of amino acid residues 447-491 of the extracellular domain of the receptor.
  • the anti-EGFR ADC of the present invention can kill tumors with EGFR mutations that are unresponsive to existing EGFR-targeted therapies, and has the potential to expand the indications for EGFR-targeted therapy. Moreover, it also has a killing effect on tumors resistant to existing EGFR-targeted drugs (e.g., tyrosine kinase inhibitors), and is expected to overcome the drug resistance problem of existing EGFR-targeted drugs.
  • existing EGFR-targeted drugs e.g., tyrosine kinase inhibitors
  • the anti-EGFR ADC of the present invention does not bind or weakly binds to normal tissues or cells (e.g., skin tissue) and does not interfere with the normal biological function of EGFR. Therefore, it will not damage normal tissues and cells expressing EGFR and is expected to solve the non-tumor targeting toxicity problem of existing EGFR-targeting drugs.
  • the anti-EGFR ADC of the present invention has a significantly reduced Fc receptor binding affinity, thereby significantly reducing the risk of Fc receptor-mediated toxic side effects.
  • the ADC can be represented by equation (I):
  • Ab represents the anti-EGFR antibody or its antigen-binding fragment of the present invention
  • D indicates a small molecule toxic compound (Drug);
  • L represents a cleavable linker that couples D to Ab
  • p represents the number of (L-D) copies coupled to Ab, ranging from 2 to 8.
  • the Ab in the ADC is the anti-EGFR antibody or its antigen-binding fragment that can specifically bind to the epitopes of amino acid residues 447-491 of the extracellular domain of EGFR, and it includes the VH and VL of the anti-EGFR antibody or its antigen-binding fragment as shown in Table 2.
  • the anti-EGFR antibody or its antigen-binding fragment in the ADC comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO:7, HCDR2 having the amino acid sequence shown in SEQ ID NO:15, HCDR3 having the amino acid sequence shown in SEQ ID NO:26 or 27, LCDR1 having the amino acid sequence shown in SEQ ID NO:33, LCDR2 having the amino acid sequence shown in SEQ ID NO:37, and LCDR3 having the amino acid sequence shown in SEQ ID NO:57, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.
  • the anti-EGFR antibody or its antigen-binding fragment in the ADC comprises: a VH containing the amino acid sequence shown in SEQ ID NO: 76 or 77 and/or a VL containing the amino acid sequence shown in SEQ ID NO: 99, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of the VH and VL, respectively.
  • the anti-EGFR antibody or its antigen-binding fragment in the ADC may also include constant regions, which include heavy chain constant regions and light chain constant regions, such as heavy chain constant regions from human IgG, especially human IgG1 or IgG4, and human ⁇ chain and ⁇ chain constant regions.
  • the heavy chain constant region of the anti-EGFR antibody or its antigen-binding fragment in the ADC includes an engineered IgG Fc region comprising human IgG1 Fc with an L234F/L235E/P331S (EU numbering system) amino acid substitution, which has reduced binding affinity for one or more Fc ⁇ Rs (e.g., Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIIa[158F], and Fc ⁇ RIIIa[158V]).
  • Fc ⁇ Rs e.g., Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIIa[158F], and Fc ⁇ RIIIa[158V]
  • the introduction of the L234F/L235E/P331S mutation into the heavy chain constant region of the anti-EGFR antibody or its antigen-binding fragment in the ADC results in the ADC being substantially non-binding to Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIIa(158F), and/or Fc ⁇ RIIIa(158V).
  • introducing the L234F/L235E/P331S mutation into the heavy chain constant region of the anti-EGFR antibody or its antigen-binding fragment in the ADC can render the ADC substantially non-binding to C1q.
  • introducing the L234F/L235E/P331S mutation into the heavy chain constant region of the anti-EGFR antibody or its antigen-binding fragment in the ADC does not affect the binding affinity of the ADC to FcRn.
  • the anti-EGFR antibody or its antigen-binding fragment in the ADC includes a light chain constant region selected from the ⁇ chain or ⁇ chain constant region, preferably the ⁇ chain constant region.
  • the heavy chain constant region of the anti-EGFR antibody or its antigen-binding fragment in the ADC includes an amino acid sequence as shown in SEQ ID NO:111 having the L234F/L235E/P331S mutation, and the light chain constant region includes an amino acid sequence as shown in SEQ ID NO:112.
  • the ADC specifically binds to EGFR and has no cross-binding activity with other members of the ErbB/HER family.
  • the "D” in the ADC is a small molecule toxic compound that, when not conjugated to the anti-EGFR antibody of this invention, exhibits cytotoxic or cell growth-inhibiting effects on both tumor cells and normal cells. However, after conjugation to form an ADC, the small molecule toxic compound only exhibits cytotoxic and/or bystander killing effects after the ADC is internalized and transported into the lysosome of the target cell, and dissociated from the ADC through enzymatic hydrolysis, oxidation, or any other mechanism.
  • the small molecule toxic compound includes cytotoxins, chemotherapeutic drugs, and radioactive isotopes.
  • the cytotoxic compound comprises a tubulin inhibitor and a DNA damaging agent.
  • the tubulin inhibitor includes eribulin, auristatins derivatives (e.g., MMAE, MMAF, MMAD), tubulysins, cryptomycins, and maytansinoids derivatives (e.g., DM1, DM2, DM3, DM4).
  • the DNA damaging agent includes topoisomerase inhibitors (e.g., camptothecin derivatives SN-38, exatecan, and DXd), pyrrolobenzodiazepines (PBD), and calic acid and its derivatives (e.g., N-acetylcalic acid [CMC]), and duocarmycin.
  • a preferred small molecule toxic compound is eribulin.
  • eribulin refers to a synthetic analogue of leptospirin B, a macrocyclic compound isolated from the marine sponge *Halichondria okadais*. Eribulin acts as a microtubule dynamics inhibitor, inhibiting the formation of the mitotic spindle by binding to tubulin, thus arresting the cell cycle at the G2/M phase.
  • exemplary structures of eribulin or its analogues and methods of their synthesis are described in WO1999065894, WO2004034990, ZL201910197071.8 and/or ZL201910509222.9, the disclosures of which are incorporated herein by reference.
  • the small molecule toxin compound is eribulin, which has a large clinical therapeutic window and low off-target cytotoxicity.
  • the chemotherapeutic agent may be a natural or synthetic compound, including but not limited to alkyl chemotherapeutic agents and other compounds in alkylated form (e.g., nitrogen mustard, ethyleneimine compounds, alkyl sulfonates, nitrosoureas, cisplatin, dacarbazine), antimetabolites (e.g., folic acid, purine or pyrimidine antagonists), mitotic inhibitors (e.g., vinca alkaloids and derivatives of podophyllotoxin), and cytotoxic antibiotics (e.g., anthracycline antibiotics including daunorubicin and doxorubicin, as well as actinomycin and bleomycin).
  • alkyl chemotherapeutic agents and other compounds in alkylated form e.g., nitrogen mustard, ethyleneimine compounds, alkyl sulfonates, nitrosoureas, cisplatin, dacarbazine
  • antimetabolites
  • the radioactive isotopes include, but are not limited to, 3H , 14C , 15N , 35S , 90Y , 99Tc , 111In , 125I , 131I , 89Sr , 177Lu , and 223Ra .
  • the cleavable linker is stable outside the cell, ensuring the structural stability of the ADC in vitro or in blood circulation, preventing systemic toxicity (off-target effects) due to the untargeted or random release of small molecule toxin compounds. Simultaneously, upon endocytosis into EGFR-overexpressing tumor cells, it can rapidly cleave and release small molecule toxin compounds to kill the tumor cells.
  • the cleavable linker refers to any linker containing a cleavable portion.
  • cleavable portion refers to any chemical bond that can be cleaved, such as those well known in the art, including but not limited to bonds unstable to acids, proteases/peptidases, light, esterases, and disulfide bonds.
  • Linkers containing a cleavable portion allow small molecule toxin compounds to dissociate from the ADC via cleavage at specific sites within the linker.
  • the cleavable linker comprises a cleavable peptide moiety, which, upon cleavage by an intracellular protease (e.g., an endosome, lysosomal, or tumor-associated protease), dissociates from the ADC to release a small molecule toxin compound that kills tumor cells.
  • an intracellular protease e.g., an endosome, lysosomal, or tumor-associated protease
  • the cleavable peptide moiety comprises an amino acid unit that can be cleaved by lysosomal cysteine cathepsins (e.g., cathepsins B, C, F, H, K, L, O, S, V, X, or W).
  • the amino acid unit may comprise naturally occurring amino acid residues and/or non-naturally occurring amino acid analogs, such as citrulline (Cit).
  • the amino acid unit comprises a dipeptide, tripeptide, or tetrapeptide, for example, a Phe-Lys, Val-Cit (VC), Glu-Val-Cit, or Gly-Gly-Phe-Gly (GGFG) amino acid sequence that can be cleaved by cathepsin B, with preferred amino acid units including GGFG.
  • the cleavable linker may include at least one spacer that couples a small molecule toxin compound (D) to the anti-EGFR antibody (Ab) of this application.
  • the spacer region may be hydrophilic to increase the hydrophilicity of the ADC, thereby improving its stability, reducing ADC product aggregation, and decreasing immunogenicity.
  • Exemplary spacers contain one or more polyethylene glycol (PEG) groups, such as 1, 2, 3, 4, 5, or 6 PEG groups, preferably 2 PEG groups.
  • PEG polyethylene glycol
  • the spacer region is via a maleic anhydride (Mal) group or... Attached to antibodies, wherein, via Mal or The spacer region attached to the antibody may also be referred to in this article as the "Mal-spacer region” or "4-(5-thio-1,3,4-oxadiazol-2-yl)benzamide-spacer region".
  • the Mal-spacer region or The spacer region contains one or more PEG portions (e.g., 2 PEGs).
  • the pyrolytic connector includes a Mal-spacer region or And a cleavable peptide moiety.
  • the spacer region comprises a PEG moiety (e.g., two PEGs), and the cleavable peptide moiety comprises an amino acid unit (e.g., a tetrapeptide GGFG).
  • the cleavable linker comprises a covalently linked Mal-spacer-amino acid unit.
  • the amino acid unit is Phe-Lys, VC, Glu-Val-Cit, or GGFG, with GGFG being the preferred amino acid unit.
  • the spacer region is (PEG) m , where m is an integer from 1 to 6, preferably 2.
  • the cleavable linker comprises Mal-(PEG) 2 and GGFG. In some embodiments, the cleavable linker comprises... And GGFG.
  • the cleavable linker comprises: a Mal-spacer region-cleavable peptide moiety.
  • the pyrolytic connector comprises the structure: Mal-(PEG) 2 -GGFG.
  • the cleavable linker comprises: a 4-(5-thio-1,3,4-oxadiazol-2-yl)benzamide-spacer region-cleavable peptide moiety.
  • the pyrolytic connector comprises: 4-(5-thio-1,3,4-oxadiazol-2-yl)benzamido-(PEG) 2 -GGFG.
  • the cis-butenylimide group or 4-(5-thio-1,3,4-oxadiazol-2-yl)benzamide group in the cleavable linker is coupled to one or more amino acid residues of the antibody moiety in the ADC of the present invention.
  • the cis-butenylimide group or 4-(5-thio-1,3,4-oxadiazol-2-yl)benzamide group can be coupled to the antibody by reacting with thiol groups in the antibody, including one or more cysteine residues coupled to the antibody.
  • the cis-butenylimide group in the cleavable linker reacts with the thiol group of the cysteine residue of the antibody to form a thioether bond, thereby coupling the cleavable linker to the antibody.
  • the 4-(5-thio-1,3,4-oxadiazol-2-yl)benzamide group in the cleavable linker reacts with the thiol group of the cysteine residue of the antibody to form a disulfide bond, thereby coupling the cleavable linker to the antibody.
  • the cis-butenylimide group or 4-(5-thio-1,3,4-oxadiazol-2-yl)benzamide group in the cleavable linker can react with the thiol group of cysteine residues at specific positions in the constant region and/or variable region of the antibody. Furthermore, the cis-butenylimide group or 4-(5-thio-1,3,4-oxadiazol-2-yl)benzamide group in the cleavable linker can couple with free cysteine residues released after reduction of disulfide bonds and/or interchain disulfide bonds in the hinge region of the antibody.
  • the maleic anhydride group or 4-(5-thio-1,3,4-oxadiazol-2-yl)benzamide group in the cleavable linker is coupled to the cysteine residues in the antibody hinge region.
  • This can generate multiple free cysteine residues by chemically reacting the interchain disulfide bonds in the antibody hinge region (e.g., reduction, pH adjustment, or hydrolysis).
  • engineered cysteine residues can be generated by using DNA recombination technology (e.g., by substituting or inserting cysteine residues) from amino acid residues at one or more specific sites in the antibody constant region.
  • the cleavable portion of the cleavable linker (e.g., a cleavable peptide) can be directly conjugated to the small molecule toxin compound portion of the ADC, wherein the small molecule toxin compound is eribulin or a derivative thereof.
  • the cleavable peptide comprises an amino acid unit containing GGFG
  • the tetrapeptide is conjugated to the C-35 amine of eribulin via a carboxyl group to construct an ADC containing the structure Mal-(PEG) 2 -GGFG-Eribulin or 4-(5-thio-1,3,4-oxadiazol-2-yl)benzamido-(PEG) 2- GGFG-Eribulin.
  • the ADC structure of the present invention comprises the anti-EGFR antibody of the present invention conjugated to eribulin or a derivative thereof via a cleavable linker, said cleavable linker comprising a Mal-spacer-cleavable peptide moiety or a 4-(5-thio-1,3,4-oxadiazol-2-yl)benzamide-spacer-cleavable peptide moiety.
  • the ADC structure comprises the anti-EGFR antibody of the present invention conjugated to the C-35 amine of eribulin via a cleavable linker, said cleavable linker comprising Mal-(PEG) 2 -GGFG and 4-(5-thio-1,3,4-oxadiazol-2-yl)benzamide-(PEG) 2 -GGFG, wherein free cysteine residues released after reduction of the disulfide bonds and/or interchain disulfide bonds in the antibody hinge region are conjugated to the maleic diamiimide group or the 4-(5-thio-1,3,4-oxadiazol-2-yl)benzamide group of the cleavable linker.
  • the ADC of the present invention comprises a portion derived from a drug payload (i.e., an LD intermediate compound):
  • the -LD portion includes:
  • the ADC of the present invention comprises an anti-EGFR antibody as described in the present invention conjugated to a drug payload comprising Mal-(PEG) 2 -GGFG-Eribulin and 4-(5-thio-1,3,4-oxadiazol-2-yl)benzamide-(PEG) 2 -GGFG-Eribulin.
  • the ADC comprises the anti-EGFR antibody as described in the present invention conjugated to the maleic bisimide group or the 4-(5-thio-1,3,4-oxadiazol-2-yl)benzamide group of the drug payload via its cysteine residues.
  • the free cysteine residues released after reduction of the disulfide bonds and/or interchain disulfide bonds in the hinge region of the antibody are conjugated to the maleic bisimide group or the 4-(5-thio-1,3,4-oxadiazol-2-yl)benzamide group of the drug payload.
  • the present invention provides an ADC having the following formula:
  • Ab represents the anti-EGFR antibody or its antigen-binding fragment described in this invention.
  • p is approximately 2 to 8.
  • p is also referred to herein as the Drug-to-Antibody Ratio (DAR) or the number of payload molecules, i.e., the number of small molecule toxin compounds conjugated to the antibody, and p is approximately 2 to 8, for example, 4 to 8.
  • DAR Drug-to-Antibody Ratio
  • the anti-EGFR antibody contained in the ADC of the present invention, conjugated to eribulin via a cleavable linker, has a desirable DAR value, meaning that the ADC not only possesses sufficient cytotoxic activity but also sufficient stability.
  • a higher DAR value (e.g., p>8) may cause the ADC to aggregate and precipitate in an aqueous environment due to hydrophobic interactions caused by the strong hydrophobicity of the small molecule toxin compounds, potentially leading to safety risks in vivo;
  • a lower DAR value (e.g., p ⁇ 2) may result in lower cytotoxic activity of the ADC against tumor cells, affecting efficacy.
  • the optimal DAR value is approximately 4 to 8.
  • the average DAR value (i.e., average effective number of molecules or average p) of the anti-EGFR ADC of the present invention can be obtained by calculation using the results of conventional analytical methods known in the art (e.g., reversed-phase LC-MS mass spectrometry and/or HIC-HPLC).
  • the present invention provides a nucleic acid encoding an anti-EGFR antibody or an antigen-binding fragment thereof.
  • the present invention also includes polynucleotide variants encoding the amino acid sequence described herein.
  • the amino acid sequence corresponding to that described in this invention, the probe or primer used for nucleic acid isolation, or the nucleotide sequence available from a database can be obtained by back-translating the amino acid sequence.
  • a polymerase chain reaction (PCR) procedure can be used to isolate and amplify DNA sequences encoding the anti-EGFR antibody or its antigen-binding fragment described in this invention.
  • Oligonucleotides defining the desired ends of the DNA fragment combination are used as 5' and 3' primers.
  • the oligonucleotides may additionally contain recognition sites for restriction endonucleases to facilitate the amplification of the DNA fragment combination for insertion into the expression vector.
  • the nucleic acid molecules described in this invention include single-stranded and double-stranded DNA and RNA, and corresponding complementary sequences, including isolated nucleic acid molecules, preferably derived from DNA or RNA isolated at least once in substantially pure form and in quantities or concentrations that can be identified, manipulated, and recovered by standard biochemical methods (e.g., the methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989, 2nd edition, Cold Spring Harbor Laboratory).
  • sequences comprise open reading frames provided and/or constructed as internal untranslated sequences or intron breaks that are not commonly found in eukaryotic genes.
  • the untranslated DNA sequence may be present at the 5' or 3' of the open reading frame, wherein the sequence does not interfere with the manipulation or expression of the coding region.
  • the anti-EGFR antibody or its antigen-binding fragment, or a polypeptide containing amino acid residues 447-491 of the EGFR extracellular domain, of the present invention can be prepared by the following steps: site-specific mutation of nucleotides in the DNA encoding the anti-EGFR antibody or its antigen-binding fragment, or the polypeptide containing amino acid residues 447-491 of the EGFR extracellular domain, using PCR or other techniques known to those skilled in the art, to produce DNA encoding a variant, followed by expression of the target recombinant protein in a cell culture as outlined herein.
  • the anti-EGFR antibody or its antigen-binding fragment, or the polypeptide containing amino acid residues 447-491 of the EGFR extracellular domain can also be prepared using established in vitro synthesis techniques.
  • the anti-EGFR antibody or its antigen-binding fragment of the present invention can be encoded by an extremely large number of nucleic acids, each of which is within the scope of the present invention and can be prepared using standard techniques. Therefore, by identifying specific amino acid sequences, those skilled in the art can easily modify their respective coding sequences with one or more codons to prepare many different nucleic acids without altering the amino acid sequence of the anti-EGFR antibody or its antigen-binding fragment of the present invention, or the polypeptide sequence containing amino acid residues 447-491 of the EGFR extracellular domain.
  • the present invention also provides an expression vector for nucleic acids encoding the anti-EGFR antibody or its antigen-binding fragment thereof, or a polypeptide containing amino acid residues 447-491 of the extracellular domain of EGFR.
  • Nucleic acid encoding the anti-EGFR antibody of the present invention or its antigen-binding fragment, or a polypeptide containing amino acid residues 447-491 of the EGFR extracellular domain can be cloned into a suitable vector for introduction into host cells for expression of the target protein.
  • Vector components typically include, but are 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 target protein in the vector is operatively linked to the promoter.
  • the term “operationally linked” refers to a functional link between a nucleic acid expression control sequence (e.g., an array of promoters, signal sequences, or transcription factor binding sites) and another nucleic acid sequence, and thus the control sequence controls the transcription and/or translation of the other nucleic acid sequence.
  • a nucleic acid expression control sequence e.g., an array of promoters, signal sequences, or transcription factor binding sites
  • Suitable vectors include plasmids, phagemids, Cos plasmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as ⁇ phage or M13 phage, and animal viruses.
  • Animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40).
  • Vectors can contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain replication initiation sites. Vectors may also include components that facilitate their entry into cells, including but not limited to viral particles, liposomes, or protein coats.
  • the present invention also provides a host cell comprising a nucleic acid or expression vector encoding the anti-EGFR antibody of the present invention or its antigen-binding fragment, or a polypeptide containing amino acid residues 447-491 of the extracellular domain of EGFR.
  • the cells can be eukaryotic cells, such as mammalian host cells, including but not limited to SV40-transformed monkey kidney cell line CV1 (COS-7, ATCC, CRL-1651), human embryonic kidney cell line (293 or 293 cell subclones in suspension culture, Graham et al., J Gen Virol 1977, 36:59-74), juvenile hamster kidney cells (BHK-21, ATCC, CCL-10), Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc Natl Acad Sci USA 1980, 77:4216-4220), and mouse testicular supporting cells (TM4, Mather, Biol Reprod 1980, 23:243-251).
  • mammalian host cells including but not limited to SV40-transformed monkey kidney cell line CV1 (COS-7, ATCC, CRL-1651), human embryonic kidney cell line (293 or 293 cell subclones in suspension culture, Graham et al., J Gen Virol 1977, 36:59-74), juvenile hamster kidney cells
  • Monkey kidney cells (CV1, ATCC, 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 hepatocytes (BRL 3A, ATCC, CRL-1442), human lung cells (W138, ATCC, CCL-75), human liver cancer cell line (HepG2, ATCC, HB-8065), mouse mammary tumor (MMT060562, ATCC, CCL-51), TRI cells (Mather et al., Ann NY Acad Sci 1982, 383:44-68), MRC5 cells, or FS4 cells.
  • CV1 Monkey kidney cells
  • VOD-76 African green monkey kidney cells
  • HELA human cervical cancer cells
  • BDL 3A Buffalo rat hepatocytes
  • human lung cells W138, ATCC, CCL-75
  • human liver cancer cell line HepG2, ATCC, HB-
  • the present invention provides a method for preparing the anti-EGFR antibody or its antigen-binding fragment, or a polypeptide containing amino acid residues 447-491 of the extracellular domain of EGFR, using the host cells described herein.
  • the method includes transfecting a nucleic acid or expression vector encoding the anti-EGFR antibody of the present invention or its antigen-binding fragment, or a polypeptide containing amino acid residues 447-491 of the EGFR extracellular domain, into host cells, and culturing the host cells in a culture medium for a period of time to express the anti-EGFR antibody of the present invention or its antigen-binding fragment, or the polypeptide containing amino acid residues 447-491 of the EGFR extracellular domain.
  • Commercially available culture media can be used without limitation.
  • the expressed anti-EGFR antibody or its antigen-binding fragment, or a polypeptide containing amino acid residues 447-491 of the EGFR extracellular domain can be secreted into the culture medium for growing host cells.
  • the antibody or polypeptide can be recovered from the culture medium using standard protein purification methods, such as removing impurities by centrifugation or ultrafiltration, or purifying the product by affinity chromatography; other purification techniques can also be used, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography.
  • the ADC of the present invention can be prepared by any method known in the art, including but not limited to: (1) reacting the nucleophilic or electrophilic group of an antibody with a cleavable linker to form an antibody-cleavable linker intermediate (Ab-L) via covalent bonds, and then reacting it with a small molecule toxin compound (D), wherein the intermediate product Ab-L may be purified before reacting with the small molecule toxin compound, or may not be purified; (2) reacting the nucleophilic or electrophilic group of a small molecule toxin compound with a cleavable linker to form a small molecule toxin compound-cleavable linker intermediate (D-L) via covalent bonds, and then reacting it with the nucleophilic or electrophilic group of an antibody, wherein the intermediate product D-L may be purified before reacting with the antibody, or may not be purified; or (3) mixing and reacting the antibody, the cleavable linker and the small molecule tox
  • the antibody is placed under reducing conditions prior to the conjugation reaction to generate one or more free cysteine residues.
  • a reducing agent e.g., dithiothreitol [DDT], 2-mercaptoethanol, or tris(2-carboxyethyl)phosphine [TCEP]
  • DDT dithiothreitol
  • TCEP tris(2-carboxyethyl)phosphine
  • the reducing agent preferentially reduces the disulfide bonds in the antibody hinge region and/or the interchain disulfide bonds between the light and heavy chains, while the intrachain disulfide bonds of the antibody remain intact.
  • reacting the antibody with the reducing agent TCEP in a buffer containing a chelating agent yields a partially or completely reduced antibody with free thiol groups.
  • the chelating agent includes, but is not limited to, ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA), and the buffer includes, but is not limited to, histidine-hydrochloric acid, sodium phosphate, sodium borate, and sodium acetate solutions.
  • the partially or fully reduced antibody with free thiol groups obtained above can react with the reactive functional groups (e.g., maleimide, 4-(5-thio-1,3,4-oxadiazol-2-yl)benzamide) of a cleavable linker (L) or a small molecule toxin compound-cleavable linker intermediate (D-L) to form a covalent bond (e.g., a thioether bond, a dithioether bond) to obtain an antibody-cleavable linker intermediate (Ab-L) or an ADC.
  • the small molecule toxin compound-cleavable linker intermediate (D-L) is used interchangeably with the drug payload.
  • the purification method for the ADC prepared by the above method can be any method or any combination of methods known in the art for purifying proteins, including but not limited to affinity chromatography, ion exchange chromatography, mixed-mode chromatography (e.g., ceramic hydroxyapatite chromatography), hydrophobic interaction chromatography, size exclusion chromatography, dialysis, filtration, selective precipitation, or any combination thereof.
  • affinity chromatography ion exchange chromatography
  • mixed-mode chromatography e.g., ceramic hydroxyapatite chromatography
  • hydrophobic interaction chromatography e.g., size exclusion chromatography
  • dialysis e.g., dialysis, filtration, selective precipitation, or any combination thereof.
  • compositions comprising the anti-EGFR antibody or its antigen-binding fragment thereof, or an anti-EGFR ADC, of the present invention, and a pharmaceutically acceptable carrier.
  • the composition comprises an effective amount of said anti-EGFR antibody or its antigen-binding fragment thereof, or an ADC.
  • the pharmaceutical composition may contain any kind of pharmaceutically acceptable carrier.
  • Carriers that may be used include excipients, surfactants, thickeners or emulsifiers, solid binders, dispersants or suspending agents, solubilizers, colorants, flavoring agents, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, or combinations thereof.
  • Gennaro ed., Remington: The Science and Practice of Pharmacy, 20th edition, 2003 (Lippincott Williams & Wilkins), the disclosure of which is incorporated herein by reference.
  • the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration.
  • parenteral administration refers to non-enteral and non-local routes of administration, including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions.
  • the antibodies described in this invention can also be administered via non-parenteral routes (e.g., local, epidermal, or mucosal administration), such as intranasal, oral, vaginal, rectal, sublingual, or local administration.
  • non-parenteral routes e.g., local, epidermal, or mucosal administration
  • the active ingredient can be coated in the material to protect it from acids/alkalis and other natural conditions that may inactivate it.
  • compositions can be sterile aqueous solutions or dispersants. They can also form microemulsions, liposomes, or other ordered structural combinations suitable for high drug concentrations.
  • compositions formed with a pharmaceutically acceptable carrier contain about 0.01% to about 99% of the active ingredient, preferably about 0.1% to about 70%, more preferably about 1% to about 30% of the active ingredient.
  • the dosage regimen can be adjusted to provide the optimal expected response (e.g., therapeutic response). For example, it can be administered as a single dose, in multiple doses, or the dose can be reduced or increased proportionally depending on the treatment outcome.
  • Formulating the parenteral administration composition in unit dosage form is particularly advantageous, as it facilitates administration and ensures uniform dosage.
  • unit dosage form refers to physically discrete units suitable as unit doses for treating subjects; each unit dose contains a predetermined amount of the active ingredient, which is determined by calculating the expected therapeutic effect when the active ingredient is administered with the desired drug carrier.
  • the anti-EGFR antibody or anti-EGFR ADC of this invention can also be administered as a sustained-release formulation, thereby reducing the frequency of administration.
  • the "therapeutic effective dose" of the anti-EGFR antibody or its antigen-binding fragment, or the anti-EGFR ADC of the present invention preferably reduces the severity of disease symptoms, increases the frequency and duration of disease progression-free periods, or prevents bodily damage or disability caused by disease.
  • the "therapeutic effective dose” for tumor-bearing subjects, relative to untreated subjects preferably inhibits tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80%.
  • the therapeutic effective dose of the therapeutic ADC can reduce tumor size or improve symptoms in subjects, typically humans or other mammals.
  • the "therapeutic effective dose” can also be determined differently based on various factors, including but not limited to formulation, administration method, age, body size, weight, patient sex or pathological condition, diet, administration time, administration interval, route of administration, excretion rate, and response sensitivity.
  • the pharmaceutical composition may be selected as a controlled-release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable and biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, for example, Robinson, ed., Sustained and Controlled Release Drug Delivery Systems, 1978 (Marcel Dekker).
  • Therapeutic pharmaceutical compositions can be delivered by medical devices selected from: (1) needle-free subcutaneous injection devices (e.g., US5399163, US5383851, US5312335, US5064413, US4941880, US4790824 and US4596556); (2) micro-infusion pumps (US4487603); (3) transdermal devices (US4486194); (4) infusion devices (US4447233 and US4447224); and (5) permeation devices (US4439196 and US4475196); the disclosures of which are incorporated herein by reference.
  • needle-free subcutaneous injection devices e.g., US5399163, US5383851, US5312335, US5064413, US4941880, US4790824 and US4596556
  • micro-infusion pumps e.g., US4487603
  • transdermal devices e.g., US44861914
  • infusion devices e.g., US4447233 and US4447224
  • permeation devices e.g
  • the anti-EGFR antibody or its antigen-binding fragment, or the anti-EGFR ADC of the present invention can be formulated to ensure biodistribution in vivo.
  • the therapeutic antibody or ADC of the present invention can cross the blood-brain barrier, it can be formulated as a liposome, which may additionally contain a targeting portion to enhance selective delivery to specific cells or organs.
  • the present invention provides a kit containing an effective amount of the anti-EGFR antibody or its antigen-binding fragment thereof, an anti-EGFR ADC, or a pharmaceutical composition thereof, and optionally at least one other tumor therapeutic agent (i.e., the kit may contain or not contain at least one other tumor therapeutic agent).
  • the present invention relates to a method for treating EGFR-related diseases or conditions (especially those related to EGFR overexpression), the method comprising administering an effective amount of the anti-EGFR ADC, or pharmaceutical composition, or kit of the present invention to a subject in need.
  • the present invention relates to the use of the aforementioned ADC, pharmaceutical composition, or kit in the preparation of a medicament for treating EGFR-related diseases or conditions.
  • diseases or conditions include EGFR-overexpressing cancers and other diseases or conditions related to EGFR overexpression.
  • the cancers that overexpress EGFR include, but are not limited to, lung cancer (including non-small cell lung cancer and small cell lung cancer), head and neck cancer (e.g., squamous cell carcinoma of the head and neck), glioblastoma, colorectal cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, kidney cancer or renal cell carcinoma, liver cancer, esophageal cancer, gallbladder cancer, pancreatic cancer, gastric cancer, thyroid cancer, bladder cancer, skin cancer, nasopharyngeal carcinoma, melanoma, and other solid cancers.
  • the cancers that overexpress EGFR also include cancers at various stages, such as early-stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, or cancer in remission.
  • the subjects can be humans, non-human primates, or other mammals such as dogs, mice, and rats.
  • cancers expressing EGFR can be characterized by the level of EGFR protein expressed on the surface of cancer/tumor cells (i.e., by "EGFR status").
  • EGFR expression levels can be determined by immunohistochemistry (IHC), for example, using the EGFR pharmDxTM kit (DakoCytomation).
  • EGFR fluorescence staining levels on cell surfaces can also be determined by flow cytometry (FACS), and the level of the EGFR gene encoding tumor cells overexpressing EGFR can be determined by RNASeq or qPCR.
  • EGFR-overexpressing cancer refers to cancer/tumor cells with significantly higher EGFR expression levels on their surface than normal tissues or cells (e.g., skin tissue).
  • EGFR expression abundance can be assessed by measuring EGFR fluorescence staining levels on the cell surface using FACS.
  • the EGFR expression levels on the surface of the MCF-10A cells and the engineered cell line 293T-EGFR#16-2D6 described in this invention are comparable to those of normal skin tissue.
  • the EGFR-overexpressing cancers include both wild-type EGFR-overexpressing cancers and mutant EGFR-overexpressing cancers, provided that the mutation does not affect the binding of the anti-EGFR antibody or ADC to its extracellular domain epitope of amino acid residues 447-491.
  • cancers with mutations in the intracellular tyrosine kinase domain of EGFR including primary EGFR mutations and acquired EGFR mutations caused by tyrosine kinase inhibitors), including but not limited to one or more of L688P, Q701H, E709V, L718Q, Q724S, K745N, L747P, D761Y, M766Q, C781R, exon 19 deletion, exon 20 insertion, L858R, T790M, and C797S.
  • L688P Q701H, E709V, L718Q, Q724S, K745N, L747P, D761Y, M766Q, C781R, exon 19 deletion, exon 20 insertion, L858R, T790M, and C797S.
  • the cytotoxic activity of the anti-EGFR ADC or its pharmaceutical composition against EGFR-expressing tumors or cancers is positively correlated with the abundance of EGFR expression on the tumor cell surface.
  • the anti-EGFR ADC or its pharmaceutical composition has a significant direct cytotoxic effect against EGFR-overexpressing tumors or cancers, while the ADC has almost no cytotoxic activity when the EGFR expression level on the cell surface is as low as that of normal skin tissue.
  • the anti-EGFR ADC or its pharmaceutical composition has a cytotoxic effect against tumors or cancers with EGFR intracellular tyrosine kinase domain mutations and EGFR overexpression, exemplarily a deletion of EGFR exon 19.
  • administering an effective amount of the anti-EGFR ADC of the present invention or its pharmaceutical composition or kit to a subject in need can reduce the tumor volume of the subject, inhibit tumor growth, prolong the subject's disease-free survival or progression-free survival, increase the subject's overall survival, reduce tumor metastasis, or improve the subject's quality of life.
  • EGFR-related diseases include, but are not limited to, autoimmune diseases, psoriasis, or inflammatory arthritis (e.g., rheumatoid arthritis, systemic lupus erythematosus-associated arthritis, psoriatic arthritis).
  • autoimmune diseases e.g., rheumatoid arthritis, systemic lupus erythematosus-associated arthritis, psoriatic arthritis.
  • other EGFR-related diseases include proliferative disorders, which, in addition to cancer, include: adrenal hyperplasia (e.g., Cushing's disease), congenital adrenal hyperplasia, endometrial hyperplasia, benign prostatic hyperplasia, breast hyperplasia, endometrial hyperplasia, focal epithelial hyperplasia (e.g., Heck's disease), sebaceous gland hyperplasia, compensatory liver hyperplasia, and any other proliferative disorders.
  • adrenal hyperplasia e.g., Cushing's disease
  • congenital adrenal hyperplasia e.g., endometrial hyperplasia
  • endometrial hyperplasia benign prostatic hyperplasia
  • breast hyperplasia e.g., endometrial hyperplasia
  • focal epithelial hyperplasia e.g., Heck's disease
  • sebaceous gland hyperplasia e.g., compensatory liver hyperplasia
  • the present invention also relates to a method for treating cancers resistant to existing EGFR-targeted therapies, the method comprising administering an effective amount of the anti-EGFR ADC of the present invention, or a pharmaceutical composition or cartridge thereof, to a patient in need.
  • the present invention relates to the use of said ADC, or a pharmaceutical composition or cartridge thereof, in the preparation of a medicament for treating cancers resistant to existing EGFR-targeted therapies.
  • the present invention relates to said ADC, or a pharmaceutical composition or cartridge thereof, for treating cancers resistant to existing EGFR-targeted therapies.
  • the patient does not respond or responds poorly to one or more existing EGFR-targeted therapies, including anti-EGFR antibodies (e.g., Cetuximab, Panitumumab, Nimotuzumab, Necitumumab) and tyrosine kinase inhibitors (e.g., Afatinib, Erlotinib, Gefitinib, Osimertinib).
  • Non-response is manifested as tumor growth, increased tumor volume, formation of metastases, or an increased number of metastases.
  • Non-response can also be a shortened time to metastasis or disease progression. Poor response refers to tumor growth or metastasis in the patient during or shortly after receiving standard EGFR-targeted therapy.
  • the present invention also relates to a method for treating patients who are unsuitable for existing EGFR-targeted therapies or are refractory to treatment, or who have relapsed after receiving existing EGFR-targeted therapies, the method comprising administering an effective amount of the anti-EGFR ADC of the present invention or a pharmaceutical composition or kit thereof to the patient in need.
  • the present invention relates to the use of said ADC or pharmaceutical composition or kit thereof in the preparation of a medicament for treating patients who are unsuitable for existing EGFR-targeted therapies or are refractory to treatment, or who have relapsed after receiving existing EGFR-targeted therapies.
  • the present invention relates to the anti-EGFR ADC of the present invention or a pharmaceutical composition or kit thereof for treating patients who are unsuitable for existing EGFR-targeted therapies or are refractory to treatment, or who have relapsed after receiving existing EGFR-targeted therapies.
  • the existing EGFR-targeted therapies include treatment using at least one of the above-described anti-EGFR antibodies and tyrosine kinase inhibitors.
  • the anti-EGFR ADC of the present invention can be used to treat progressive cancer when a patient has experienced disease progression with existing EGFR-targeted therapies.
  • anti-EGFR antibody anti-EGFR ADC
  • pharmaceutical composition or kit of the present invention can be used alone or in combination with other types of cancer therapies known in the art, such as surgery, chemotherapy, radiotherapy, gene therapy, immunotherapy, photodynamic therapy, radiofrequency ablation, etc.
  • the present invention also relates to the use of anti-EGFR antibodies or antigen-binding fragments thereof for detecting and/or measuring EGFR in samples (e.g., biological samples), and for screening patients with EGFR-related diseases or conditions who respond to the anti-EGFR ADC treatment of the present invention.
  • the anti-EGFR antibody or its antigen-binding fragment can be used to diagnose conditions or diseases (e.g., cancer) of EGFR aberration (e.g., overexpression) to facilitate the determination of treatment regimens.
  • the antibody can be conjugated to a detectable marker or reporter molecule, and the labeled antibody can be incubated with a biological sample derived from a patient to diagnose EGFR expression.
  • the detectable marker or reporter molecule can be a radioactive isotope, such as 3H , 14C , 32P , 35S , or 125I ; a fluorescent material, such as umbelliferone, luciferin, rhodamine, luciferin isothiocyanate, luciferin dichlorotriazine, dansyl chloride, or phycoerythrin; a chemiluminescent material, such as luminol; a bioluminescent material, such as luciferase, luciferin, or jellyfish luminescent protein; or an enzyme, such as alkaline phosphatase, ⁇ -galactosidase, acetylcholinesterase, horseradish peroxidase, or luciferase.
  • a radioactive isotope such as 3H , 14C , 32P , 35S , or 125I
  • a fluorescent material such as umbelliferone, luci
  • Specific exemplary assays that can be used to detect or measure EGFR in a sample include immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoPET (e.g., 89Zr , 64Cu , etc.), and fluorescence activated cell flow cytometry (FACS).
  • IHC immunohistochemistry
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • immunoPET e.g., 89Zr , 64Cu , etc.
  • FACS fluorescence activated cell flow cytometry
  • Example 1 Construction and screening of anti-human EGFR antibody libraries, and identification of antibody binding properties and biological activity.
  • mice Three BALB/c mice were immunized with recombinant human EGFR extracellular domain protein (sequence derived from positions 25-645 of Swiss-Prot accession number P00533, or positions 25-645 of the amino acid sequence shown in SEQ ID NO:105, or positions 1-621 of the amino acid sequence shown in SEQ ID NO:106) and human skin squamous cell carcinoma A431 cells via alternating subcutaneous injection. Serum was collected after three immunizations, and the antibody titer in the serum was detected using ELISA with the EGFR recombinant protein as the antigen. The results showed that the serum antibody titers of all three immunized mice exceeded 1:320,000.
  • mice antibody heavy chain variable region and light chain variable region gene fragments were amplified separately by PCR using specific primers.
  • the light and heavy chain variable region gene fragments were then spliced and amplified separately by overlap PCR (SOE-PCR) to obtain single-chain antibody (scFv) variable region gene fragments.
  • SOE-PCR overlap PCR
  • the scFv sequence was ligated into a phage display vector by enzyme digestion.
  • the ligation product was then electroporated into TG1 competent cells. After infection with helper phage M13KO7, three mouse immune libraries were obtained.
  • RNA was extracted from commercially available human PBMCs (iXCells Biotechnologies, USA) and a fully human antibody natural library was constructed.
  • EGFR extracellular domain proteins as target antigens, three mouse immune libraries and one fully human antibody natural library were subjected to an adsorption-elution-amplification process to screen for anti-EGFR antibodies. After 3-5 rounds of screening, and identification by bacterial PCR and monoclonal phage ELISA, positive clones were sequenced, yielding 31 anti-EGFR antibodies with distinct sequences (including 24 mouse antibodies and 7 fully human antibodies).
  • the light and heavy chain genes of the 31 antibodies were homologously recombinated with linearized expression vectors (the heavy chain variable region VH was ligated to a pcDNA3.1 vector containing the human IgG1 constant region, and the light chain variable region VL was ligated to a pcDNA3.1 vector containing the human CK constant region).
  • Corresponding antibody expression plasmids were obtained through colony PCR and DNA sequencing analysis.
  • Antibody expression plasmids containing light and heavy chain gene fragments were transiently transfected into ExpiCHO-S cells (Thermo), and then expressed by serum-free culture.
  • Antibody proteins including chimeric antibody mAb1135 and fully human antibody hAb173 were purified by Protein A affinity chromatography using the AKTA Pure system.
  • an expression plasmid (Yiqiao Shenzhou) encoding the full-length human EGFR protein was transfected into 293T cells. After selection with hygromycin, a stable EGFR-expressing 293T-EGFR(POOL) engineered cell subpopulation was obtained. Subsequently, AF488-labeled Cetuximab was used as the detection antibody, and A431 cells overexpressing EGFR and MCF10A cells with EGFR expression levels equivalent to normal skin tissue were used as control cell lines.
  • the binding activity of anti-EGFR antibodies to cells expressing different levels of EGFR was detected by flow cytometry, including EGFR-overexpressing 293T-EGFR#2 cells and 293T-EGFR#16-2D6 cells with EGFR expression levels comparable to normal skin tissue. Cetuximab was used as the control antibody.
  • the specific method was as follows: Cells were resuspended in pre-chilled FACS buffer (PBS + 1% BSA) to a concentration of 6–10 ⁇ 106 cells/mL, and 50 ⁇ L/well was added to each well of a 96-well U-shaped plate.
  • mAb1135 and hAb173 showed cell binding activity dependent on the expression abundance of EGFR on the cell surface. They exhibited strong binding to 293T-EGFR#2 cells, with binding activity comparable to Cetuximab, while showing very weak binding activity to 293T-EGFR#16-2D6 cells.
  • the control antibody Cetuximab showed strong binding activity to both 293T-EGFR#2 and 293T-EGFR#16-2D6 cells, independent of EGFR expression abundance.
  • this embodiment utilizes flow cytometry to conduct an antibody competitive binding experiment.
  • the specific steps are as follows: First, A431 cells were collected and resuspended in pre-chilled FACS buffer to a concentration of 6–10 ⁇ 106 cells/mL, and 50 ⁇ L/well was added to each well of a 96-well U-shaped plate.
  • a serially diluted mAb1135 and hAb173 antibody were each mixed with an equal volume of 1 ⁇ g/mL biotin-labeled hAb173 antibody, and then 50 ⁇ L/well was added to each well of A431 cells for incubation.
  • AF488-labeled streptavidin Jackson Immuno
  • flow cytometry was used to detect the binding of the biotin-labeled hAb173 antibody on A431 cells to determine whether there was competition between the two antibodies for EGFR binding.
  • mAb1135 and hAb173 were able to compete with each other completely on A431 cells, indicating that the two antibodies bind to the same or overlapping epitopes of EGFR on the surface of A431 cells.
  • this embodiment considering the similarity in primary and tertiary structures between the extracellular domains of EGFR and HER4, designed and prepared a series of EGFR/HER4 chimeric antigen peptides.
  • the specific method is as follows: the gene sequences encoding the extracellular subdomains of human EGFR and human HER4 were recombined and spliced, and homologous recombination was performed and ligated into the pcDNA3.1 expression vector to construct an EGFR/HER4 extracellular domain chimeric protein expression plasmid.
  • EGFR/HER4 chimeric recombinant proteins each recombinant protein has a 6 ⁇ His tag at its C-terminus.
  • the human EGFR/HER4 chimeric recombinant proteins EGFR-I, EGFR-II, EGFR-III, and EGFR-IV represent the replacement of the D1, D2, D3, and D4 sequences of the human HER4 extracellular region with the corresponding human EGFR extracellular region subdomain sequences.
  • EGFR-I represents the replacement of the D1 sequence of the HER4 extracellular region with the D1 sequence of the EGFR extracellular region.
  • human EGFR/HER4 chimeric recombinant proteins HER4-I, HER4-II, HER4-III, and HER4-IV represent the replacement of the D1, D2, D3, and D4 sequences of the human EGFR extracellular region with the corresponding human HER4 extracellular region subdomain sequences.
  • HER4-I represents the replacement of the D1 sequence of the EGFR extracellular region with the D1 sequence of the HER4 extracellular region ( Figure 4).
  • the subdomain containing the antigen-binding epitope of the anti-EGFR antibody was identified by ELISA.
  • the control antibody used was Cetuximab.
  • the specific method is as follows: The test antibody and control antibody were diluted with PBS, and 100 ⁇ L was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. After washing with PBS, 200 ⁇ L of blocking buffer (PBST [PBS + 0.1% Tween-20] containing 1% BSA) was added to each well, and the plate was incubated at room temperature for 1 hour.
  • PBST PBS + 0.1% Tween-20
  • EGFR, HER4, or EGFR/HER4 chimeric recombinant protein including EGFR-I, EGFR-II, EGFR-III, and EGFR-IV, and HER4-I, HER4-II, HER4-III, and HER4-IV
  • 100 ⁇ L of anti-6 ⁇ His-HRP antibody (Proteintech) diluted 1:10,000 in blocking buffer was added to each well, and the plate was incubated at room temperature for 1 hour.
  • the recognition epitope of hAb173 is located at D3 of the EGFR extracellular region
  • the recognition epitope of mAb1135 is located at the junction of D3 and D4 of the EGFR extracellular region
  • the recognition epitope of the positive control Cetuximab is located at D3 of the EGFR extracellular region.
  • the binding activity of chimeric antibody mAb1135 and fully human antibody hAb173 to members of the human ErbB/HER family was detected by ELISA.
  • the specific method is as follows: Recombinant proteins of the extracellular domains of human ErbB/HER family members (EGFR, HER2, HER3, and HER4) were diluted with PBS and added at 100 ⁇ L/well to a 96-well ELISA plate, incubated overnight at 4°C. After washing with PBS, 200 ⁇ L of blocking buffer (PBST containing 1% BSA) was added to each well, and the plate was incubated at room temperature for 1 hour.
  • PBST blocking buffer
  • OD450 values were then read using a multi-mode microplate reader. Analysis of the raw data using GraphPad Prism 9 software showed that mAb1135 and hAb173 specifically bound to EGFR, but showed no cross-binding with other members of the ErbB/HER family (data not shown).
  • the binding kinetics of chimeric antibody mAb1135 and fully human antibody hAb173 to recombinant EGFR protein were detected using a ForteBio molecular interaction analyzer. Cetuximab was used as the control antibody.
  • the specific method was as follows: The test antibody was diluted to 5 ⁇ g/mL with PBS, and 200 ⁇ L was added to each well of a 96-well black-walled plate. The recombinant EGFR protein was then diluted to 200 nM with PBS, and after a 1:2 serial dilution, 200 ⁇ L was added to each well of the 96-well black-walled plate.
  • the 96-well black-walled plate containing the test sample and the Protein A detection probe were placed in the ForteBio analyzer.
  • the program was set to allow the Protein A probe to capture the test antibody, followed by antibody-antigen binding/dissociation detection. After each binding/dissociation cycle, the probe was immersed in 10 mM glycine buffer (pH 1.5) to regenerate the probe before starting the next cycle.
  • analysis software was used to fit and calculate the binding constant ( ka ) and dissociation constant ( dis ) between the antibody and the antigen, and the affinity constant ( KD ) was also calculated.
  • the test results are shown in Table 3.
  • the binding affinity constant ( KD ) of mAb1135 and hAb173 to the EGFR recombinant protein was approximately ⁇ 1.5 ⁇ 10 ⁇ 8 M, which was not significantly different from that of Cetuximab.
  • Full R2 represents the similarity between the fitted curve and the measured curve.
  • the binding activity of the humanized antibodies to recombinant EGFR protein was detected using ELISA, with Cetuximab as the control antibody. The results showed that the binding activity of the humanized antibodies Hu1135-H1L1 and Hu1135-H3L1 to recombinant EGFR protein was significantly lower than that of the chimeric antibody mAb1135 (data not shown). Furthermore, following the method described in Section 1.4 of Example 1, the binding activity of the above-mentioned humanized antibodies to A431 cells was detected using flow cytometry. The results showed that although the humanized antibodies Hu1135-H1L1 and Hu1135-H3L1 retained their binding properties to A431 cells, their binding activity was lower than that of the chimeric antibody mAb1135 (data not shown).
  • antibody Hu1135-H1L1 was selected for amino acid sequence optimization.
  • a reversion mutation was performed on the heavy chain backbone region of Hu1135-H1L1.
  • Different reversion mutations were introduced into the expression vector of Hu1135-H1L1 using PCR (as shown in Table 4).
  • the light and heavy chain expression plasmids were transiently transfected into ExpiCHO-S cells to express the respective reversion mutant antibodies.
  • the antibodies in the culture supernatant were purified by Protein A affinity chromatography using the AKTA Pure system to obtain purified antibody protein.
  • the expression levels of the 94 mutant antibodies and their parent antibody Hu1135-H1L1-BM02 were detected using a ForteBio assay instrument.
  • the specific method is as follows: After transient transfection expression in ExpiCHO-S cells, the culture supernatant was harvested and added to 96-well black-walled plates at 200 ⁇ L/well. The 96-well black-walled plates containing the test samples and Protein A probes were placed in the ForteBio assay instrument. The binding rate of each antibody molecule to the Protein A probe in the culture supernatant was detected using a preset quantitative program. After each detection cycle, the probe was immersed in 10 mM glycine buffer (pH 1.5) to regenerate the probe before starting the next detection cycle. After the detection was completed, the expression level of each antibody was calculated using the workbench analysis software based on the standard curve.
  • L represents light chains
  • H represents heavy chains
  • N/A indicates not applicable
  • Full R2 represents the similarity between the fitted curve and the measured curve.
  • the mutant antibodies included BMO2-H03, BMO2-H04, BMO2-H11, BMO2-H12, BMO2-H17, BMO2-H29, BMO2-H32, BMO2-H35, and BMO2- H54, BM02-H55, BM02-L58, BM02-L59, BM02-L60, BM02-L61, BM02-L62, BM02-L63, BM02-L65, BM02-L71, BM02-L76, BM02-L77, BM02-L78, BM02-L79, BM02-L80, BM02-L82, BM02-L83, BM02-L84 and BM02-L85.
  • the nine preferred single-point mutations were combined and introduced into the variable region sequence of antibody Hu1135-H1L1-BM02 to construct antibody molecules containing different combinations of mutations (as shown in Table 6), and antibody samples were prepared according to the method described in item 2.2 of this embodiment.
  • the binding activity of the aforementioned anti-EGFR humanized antibodies BM02-H302-L202 and BM02-H303-L202 to 293T engineered cell lines expressing different abundances of EGFR (including 293T-EGFR#2 and 293T-EGFR#16-2D6) and their endocytosis efficiency were detected by flow cytometry.
  • Cetuximab was used as the control antibody.
  • the specific method was as follows: cells were collected after trypsin digestion and washed once with pre-chilled FACS buffer, then resuspended at 6–10 ⁇ 106 cells/mL.
  • both BM02-H302-L202 and BM02-H303-L202 were rapidly and efficiently endocytosed by the EGFR-overexpressing engineered cell line 293T-EGFR#2, with endocytosis efficiency comparable to Cetuximab, both exceeding 60%.
  • endocytosis efficiency comparable to Cetuximab, both exceeding 60%.
  • BM02-H302-L202 nor BM02-H303-L202 could effectively bind to EGFR on the cell surface or induce EGFR endocytosis, compared to the control antibody Cetuximab.
  • the exemplary antibody BM02-H302-L202 was constructed by introducing the L234F/L235E/P331S (EU Numbering) mutation (hereinafter referred to as "TM mutation") into the Fc region of the exemplary antibody BM02-H302-L202 to reduce its binding to Fc ⁇ Rs and C1q, thereby eliminating the Fc-mediated effector function.
  • TM mutation L234F/L235E/P331S (EU Numbering) mutation
  • A431 cells were collected by centrifugation, resuspended in ADCC detection buffer (RPMI-1640 medium containing 0.5% FBS), and added at 50 ⁇ L/well to a 96-well white-walled plate to achieve a target cell count of 1.0 ⁇ 104 in each well.
  • ADCC detection buffer RPMI-1640 medium containing 0.5% FBS
  • the antibody to be tested was serially diluted 5-fold from 100 nM using ADCC detection buffer, and 25 ⁇ L/well was added to the above 96-well plate. Effector cells were collected by centrifugation and resuspended in ADCC detection buffer. 25 ⁇ L/well was added to each well of the 96-well plate to ensure an effector cell count of 1.5 ⁇ 105 . The plate was then incubated overnight at 37°C with 5% CO2 . The next day, the plate was brought to room temperature for equilibration, and Bio-Lite Luciferase Reagent (Nanjing Novozymes) was added. The chemiluminescence values were read using a multi-mode microplate reader. The results are shown in Figure 11. Unlike the control antibody Cetuximab, the L202-H302TM antibody, which contains a TM mutation in the Fc region, did not exhibit ADCC activity.
  • the antigen-binding properties of the L202-H302TM antibody were verified by ELISA according to the method described in item 1.6 of Example 1.
  • the results showed that the L202-H302TM antibody retained the binding specificity of its parent antibody mAb1135, that is, it only binds to EGFR, and has no cross-binding activity with other members of the ErbB/HER family (data not shown).
  • the binding activity of the L202-H302TM antibody and the control antibody Cetuximab to cells expressing different levels of EGFR was detected by flow cytometry.
  • the expression levels of EGFR on the surface of each cell and the cell-binding activities of the antibodies are shown in Table 7 and Figure 12. Since the EGFR expression level of MCF-10A cells is comparable to that of normal skin tissue, cells with significantly higher EGFR expression levels than those of MCF-10A cells can be considered as EGFR overexpression (e.g., the MFI value measured by FACS is 10 times or higher than that of MCF-10A cells).
  • Antibody L202-H302TM exhibited significant binding activity to EGFR-overexpressing cell lines A431, HCC827, 293T-EGFR#2, FaDu, and JIMT-1. It showed weak binding to NCI-N87 cells, whose EGFR expression level was higher than MCF-10A cells but less than 10-fold (MFI value), and virtually no binding to 293T-EGFR#16-2D6 and MCF-10A cells. However, Cetuximab bound with strong activity to all of the aforementioned cell lines, regardless of the abundance of EGFR expression on the cell surface.
  • Immunohistochemistry was used to detect the binding activity of the anti-EGFR antibody L202-H302TM with normal human tissues and human lung cancer tissues.
  • the frozen section microarrays of normal human tissues containing 30 different normal tissues, each with 3 different subjects; #T6234701-1) and the frozen section microarrays of human lung cancer tissues (#T6235152-5) were both derived from BioChain (Newark, CA, USA).
  • the positive control antibody used was Cetuximab, and the negative control antibody was an IgG isotype antibody (SouthernBiotech, #0151K-28). Staining and detection were performed using the Biocare IntelliPATH automated staining platform and standard methods.
  • the IHC results are shown in Figure 13.
  • the positive control antibody Cetuximab showed strong positive staining of the cell membrane and cytoplasm in normal human epidermal tissue;
  • L202-H302TM showed only weak positive staining of the cytoplasm in epidermal tissue, with no staining on the cell membrane;
  • the negative control antibody IgG isotype antibody
  • Both L202-H302TM and the positive control Cetuximab showed strong positive staining of the cell membrane and cytoplasm in EGFR-overexpressing lung cancer tissue; the negative control antibody (IgG isotype antibody) showed no staining in lung cancer epithelial cells, only limited mild nonspecific staining in the mesenchymal matrix.
  • the IHC results indicate that the L202-H302TM antibody can selectively bind to EGFR-overexpressing lung cancer cells, but not to any normal tissue (including skin tissue).
  • this embodiment designed a series of mutations targeting the D3-D4 junction of the EGFR extracellular domain to construct antigen mutants. These mutations involved replacing amino acid residues at positions 447-508 of the EGFR extracellular domain with corresponding amino acid residues from HER4.
  • the specific method is as follows: Using an expression plasmid expressing the EGFR extracellular domain protein as a template, amino acid mutations as shown in Table 8 were performed by PCR to construct EGFR mutant protein expression plasmids containing different amino acid mutations at the D3-D4 junction of the EGFR extracellular domain.
  • the binding activity of anti-EGFR antibodies L202-H302TM and hAb173 to antigen mutant proteins containing different mutations at the D3 and D4 junctions of the extracellular region of EGFR was detected by ELISA. Cetuximab was used as the control antibody.
  • the specific method was as follows: EGFR and each antigen mutant protein were diluted with PBS, and 100 ⁇ L was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. After washing with PBS, 200 ⁇ L of blocking buffer (PBST containing 1% BSA) was added to each well, and the plate was incubated at room temperature for 1 hour.
  • PBST blocking buffer
  • amino acid mutations in the EGFR extracellular domain peptides at positions 447-462, 486-491, and positions 463 and 465 in the 462-469 peptide domain resulted in a decrease of at least 50% in the binding activity of antibody L202-H302TM with EGFR.
  • Amino acid mutations in the EGFR extracellular domain peptides at positions 452-456, 469-482, and position 467 in the 462-469 peptide domain resulted in a decrease of at least 50% in the binding activity of antibody hAb173 with EGFR.
  • the antigen-binding epitope of antibody L202-H302TM includes amino acid sequences 447-462, 463, 465, and 486-491 of the EGFR extracellular domain; while the antigen-binding epitope of antibody hAb173 includes amino acid sequences 452-456, 467, and 469-482 of the EGFR extracellular domain.
  • the peptide segment at positions 462-469 of the EGFR extracellular domain also contains key amino acids affecting the binding of Cetuximab, the contact site differs from that of L202-H302TM and hAb173, as shown in Figure 14.
  • the key binding site for Cetuximab in this peptide segment is serine residue 468 (S468).
  • the anti-EGFR antibody L202-H302TM can specifically recognize the EGFR extracellular domain epitope of amino acid residues 447-491
  • the EGFR (447-462)/HER4 chimeric antigen peptide named HER4-15
  • the EGFR (447-491)/HER4 chimeric antigen peptide named HER4-45
  • the binding affinity of antibody L202-H302TM to HER4-15 and HER4-45 chimeric antigen peptides was detected according to the method described in section 1.7 of Example 1, wherein the control antigens included recombinant proteins containing the extracellular domains of EGFR and HER4.
  • the results are shown in Table 9.
  • the binding affinity of the HER4-15 chimeric antigen peptide to L202-H302TM was approximately 1.5 ⁇ 10 ⁇ 6 M. Although this is an order of magnitude lower than that of the EGFR extracellular domain protein, it still showed significant binding activity to antibody L202-H302TM relative to HER4, indicating that the mutated amino acid sequence it contains can directly or indirectly participate in the interaction with the L202-H302TM antibody.
  • the binding affinity of L202-H302TM to the HER4-45 chimeric antigen peptide was approximately 2 ⁇ 10 ⁇ 7 M, comparable to its binding affinity to EGFR.
  • the antigen-binding epitope of the anti-EGFR antibody L202-H302TM is the amino acid sequence of EGFR from position 447 to 491, and the specific amino acid sequence is: YANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGC (SEQ ID NO: 107).
  • Full R- squared represents the similarity between the fitted curve and the measured curve.
  • Example 4 Study and verification of the correlation between antigen-binding epitopes of anti-EGFR antibodies and their cell-binding activity.
  • a mouse immune library as described in section 1.1 of Example 1 was panned to screen for antibodies that specifically recognize the epitopes of amino acid residues 447-491 of the EGFR extracellular domain.
  • the specific steps are as follows: In the first and second rounds of panning, HER4 extracellular domain recombinant protein was added to the immune library at a final concentration of 20 ⁇ g/mL, and then coated with EGFR extracellular domain recombinant protein for panning to enrich anti-EGFR antibodies.
  • HER4-45 chimeric antigen or HER4-15 chimeric antigen was coated in 96-well plates, and HER4 extracellular domain recombinant protein was added to the immune library at a final concentration of 20 ⁇ g/mL to enrich antibodies that specifically recognize the epitopes of the EGFR extracellular domain.
  • HER4-45 chimeric antigen or HER4-15 chimeric antigen were coated in 96-well plates for monoclonal screening and sequencing analysis, ultimately yielding mouse anti-human EGFR antibodies E45-mab304 and E45-mab309 that recognize the epitopes of amino acid residues 447-491 of the EGFR extracellular domain.
  • the antigen-binding epitope of antibody E45-mab304 contains amino acid residue 465 and amino acid sequence 469-477 of the EGFR extracellular domain
  • the antigen-binding epitope of antibody E45-mab309 contains amino acid sequence 447-462, amino acid residue 465, and amino acid sequence 486-491 of the EGFR extracellular domain.
  • PI3K/AKT is a key signaling molecule in the downstream signal transduction pathway mediated by EGFR. Detecting AKT phosphorylation levels can assess the impact of antibodies specifically binding to the EGFR extracellular domain epitopes (amino acid residues 447-491) on the EGFR signal transduction pathway.
  • the specific method is as follows: A431 cells were seeded into 6-well plates. After the cells reached approximately 80% confluence, the culture medium was discarded, and serum-free DMEM was added for overnight starvation.
  • test antibody including L202-H302TM, E45-mab304, or E45-mab309
  • the positive control antibody Cetuximab were added to each well to a final concentration of 10 ⁇ g/mL.
  • EGF NOVUS
  • the wells were washed with pre-chilled PBS, and then 150 ⁇ L/well of RIPA cell lysis buffer (Porteintech) was added for total protein extraction. Cell lysates from each well were collected in 1.5-mL EP tubes and centrifuged to obtain the supernatant.
  • Protein concentration in each lysate was determined using BCA, and protein electrophoresis samples were prepared using reducing buffer, followed by SDS-PAGE gel electrophoresis. After electrophoresis, protein bands were transferred to a PVDF membrane, which was then blocked with 5% skim milk at room temperature. Rabbit anti-p-AKT (Ser465) antibody (CST) or rabbit anti-pan-AKT antibody (CST) was added at a 1:1000 dilution, and the membrane was incubated overnight at 4°C. After washing the PVDF membrane with PBST, goat anti-rabbit IgG-HRP antibody (Jacskon Immuno) diluted 1:5000 in 5% skim milk was added, and the membrane was incubated at room temperature for 1 hour.
  • Anti-EGFR antibody L202-H302TM was conjugated to a small molecule toxin compound (e.g., eribulin) via cleavable linkers (including Mal-(PEG) 2 -GGFG linkers and 4-(5-thio-1,3,4-oxadiazol- 2 -yl)benzamido-(PEG)2-GGFG linkers) to prepare anti-EGFR ADCs (named L202-H302TM-ADC and EGFR-BP182p-ADC, respectively).
  • the eribulin was prepared according to the methods described in ZL201910197071.8 or ZL201910509222.9, and WO1999065894.
  • the preparation method of Mal-(PEG) 2- GGFG-Eribulin drug payload is as follows: Appropriate amounts of NH2- (PEG) 2- COOH and maleic anhydride are added to acetic acid, heated under reflux overnight, and the acetic acid is removed by rotary evaporation. The residue is then prepared by HPLC and lyophilized to obtain Mal-(PEG) 2 -COOH. Using 2Cl Trt Resin as the solid-phase support, Fmoc protection is removed with 20% piperidine/DMF (v/v) solution.
  • Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, and Fmoc-Gly-OH are sequentially coupled.
  • Fmoc-GGFG-OH and eribulin were placed in a single-necked flask, dissolved in an appropriate amount of DMF, and cooled to 0°C.
  • L202-H302TM-ADC The preparation method of L202-H302TM-ADC is as follows: TCEP (6 mM) was slowly added to a solution of anti-EGFR antibody L202-H302TM (2 mM) under an ice-water bath (antibody preparation buffer consisted of 50 mM Histidine-HCl and 8% sucrose, pH 5.5). The final reaction concentrations of TCEP and L202-H302TM antibody were 0.15 mM and 0.05 mM, respectively. After mixing, the mixture was shaken at 25°C for approximately 1.5 hours for reduction.
  • the drug-antibody ratio (DAR) and heterogeneity of the prepared ADC were analyzed using HIC-HPLC (BioCore HIC-Butyl 5 ⁇ m/4.6 ⁇ 100 mm, NanoChrom), and the purity of the ADC was analyzed using SEC-HPLC (Zenix-C SEC-300, Cyfineon Technologies).
  • the 4-(5-methylsulfonyl-1,3,4-oxadiazol-2-yl)benzamido-(PEG) 2 -GGFG-Eribulin drug payload molecule was provided by Borui Biopharmaceutical (Suzhou) Co., Ltd.
  • the preparation method of EGFR-BP182p-ADC is as follows: ZnCl2 (3.0mM) and TCEP (6.0mM) were slowly added to the anti-EGFR antibody L202-H302TM solution (1.5mM) under ice-water bath (antibody preparation buffer was 50mM Histidine-HCl, 8% sucrose and 0.05% PS80, pH 5.5). The final concentrations of ZnCl2 , TCEP and L202-H302TM antibody were 0.10mM, 0.20mM and 0.05mM, respectively. After mixing, the mixture was shaken at 10°C for about 16 hours for reduction.
  • the average DAR value of the prepared ADC is calculated using the following formula:
  • the average DAR value is calculated as follows: [AUC +1 + 2(AUC +2 ) + 3(AUC +3 ) + ... + n(AUC +n )] / ⁇ AUC_total ]; where AUC +1 is the area under the curve (AUC) corresponding to the ADC coupled with one small molecule toxin compound, AUC +2 is the AUC corresponding to the ADC coupled with two small molecule toxin compounds, and so on. ⁇ AUC_total is the AUC of all peaks.
  • the purity of the ADCs prepared in this example is >95%, with the average DAR value of L202-H302TM-ADC being approximately 4.5 and the average DAR value of EGFR-BP182p-ADC being approximately 4.0.
  • L202-H302TM-ADC binding activity and specificity of L202-H302TM-ADC with EGFR were detected using ELISA.
  • the antigens tested included recombinant proteins of the extracellular domains of EGFR, HER2, HER3, and HER4.
  • the control antibodies included the corresponding naked antibodies (L202-H302TM) and Cetuximab.
  • the results are shown in Figure 19.
  • the binding activity of L202-H302TM-ADC with EGFR was comparable to that of its corresponding naked antibody L202-H302TM.
  • L202-H302TM-ADC showed no cross-binding activity with other members of the ErbB/HER family, indicating that the anti-EGFR ADC maintained the antigen-binding activity and specificity of the corresponding naked antibody.
  • a series of cell lines expressing different levels of EGFR were selected to evaluate the in vitro cytotoxic activity of the anti-EGFR ADC.
  • the EGFR expression abundance of each selected cell line is shown in Table 7.
  • the cells with EGFR expression abundance from high to low are A431, HCC827, 293T-EGFR#2, FaDu, JIMT-1, NCI-N87, 293T-EGFR#16-2D6 and MCF-10A.
  • the EGFR expression level on the surface of 293T-EGFR#16-2D6 and MCF-10A cells is comparable to that of normal skin tissue, while the EGFR expression abundance of A431, HCC827, 293T-EGFR#2, FaDu and JIMT-1 cells is significantly higher than that of 293T-EGFR#16-2D6 and MCF-10A cells, and therefore they are considered to be EGFR overexpressing cells.
  • the specific method for detecting the in vitro cytotoxic activity of anti-EGFR ADCs is as follows: Cell lines were collected by trypsin digestion, resuspended in medium containing 10% FBS, and seeded into 96-well white-walled plates at 5000 or 10000 cells per well. The plates were incubated at 37°C/5% CO2 until complete cell adhesion. Serially diluted ADCs or the small molecule toxin compound eribulin were added. Depending on the growth rate of each cell line, the plates were incubated at 37°C/5% CO2 for 3–5 days.
  • the plates were then removed from the incubator and allowed to equilibrate to room temperature.
  • CellCount Lite 2.0 reagent was added, and the mixture was shaken for 2–5 minutes to allow for complete cell lysis.
  • the chemiluminescence values were read using a multi-mode microplate reader. The obtained data were analyzed using GraphPad Prism 9 software. The detection results were expressed as a relative percentage of the chemiluminescence values of the corresponding control wells, wherein the cells in the corresponding control wells were not treated with ADC or small molecule toxins during the experiment.
  • the ADC showed no killing activity, while the small molecule toxin compound Eribulin had the same killing activity against the engineered cell lines 293T-EGFR#16-2D6 and 293T-EGFR#2 derived from the same parent cell line, regardless of their EGFR expression levels.
  • the bystander killing effect of L202-H302TM-ADC was detected by co-culturing EGFR-overexpressing human breast cancer cell line MDA-MB-468 and EGFR-non-expressing Jurkat cell line.
  • the specific method was as follows: MDA-MB-468 cells and GFP-expressing Jurkat cells were collected and individually or jointly inoculated into 24-well cell culture plates at a final concentration of 1 ⁇ 105 cells/well.
  • a blank control group i.e., RPMI-1640 medium only, designated Medium
  • L202-H302TM-ADC Under monoculture, L202-H302TM-ADC showed significant killing activity against MDA-MB-468 cells, but almost no killing effect on Jurkat cells. However, under co-culture of the two cell types, L202-H302TM-ADC caused significant killing effects on both MDA-MB-468 and Jurkat cells. In summary, the experimental results indicate that L202-H302TM-ADC has a bystander killing effect.
  • Example 6 Detection of anti-EGFR ADC anti-tumor activity in a mouse subcutaneous xenograft model
  • This embodiment tested the antitumor activity of L202-H302TM-ADC in two mouse subcutaneous xenograft tumor models constructed based on human tumor cell lines: an EGFR-overexpressing A431 skin cancer tumor model and an EGFR-overexpressing JIMT-1 breast cancer tumor model.
  • the specific method is as follows: After the cell lines (A431 and JIMT-1) reached the logarithmic growth phase, the cells were collected and seeded subcutaneously into the right forelimb dorsal region of immunodeficient mice (BALB/c Nude or SCID Beige) at a density of 5 ⁇ 106 cells per mouse.

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Abstract

La présente invention concerne un anticorps anti-EGFR ou un fragment de liaison à l'antigène de celui-ci, un conjugué anticorps anti-EGFR-médicament (ADC) construit à partir de celui-ci, et une composition pharmaceutique et un kit le comprenant. La présente invention concerne en outre une utilisation de l'anticorps, de l'ADC, de la composition pharmaceutique et du kit pour le traitement de maladies associées à une expression anormale de l'EGFR.
PCT/CN2025/095291 2024-05-16 2025-05-16 Nouvel anticorps anti-egfr, conjugué de médicament et son utilisation Pending WO2025237392A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101029082A (zh) * 2006-03-03 2007-09-05 广州枫岛医药科技有限公司 一种人源化抗egfr单克隆抗体的重组制备方法
CN101233155A (zh) * 2004-03-19 2008-07-30 伊姆克罗尼系统公司 人抗表皮生长因子受体抗体
EP2332990A1 (fr) * 2004-03-19 2011-06-15 Imclone LLC Anticorps de récepteur de facteur de croissance anti-épidermique humain
AU2011357568A1 (en) * 2011-01-27 2013-08-15 Hefei Tairui Biotechnology Co., Ltd Humanized anti-EGFR antibody L4-H3 and coding gene thereof
CN116333120A (zh) * 2021-12-16 2023-06-27 徕特康(苏州)生物制药有限公司 抗表皮生长因子受体抗体及其制备方法和用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101233155A (zh) * 2004-03-19 2008-07-30 伊姆克罗尼系统公司 人抗表皮生长因子受体抗体
EP2332990A1 (fr) * 2004-03-19 2011-06-15 Imclone LLC Anticorps de récepteur de facteur de croissance anti-épidermique humain
CN101029082A (zh) * 2006-03-03 2007-09-05 广州枫岛医药科技有限公司 一种人源化抗egfr单克隆抗体的重组制备方法
AU2011357568A1 (en) * 2011-01-27 2013-08-15 Hefei Tairui Biotechnology Co., Ltd Humanized anti-EGFR antibody L4-H3 and coding gene thereof
CN116333120A (zh) * 2021-12-16 2023-06-27 徕特康(苏州)生物制药有限公司 抗表皮生长因子受体抗体及其制备方法和用途

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