WO2021160138A1 - Protéine de liaison à l'antigène de l'anti-récepteur du facteur de croissance épidermique et son application - Google Patents

Protéine de liaison à l'antigène de l'anti-récepteur du facteur de croissance épidermique et son application Download PDF

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WO2021160138A1
WO2021160138A1 PCT/CN2021/076393 CN2021076393W WO2021160138A1 WO 2021160138 A1 WO2021160138 A1 WO 2021160138A1 CN 2021076393 W CN2021076393 W CN 2021076393W WO 2021160138 A1 WO2021160138 A1 WO 2021160138A1
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seq
amino acid
antigen binding
binding protein
acid sequence
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PCT/CN2021/076393
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English (en)
Chinese (zh)
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孙非
张喜田
梁重阳
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张喜田
孙非
张欣
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Publication of WO2021160138A1 publication Critical patent/WO2021160138A1/fr

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    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL

Definitions

  • This application relates to the field of biomedicine, in particular to an anti-epidermal growth factor receptor antigen binding protein. In addition, this application also relates to the preparation method and use of the antibody.
  • Growth factor receptors involved in carcinogenesis include epidermal growth factor receptor (Epidermal Growth Factor Receptor, EGFR), platelet-derived growth factor receptor (PDGFR), insulin-like growth factor receptor (IGFG), nerve growth factor receptor (NGFR) and fibroblast growth factor receptor (FGF).
  • EGFR epidermal growth factor receptor
  • PDGFR platelet-derived growth factor receptor
  • IGFG insulin-like growth factor receptor
  • NGFR nerve growth factor receptor
  • FGF fibroblast growth factor receptor
  • the high expression of EGFR may promote tumor cell proliferation, angiogenesis, adhesion, invasion and metastasis, inhibit tumor cell apoptosis, and lead to low survival rate of tumor patients, poor prognosis, poor curative effect, high possibility of tumor metastasis, and easy to cause tumor cells Resistance to various cytotoxic drugs.
  • the currently marketed EGFR inhibitors are mainly small molecules and antibody drugs. They target the EGF binding site of the extracellular part of EGFR or the active site of tyrosine kinase in the intracellular part of EGFR.
  • EGFR inhibitors target the EGF binding site of the extracellular part of EGFR or the active site of tyrosine kinase in the intracellular part of EGFR.
  • problems of strong toxic and side effects and easy development of drug resistance Therefore, there is an urgent need to develop highly efficient and novel EGFR antibodies.
  • the present application provides an isolated antigen binding protein, which can specifically bind to the epidermal growth factor receptor (EGFR) on the cell membrane to internalize the complex and enter the cell.
  • EGFR epidermal growth factor receptor
  • the present application provides an isolated antigen binding protein, which can specifically bind to the epidermal growth factor receptor (EGFR) on the cell membrane to internalize the complex.
  • EGFR epidermal growth factor receptor
  • the isolated antigen binding protein can specifically bind to the II domain of EGFR, and the II domain of EGFR comprises the amino acid sequence shown in SEQ ID NO: 13.
  • the isolated antigen binding protein can specifically bind to at least one amino acid selected from the group consisting of Ser196, Ser222, Lys269 and Ser282 on the II domain of EGFR.
  • the isolated antigen binding protein can competitively bind to the EGFR with the LZ-8 protein.
  • the isolated antigen binding protein can compete with a reference antibody for binding to the EGFR, wherein the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL, and the reference
  • the VH of the antibody includes HCDR1, HCDR2, and HCDR3, the VL of the reference antibody includes LCDR1, LCDR2, and LCDR3, and the HCDR1 of the reference antibody includes SEQ ID NO: 15, SEQ ID NO: 29, and SEQ ID NO: 43
  • the amino acid sequence shown in any one of the HCDR2 of the reference antibody comprises the amino acid sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 30 and SEQ ID NO: 44, the reference antibody
  • the HCDR3 of the reference antibody includes the amino acid sequence shown in any one of SEQ ID NO: 17, SEQ ID NO: 31 and SEQ ID NO: 45, and the LCDR1 of the reference antibody includes SEQ ID NO: 18, SEQ ID NO: 32 and The amino acid sequence shown in any one of SEQ ID NO: 46, the LCDR
  • the isolated antigen binding protein includes an antibody or antigen binding fragment thereof.
  • the antigen-binding fragment includes Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
  • the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the antibody may be a monoclonal antibody.
  • the isolated antigen binding protein comprises at least one CDR in a heavy chain variable region VH, and the VH comprises any of SEQ ID NO: 68, SEQ ID NO: 72 and SEQ ID NO: 76 The amino acid sequence shown in one item.
  • the isolated antigen binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 17, SEQ ID NO: 31, and SEQ ID NO: 45.
  • the isolated antigen binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 30, and SEQ ID NO: 44.
  • the isolated antigen binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 15, SEQ ID NO: 29, and SEQ ID NO: 43.
  • the isolated antigen binding protein includes HCDR1, HCDR2, and HCDR3, and the HCDR1 includes any one of SEQ ID NO: 15, SEQ ID NO: 29, and SEQ ID NO: 43.
  • the isolated antigen binding protein comprises HCDR1, HCDR2, and HCDR3, and the HCDR1, HCDR2, and HCDR3 are selected from any of the following amino acid sequences:
  • HCDR1 SEQ ID NO: 15, HCDR2: SEQ ID NO: 16 and HCDR3: SEQ ID NO: 17;
  • HCDR1 SEQ ID NO: 29, HCDR2: SEQ ID NO: 30 and HCDR3: SEQ ID NO: 31;
  • HCDR1 SEQ ID NO: 43
  • HCDR2 SEQ ID NO: 44
  • HCDR3 SEQ ID NO: 45.
  • the isolated antigen binding protein comprises a heavy chain variable region VH, wherein the VH includes a framework region H-FR1, and the C-terminus of the H-FR1 is directly or directly from the N-terminus of the HCDR1.
  • VH heavy chain variable region
  • H-FR1 framework region
  • the H-FR1 includes the amino acid sequence shown in any one of SEQ ID NO: 21, SEQ ID NO: 35 and SEQ ID NO: 49.
  • the isolated antigen binding protein comprises a heavy chain variable region VH, wherein the VH includes a framework region H-FR2, and the H-FR2 is located between the HCDR1 and the HCDR2, and
  • the H-FR2 includes the amino acid sequence shown in any one of SEQ ID NO: 22, SEQ ID NO: 36 and SEQ ID NO: 50.
  • the isolated antigen binding protein comprises a heavy chain variable region VH, wherein the VH includes a framework region H-FR3, and the H-FR3 is located between the HCDR2 and the HCDR3, and
  • the H-FR3 includes the amino acid sequence shown in any one of SEQ ID NO: 23, SEQ ID NO: 37 and SEQ ID NO: 51.
  • the isolated antigen binding protein comprises a heavy chain variable region VH, wherein the VH includes a framework region H-FR4, and the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, And the H-FR4 includes the amino acid sequence shown in any one of SEQ ID NO: 24, SEQ ID NO: 38, and SEQ ID NO: 52.
  • the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3, and H-FR4, wherein the H-FR1 comprises SEQ ID NO: 21, SEQ ID NO: 35 and The amino acid sequence shown in any one of SEQ ID NO: 49, and the H-FR2 includes the amino acid sequence shown in any one of SEQ ID NO: 22, SEQ ID NO: 36 and SEQ ID NO: 50, the H-FR3 includes the amino acid sequence shown in any one of SEQ ID NO: 23, SEQ ID NO: 37 and SEQ ID NO: 51, and the H-FR4 includes SEQ ID NO: 24, SEQ ID NO: 38 and SEQ ID NO: the amino acid sequence shown in any one of 52.
  • the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3, and H-FR4, and the H-FR1, H-FR2, H-FR3, and H-FR4 are selected from From any of the following amino acid sequences:
  • F-FR1 SEQ ID NO: 21, H-FR2: SEQ ID NO: 22, H-FR3: SEQ ID NO: 23 and H-FR4: SEQ ID NO: 24;
  • H-FR1 SEQ ID NO: 35
  • H-FR2 SEQ ID NO: 36
  • H-FR3 SEQ ID NO: 37
  • H-FR4 SEQ ID NO: 38;
  • H-FR1 SEQ ID NO: 49
  • H-FR2 SEQ ID NO: 50
  • H-FR3 SEQ ID NO: 51
  • H-FR4 SEQ ID NO: 52.
  • the isolated antigen binding protein comprises a heavy chain variable region VH, and the heavy chain variable region VH comprises any of SEQ ID NO: 68, SEQ ID NO: 72 and SEQ ID NO: 76 The amino acid sequence shown in one item.
  • the isolated antigen binding protein includes a heavy chain constant region, and the heavy chain constant region is derived from IgG1.
  • the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 57 or SEQ ID NO: 58.
  • the isolated antigen binding protein comprises an antibody heavy chain
  • the antibody heavy chain comprises the one shown in any one of SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64 Amino acid sequence.
  • the isolated antigen binding protein comprises at least one CDR in the light chain variable region VL, and the VL comprises any of SEQ ID NO: 69, SEQ ID NO: 73 and SEQ ID NO: 77 The amino acid sequence shown in one item.
  • the isolated antigen binding protein comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 20, SEQ ID NO: 34, and SEQ ID NO: 48.
  • the isolated antigen binding protein comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 19, SEQ ID NO: 33, and SEQ ID NO: 47.
  • the isolated antigen binding protein comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 18, SEQ ID NO: 32, and SEQ ID NO: 46.
  • the isolated antigen binding protein includes LCDR1, LCDR2, and LCDR3, and the LCDR1 includes SEQ ID NO: 18, SEQ ID NO: 32, and SEQ ID NO: 46.
  • An amino acid sequence the LCDR2 includes the amino acid sequence shown in any one of SEQ ID NO: 19, SEQ ID NO: 33 and SEQ ID NO: 47, and the LCDR3 includes SEQ ID NO: 20, SEQ ID NO: 34 and The amino acid sequence shown in any one of SEQ ID NO: 48.
  • the isolated antigen binding protein comprises LCDR1, LCDR2, and LCDR3, and the LCDR1, LCDR2, and LCDR3 are selected from any of the following amino acid sequences:
  • LCDR1 SEQ ID NO: 18, LCDR2: SEQ ID NO: 19 and LCDR3: SEQ ID NO: 20;
  • LCDR1 SEQ ID NO: 32
  • LCDR2 SEQ ID NO: 33
  • LCDR3 SEQ ID NO: 34;
  • LCDR1 SEQ ID NO: 46
  • LCDR2 SEQ ID NO: 47
  • LCDR3 SEQ ID NO: 48.
  • the isolated antigen binding protein comprises a light chain variable region VL, wherein the VL includes a framework region L-FR1, the C-terminus of the L-FR1 and the N-terminus of the LCDR1 are directly or Are connected indirectly, and the L-FR1 includes the amino acid sequence shown in any one of SEQ ID NO: 25, SEQ ID NO: 39 and SEQ ID NO: 53.
  • the isolated antigen binding protein comprises a light chain variable region VL, wherein the VL comprises a framework region L-FR2, and the L-FR2 is located between the LCDR1 and the LCDR2, and
  • the L-FR2 includes the amino acid sequence shown in any one of SEQ ID NO: 26, SEQ ID NO: 40, and SEQ ID NO: 54.
  • the isolated antigen binding protein comprises a light chain variable region VL, wherein the VL comprises a framework region L-FR3, and the L-FR3 is located between the LCDR2 and the LCDR3, and
  • the L-FR3 includes the amino acid sequence shown in any one of SEQ ID NO: 27, SEQ ID NO: 41 and SEQ ID NO: 55.
  • the isolated antigen binding protein comprises a light chain variable region VL, wherein the VL comprises a framework region L-FR4, and the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, And the L-FR4 includes the amino acid sequence shown in any one of SEQ ID NO: 28, SEQ ID NO: 42 and SEQ ID NO: 56.
  • the isolated antigen binding protein comprises L-FR1, L-FR2, L-FR3, and L-FR4, wherein the L-FR1 comprises SEQ ID NO: 25, SEQ ID NO: 39 and The amino acid sequence shown in any one of SEQ ID NO: 53, the L-FR2 includes the amino acid sequence shown in any one of SEQ ID NO: 26, SEQ ID NO: 40 and SEQ ID NO: 54, L-FR3 includes the amino acid sequence shown in any one of SEQ ID NO: 27, SEQ ID NO: 41 and SEQ ID NO: 55, and the L-FR4 includes SEQ ID NO: 28, SEQ ID NO: 42 and SEQ The amino acid sequence shown in any one of ID NO:56.
  • the L-FR1 comprises SEQ ID NO: 25, SEQ ID NO: 39 and The amino acid sequence shown in any one of SEQ ID NO: 53
  • the L-FR2 includes the amino acid sequence shown in any one of SEQ ID NO: 26, SEQ ID NO: 40 and SEQ ID NO: 54
  • L-FR3 includes the amino acid sequence shown in any one of
  • the isolated antigen binding protein comprises L-FR1, L-FR2, L-FR3, and L-FR4, and the L-FR1, L-FR2, L-FR3, and L-FR4 are selected from From any of the following amino acid sequences:
  • F-FR1 SEQ ID NO: 39
  • L-FR2 SEQ ID NO: 40
  • L-FR3 SEQ ID NO: 41
  • L-FR4 SEQ ID NO: 42;
  • L-FR1 SEQ ID NO: 53
  • L-FR2 SEQ ID NO: 54
  • L-FR3 SEQ ID NO: 55
  • L-FR4 SEQ ID NO: 56;
  • L-FR1 SEQ ID NO: 25
  • L-FR2 SEQ ID NO: 26
  • L-FR3 SEQ ID NO: 27
  • L-FR4 SEQ ID NO: 28.
  • the isolated antigen binding protein comprises a light chain variable region VL, and the light chain variable region VL comprises SEQ ID NO: 69, SEQ ID NO: 73 and SEQ ID NO: 77 The amino acid sequence shown in any item.
  • the isolated antigen binding protein includes a light chain constant region, and the light chain constant region includes the amino acid sequence shown in SEQ ID NO:59.
  • the isolated antigen-binding protein comprises an antibody light chain
  • the antibody light chain comprises any one of SEQ ID NO: 61, SEQ ID NO: 63, and SEQ ID NO: 65 Amino acid sequence.
  • the application provides a polypeptide comprising the isolated antigen binding protein.
  • the application provides an immunoconjugate, which comprises the isolated antigen binding protein or the polypeptide, and a small molecule compound.
  • the small molecule compound contains a cytotoxic agent and/or a marker.
  • the small molecule compound is coupled to the primary amine group of the upper amino acid residue of the isolated antigen binding protein.
  • the immunoconjugate is capable of internalizing cells.
  • the application provides isolated one or more nucleic acid molecules, which encode the isolated antigen binding protein.
  • the nucleic acid molecule includes SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO : 67, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 74, and SEQ ID NO: 75.
  • the application provides a vector, which comprises the nucleic acid molecule.
  • the application provides a cell, which contains the nucleic acid molecule or is based on the vector.
  • the present application provides a method for preparing the isolated antigen binding protein, the method comprising culturing the cell under conditions such that the isolated antigen binding protein is expressed.
  • the application provides a pharmaceutical composition comprising the isolated antigen binding protein, the polypeptide, the immunoconjugate, the nucleic acid molecule, the carrier and/or the cell, and optionally ⁇ pharmaceutically acceptable carrier.
  • the application provides that the isolated antigen binding protein, the polypeptide, the immunoconjugate, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition are used in the preparation of a medicine
  • the medicine is used to prevent, alleviate and/or treat diseases related to EGFR activity.
  • the disease associated with EGFR activity includes tumors.
  • the tumor includes a tumor associated with abnormal expression of EGFR.
  • the tumors related to abnormal expression of EGFR include tumors related to overexpression of EGFR.
  • the tumor comprises a solid tumor.
  • the tumor includes glioma, breast cancer, lung cancer, ovarian cancer, head and neck cancer, cervical cancer, esophageal cancer, prostate cancer, liver cancer, colon cancer, and/or gastric cancer.
  • the present application provides a method of preventing, alleviating or treating diseases related to EGFR activity, the method comprising administering the isolated antigen binding protein, the polypeptide, the immunoconjugate to a subject in need Substance, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition.
  • the disease associated with EGFR activity includes tumors.
  • the tumor includes a tumor associated with abnormal expression of EGFR.
  • the tumors related to abnormal expression of EGFR include tumors related to overexpression of EGFR.
  • the tumor comprises a solid tumor.
  • the tumor includes glioma, breast cancer, lung cancer, ovarian cancer, head and neck cancer, cervical cancer, esophageal cancer, prostate cancer, liver cancer, colon cancer, and/or gastric cancer.
  • the application provides the isolated antigen binding protein, the polypeptide, the immunoconjugate, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition for preventing , Relieve or treat diseases related to EGFR activity.
  • the disease associated with EGFR activity includes tumors.
  • the tumor includes a tumor associated with abnormal expression of EGFR.
  • the tumors related to abnormal expression of EGFR include tumors related to overexpression of EGFR.
  • the tumor comprises a solid tumor.
  • the tumor includes glioma, breast cancer, lung cancer, ovarian cancer, head and neck cancer, cervical cancer, esophageal cancer, prostate cancer, liver cancer, colon cancer, and/or gastric cancer.
  • the present application provides a method for detecting the presence and/or content of EGFR, which comprises administering the isolated antigen binding protein or the polypeptide.
  • the application provides a kit comprising the isolated antigen binding protein, the polypeptide, the immunoconjugate, the nucleic acid molecule, the carrier, the cell and/or the drug combination Things.
  • the application provides a method for internalizing a cell, which comprises administering the isolated antigen binding protein, the polypeptide, the immunoconjugate, the nucleic acid molecule, the vector, the cell And/or the pharmaceutical composition.
  • Figure 1 shows the protein electrophoresis and western blotting of the cell supernatant 5 days after transfection (lane 1: reducing conditions, lane 2: non-reducing conditions, lane P: human IgG1 as a positive control).
  • FIG. 2 shows the results of an isothermal calorimetric titration experiment (ITC) for the extracellular domain of EGFR and LZ8.
  • ITC isothermal calorimetric titration experiment
  • Figure 3 shows the separation of the EGFR extracellular domain and LZ8 complex using SDS-PAGE.
  • Figure 4 shows the determination of the interaction interface between EGFR and LZ8 using chemical cross-linking coupled mass spectrometry, and the amino acid residues involved in the interaction are represented by spheres.
  • Figure 5 shows the optimal docking configuration of the extracellular domain of EGFR and LZ8.
  • the key amino acid residues are labeled D20, K41 and S222, K269, S282, and S196 respectively.
  • Figure 6 shows the structure of Alexa Fluor 568 NHS Ester.
  • Figure 7 shows the purification of the 1Z4-AF568 conjugate.
  • Figure 8 shows the internalization of the 1Z4-AF568 conjugate.
  • the ellipse on the left is the nucleus, and the vesicle structure formed by the conjugate is shown on the right.
  • Figure 9 shows the flow cytometry analysis of the binding of 1Z4 to EGFR positive and negative cells.
  • the cells corresponding to 92.8 and 99.2 are MD-MB-453, and the cells corresponding to 9660 and 17420 are MD-MB-468.
  • Figure 10 shows the comparative analysis of the internalization intensity of the antigen binding protein 1Z4 described in this application, cetuximab and mAb806, and the oval shape is the nucleus.
  • Figure 11 shows the result of the interaction between the antigen binding protein 1Z4 described in this application and the NIH3T3 EGFR wild-type cell line, and the oval shape is the nucleus.
  • Figure 12 shows the result of the interaction of the antigen binding protein 1Z4 described in this application with the NIH3T3 EGFR mutant cell line, and the oval shape is the nucleus.
  • Figure 13 shows the result of the internalization of the antigen binding protein 1Z4 antibody described in the present application into the cell, and the oval shape is the nucleus.
  • Figure 14 shows the result of the internalization of the antigen binding protein 6F2 antibody described in the present application into the cell, and the elliptical shape is the nucleus.
  • Figure 15 shows the result of the internalization of the antigen binding protein 4B1 antibody described in the present application into the cell, and the oval shape is the nucleus.
  • EGFR epidermal growth factor receptor
  • HER-1 epidermal growth factor receptor
  • Erb-B1 epidermal growth factor receptor
  • EGFR is composed of three parts: the ligand binding domain outside the cell, the hydrophobic transmembrane domain and the kinase domain inside the cell.
  • the intracellular structure of EGFR contains a tyrosine kinase domain (tyrosine kinase domain) and a carboxy-terminal tail with multiple autophosphorylation sites, belonging to the receptor tyrosine kinase family (receptor tyrosine kinase, RTKs); EGFR
  • the extracellular region is composed of ligand binding sites and two cysteine-rich regions.
  • the extracellular domain of EGFR can be further divided into four domains: I, II (amino acids 165-310), III, and IV.
  • EGFR can bind to a variety of ligands with agonistic functions, mainly epidermal growth factor, EGF), transforming growth factor (transforming growth factor a, TGFa), EGFR ligands also include AREG, epigen (EPGN), BTC (betacellulin), eregregulin (EREG) and/or HBEGF (heparin-binding EGF).
  • EGF epidermal growth factor
  • TGFa tumor growth factor
  • EGFR ligands also include AREG, epigen (EPGN), BTC (betacellulin), eregregulin (EREG) and/or HBEGF (heparin-binding EGF).
  • EGFR regulates multiple cellular processes through signal transduction pathways mediated by tyrosine kinases, including but not limited to signal transduction pathways that activate and control cell proliferation, differentiation, cell survival, apoptosis, angiogenesis, mitogenesis, and metastasis .
  • EGFR covers any natural EGFR or modified EGFR derived from any vertebrate, including fragments thereof and related polypeptides.
  • Related polypeptides include, but are not limited to, allelic variants, splice variants, derivative variants, substitution variants, Deletion variants and/or insertion variants (including the addition of N-terminal methionine), fusion polypeptides and interspecies homologues, any of the vertebrate origins including mammals, such as primates (e.g., humans or monkeys) and rodents Class (e.g., mouse or rat).
  • EGFR can exist as a transmembrane protein or as a soluble protein.
  • the term also encompasses naturally occurring variants of EGFR, such as splice variants or allelic variants. There are four splice variants of EGFR.
  • the EGFR sequence is known in the art. Exemplary amino acid sequences of human EGFR proteins can be found under UniProt accession numbers P00533-1, P00533-2, P00533-3 and/or P00533-4.
  • EGFR II domain generally refers to the extracellular domain II of EGFR.
  • the II domain of EGFR is usually rich in cystine, and its amino acid sequence usually includes the 165th to 310th amino acids of EGFR.
  • the amino acid sequence of the II domain of EGFR may include amino acids 196 to 287 of EGFR.
  • the amino acid sequence of the II domain of EGFR may include the amino acid sequence shown in SEQ ID NO: 13.
  • LZ-8 and LZ8 are also called Ling Zhi-8 or Ganoderma lucidum protein 8, and generally refer to an immunomodulatory protein derived from Ganoderma lucidum.
  • LZ-8 also includes its allelic variants, splice variants, derivative variants, substitution variants, deletion variants and/or insertion variants (including the addition of N-terminal methionine), fusion polypeptides and interspecies homologs .
  • the LZ-8 may refer to an immunomodulatory protein derived from Ganoderma lucidum.
  • the LZ-8 may include the amino acid sequence shown in SEQ ID NO: 14.
  • the term “competitive binding” generally means that the first molecule binds to the epitope in a manner sufficiently similar to the binding of the second molecule, so that compared to the binding of the first molecule in the absence of the second molecule, In the presence of the second molecule, the result of the binding of the first molecule to its homologous epitope is detectably reduced.
  • the antigen-binding protein of the present application binds to EGFR in a binding manner sufficiently similar to that of LZ-8.
  • the antigen-binding protein of the present application Compared with the antigen-binding protein of the present application in which LZ-8 does not exist, in the presence of LZ-8, the antigen-binding protein of the present application
  • the result of the binding of the binding protein to EGFR is detectably reduced; or, compared with the LZ-8 in which the antigen binding protein of the present application does not exist, in the presence of the antigen binding protein of the present application, the binding of LZ-8 to EGFR is The result is a detectable reduction.
  • the degree to which the first molecule can interfere with the binding of the second molecule to the target can be determined using a competitive binding test, for example, a FACS test, an ELISA or a BIACORE test.
  • the second molecule when the second molecule is present in excess, it will inhibit the specific binding of the first molecule to the common epitope by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. %. In some cases, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more.
  • the term "antigen-binding protein” generally refers to a protein that includes an antigen-binding portion, and optionally a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that promotes the binding of the antigen-binding protein to the antigen.
  • the antigen binding protein may typically comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both, and functional fragments thereof.
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments, immunoconjugates, multispecific antibodies (such as bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs or fusion proteins, etc., as long as they show The required antigen binding activity can be obtained.
  • antibody generally refers to an immunoglobulin that is reactive to a specified protein or peptide or fragment thereof.
  • Antibodies can be antibodies from any class, including but not limited to IgG, IgA, IgM, IgD, and IgE, and antibodies from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4).
  • the antibody may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4.
  • the antibody may also have a light chain selected from, for example, kappa ( ⁇ ) or lambda ( ⁇ ).
  • the antibodies of the application can be derived from any species.
  • antigen-binding fragment generally refers to a certain part of an antibody molecule that contains amino acid residues that interact with an antigen and confer specificity and affinity for the antibody to the antigen.
  • antigen-binding fragments may include, but are not limited to, Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
  • Fab generally refers to a fragment containing the variable domain of the heavy chain and the variable domain of the light chain, and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab' usually refers to a fragment that is different from Fab by adding a small number of residues (including one or more cysteine from the hinge region of an antibody) to the carboxyl end of the CH1 domain of the heavy chain;
  • F(ab ') 2 generally refers to Fab' dimer antibody fragments comprising two Fab fragments by a disulfide bridge at the hinge region.
  • Fv generally refers to the smallest antibody fragment that contains a complete antigen recognition and binding site.
  • the fragment may consist of a dimer in which a heavy chain variable region and a light chain variable region are tightly non-covalently bound;
  • dsFv usually refers to a disulfide bond-stabilized Fv fragment, The bond between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.
  • dAb fragment generally refers to an antibody fragment composed of a VH domain.
  • scFv generally refers to a monovalent molecule formed by covalently connecting and pairing a heavy chain variable domain and a light chain variable domain of an antibody through a flexible peptide linker; such scFv molecules may have general Structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • variable region or “variable domain” generally refers to the domain of an antibody heavy chain or light chain involved in the binding of an antibody to an antigen.
  • variable generally refers to that certain parts of the sequence of the variable domain of an antibody change strongly, forming the binding and specificity of various specific antibodies to their specific antigens.
  • the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in the three segments of the light chain variable region and the heavy chain variable region, called the complementarity determining region (CDR) or hypervariable region (HVR), which are LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3.
  • CDR complementarity determining region
  • HVR hypervariable region
  • the more highly conserved parts of variable domains are called framework regions (FR).
  • variable domains of the natural heavy chain and light chain each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4) Most of them adopt ⁇ -sheet configuration and are connected by three CDR structure loop regions.
  • the CDRs in each chain are closely brought together by the FR region, and together with the CDRs from the other chain form the antigen binding site of the antibody.
  • the term "monoclonal antibody” generally refers to antibodies obtained from a population of substantially homogeneous antibodies, that is, the antibodies constituting the population are the same, except for possible naturally occurring mutations and/or Post-translational modification (eg isomerization, amidation) outside. Monoclonal antibodies are highly specific and are directed against a single antigenic site.
  • chimeric antibody generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species.
  • the variable region is derived from an antibody of an experimental animal such as a rodent ("parent antibody”), and the constant region is derived from a human antibody, so that the resulting chimeric antibody is compared with the parental (e.g., mouse-derived) antibody in a human individual The possibility of triggering an adverse immune response is reduced.
  • humanized antibody generally refers to an antibody in which some or all of the amino acids other than the CDR region of a non-human antibody (such as a mouse antibody) are replaced with corresponding amino acids derived from human immunoglobulin. In the CDR regions, the addition, deletion, insertion, substitution or modification of amino acids may also be allowed, as long as they still retain the ability of the antibody to bind to a specific antigen.
  • the humanized antibody may optionally comprise at least a portion of the constant region of a human immunoglobulin. "Humanized antibodies” retain antigen specificity similar to the original antibodies.
  • “Humanized” forms of non-human (e.g., murine) antibodies may contain minimally chimeric antibodies derived from non-human immunoglobulin sequences.
  • the CDR region residues in human immunoglobulin can be equipped with non-human species (donor antibody) (such as mouse, rat) with the desired properties, affinity, and/or ability.
  • donor antibody such as mouse, rat
  • Rabbit or non-human primate residues in the CDR region In some cases, the FR region residues of the human immunoglobulin can be replaced with corresponding non-human residues.
  • humanized antibodies may contain amino acid modifications that are not in the recipient antibody or in the donor antibody.
  • the term "fully human antibody” generally refers to an antibody whose all parts (including the variable and constant regions of the antibody) are encoded by genes of human origin.
  • Methods for obtaining fully human antibodies in the art include phage display technology, transgenic mouse technology, ribosome display technology, RNA-polypeptide technology, and the like.
  • binding generally refer to a measurable and reproducible interaction, such as the binding between an antigen and an antibody, which can determine the presence of a molecule
  • targets in the context of heterogeneous populations (including biological molecules).
  • an antibody binds to an epitope through its antigen binding domain, and this binding requires some complementarity between the antigen binding domain and the epitope.
  • an antibody that specifically binds to a target is an antibody that binds to this target with greater affinity, affinity, easier and/or longer duration than it binds to other targets.
  • polypeptide or “protein” are used interchangeably and generally refer to a polymer of amino acid residues.
  • the term also applies to amino acid polymers in which one or more amino acid residues are analogs or mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers.
  • the term can also include modified amino acid polymers, for example, by adding sugar residues to form glycoproteins or being phosphorylated.
  • Polypeptides and proteins can be produced by naturally occurring and non-recombinant cells or by genetically engineered or recombinant cells, and can contain molecules with the amino acid sequence of a natural protein, or deletions or additions of one or more amino acids of the natural sequence. And/or substituted molecules.
  • polypeptide and “protein” especially include the deletion, addition and/or substitution sequence of one or more amino acids of the antigen binding protein described in the present application.
  • isolated generally refers to a biological material (such as a virus, nucleic acid, or protein) that is substantially free of components that normally accompany or interact with it in the environment in which it naturally occurs.
  • the isolated biological material optionally contains additional materials that the biological material has not been found to possess in its natural environment (e.g., nucleic acid or protein).
  • isolated when referring to a protein, “isolation” generally means that the molecule is separated and separated from the entire organism in which the molecule is found to occur naturally, or that there are substantially no other biological macromolecules of the same type.
  • immunoconjugate generally refers to a substance formed by linking an antigen binding protein with other active agents.
  • Other active agents can be small molecule active agents, such as chemotherapeutics, toxins, immunotherapeutics, imaging probes Or spectroscopic probe.
  • nucleic acid generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or their analogs of any length isolated from their natural environment or artificially synthesized.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells.
  • the vector may include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of DNA or RNA transcription and/or translation.
  • the carrier also includes a carrier having a variety of the above-mentioned functions.
  • the vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, by culturing a suitable host cell containing the vector, the vector can produce the desired expression product.
  • the term "cell” generally refers to individual cells, cell lines or cell cultures that can contain or already contain the nucleic acid molecule described in this application, or can express the antigen binding protein described in this application. Things.
  • the cell may include the progeny of a single host cell. Due to natural, accidental or deliberate mutations, the progeny cells and the original parent cells may not necessarily be identical in morphology or genome, but they can express the antibodies or antigen-binding fragments described in this application.
  • the cells can be obtained by transfecting cells in vitro using the vectors described in this application.
  • the cell may be a prokaryotic cell (such as Escherichia coli), or a eukaryotic cell (such as yeast cells, such as COS cells, Chinese Hamster Ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells or Myeloma cells).
  • the cell may be a mammalian cell.
  • the mammalian cell may be a CHO-K1 cell.
  • the term "pharmaceutical composition” generally refers to a preparation that exists in a form that allows the biological activity of the active ingredient to be effective, and does not contain unacceptable toxicity to the subject to which the composition will be administered. Additional ingredients.
  • treatment generally refers to the desire to change the natural course of the individual to be treated, and may be a clinical intervention to achieve prevention and treatment or in the course of clinical pathology.
  • Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, reducing symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving the prognosis.
  • antibodies e.g., anti-PD-1 antibodies
  • administering generally refers to the subject (eg, patient) administering a certain dose of a compound (eg, an anticancer therapeutic agent) or a pharmaceutical composition (eg, a pharmaceutical composition containing an anticancer therapeutic agent) Methods. Administration can be carried out by any suitable means, including parenteral, intrapulmonary and intranasal, and (if local treatment is required) intralesional administration. Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • tumor generally refers to all neoplastic cell growth and proliferation (whether malignant or benign) as well as all precancerous and cancerous cells and tissues.
  • the tumor may be a tumor in which the EGFR of cells and tissues is abnormally expressed.
  • the tumor may be a tumor overexpressing EGFR in cells and tissues.
  • Tumors may include solid tumors and/or non-solid tumors (e.g., hematoma, lymphoma).
  • EGFR activity diseases related to EGFR activity include tumor cell proliferation, pathological new blood vessel formation (which promotes the growth of solid tumors), new blood vessel formation in the eye (diabetic retinopathy, age-induced macular degeneration, etc.) and inflammation (psoriasis, Rheumatoid arthritis, etc.).
  • pathological new blood vessel formation which promotes the growth of solid tumors
  • new blood vessel formation in the eye new blood vessel formation in the eye
  • inflammation psoriasis, Rheumatoid arthritis, etc.
  • Clinical studies have shown that many types of tumors such as glioma, breast cancer, lung cancer, ovarian cancer, head and neck squamous cell carcinoma, cervical cancer, esophageal cancer, prostate cancer, liver cancer, colon cancer, gastric cancer, etc. have high levels of EGFR Express.
  • Abnormal expression of EGFR generally refers to a measurable higher or lower level of EGFR on the surface of a certain cell compared with normal cells of the same tissue type.
  • EGFR overexpression generally refers to a measurable higher level of EGFR on the surface of a certain cell compared to normal cells of the same tissue type. This abnormal expression (e.g., overexpression) can be caused by gene amplification or increased transcription or translation.
  • abnormal expression of EGFR may be caused by mutations in EGFR
  • abnormal expression (for example, overexpression) of EGFR ligands for example, EGF and TGF ⁇
  • EGFR ligands for example, EGF and TGF ⁇
  • EGFR signal transduction system or EGFR itself Abnormal expression (for example, overexpression).
  • Abnormal expression can refer to abnormal expression (e.g., overexpression) at the protein and nucleic acid levels (due to increased transcription, post-transcriptional processing, translation, post-translational processing, altered stability and altered protein degradation), As well as local abnormal expression (for example, increased nuclear localization) and enhanced functional activity caused by changes in protein transport mode, for example, such as increased enzymatic hydrolysis of substrates.
  • EGFR expression (or, overexpression) can be determined in diagnostic or prognostic assays by evaluating the level of EGFR present on the cell surface or in cell lysates by techniques known in the art: for example, immunohistochemical assays, immunofluorescence assays , Immunoenzyme assay, ELISA, flow cytometry, radioimmunoassay, Western blot, ligand binding and/or kinase activity, etc.
  • whether overexpression can be assessed by measuring the level of nucleic acid molecules encoding EGFR in the cells, for example, by fluorescence in situ hybridization, Southern blotting, or PCR technology.
  • the level of EGFR in normal cells is compared with the level of cells affected by cell proliferation disorders (such as tumors) to determine whether EGFR is abnormally expressed.
  • Abnormal expression may be about 50%, 60%, 70%, 80%, 90% or more difference in EGFR expression level from normal cells or comparison cells.
  • Overexpression can be about 50%, 60%, 70%, 80%, 90% or more of EGFR expression levels higher than normal cells or comparative cells.
  • the term "internalization” generally refers to the process by which extracellular substances (for example, proteins, nucleic acids, or small molecules) pass through cell membranes (for example, plasma membrane, endosomal membrane, and endoplasmic reticulum membrane).
  • the internalization includes energy-dependent (ie, active) transport mechanisms (such as phagocytosis, endocytosis, pinocytosis, ligand internalization, and antibody internalization) and energy-independent (ie, passive) transport mechanisms (such as diffusion).
  • internalization may refer to receptor-mediated endocytosis, in which proteins or polypeptides are absorbed by cells by binding to receptors on the surface of cell membranes.
  • the term "between” usually means that the C-terminus of a certain amino acid fragment is directly or indirectly connected to the N-terminus of the first amino acid fragment, and the N-terminus is directly or indirectly connected to the C-terminus of the second amino acid fragment. Indirect connection.
  • the N-terminus of the L-FR2 is directly or indirectly connected to the C-terminus of the LCDR1
  • the C-terminus of the L-FR2 is directly or indirectly connected to the N-terminus of the LCDR2.
  • the N-terminus of the L-FR3 is directly or indirectly connected to the C-terminus of the LCDR2
  • the C-terminus of the L-FR3 is directly or indirectly connected to the N-terminus of the LCDR3.
  • the N-terminus of the H-FR2 is directly or indirectly connected to the C-terminus of the HCDR1
  • the C-terminus of the H-FR2 is directly or indirectly connected to the N-terminus of the HCDR2.
  • the N-terminus of the H-FR3 is directly or indirectly connected to the C-terminus of the HCDR2
  • the C-terminus of the H-FR3 is directly or indirectly connected to the N-terminus of the HCDR3.
  • the "first amino acid fragment" and the "second amino acid fragment” can be any amino acid fragment that is the same or different.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides an isolated antigen binding protein, which can specifically bind to the epidermal growth factor receptor (EGFR) on the cell membrane to internalize the cell.
  • EGFR epidermal growth factor receptor
  • the isolated antigen binding protein can specifically bind to the II domain of EGFR, and the II domain of EGFR may include the amino acid sequence shown in SEQ ID NO: 13.
  • the isolated antigen binding protein can specifically bind to at least one amino acid selected from the group consisting of Ser196, Ser222, Lys269 and Ser282 on the II domain of EGFR.
  • the isolated antigen binding protein can specifically bind to at least one amino acid on the II domain of EGFR, and the amino acid is selected from the group consisting of Ser196, Ser222, Lys269 and Ser282.
  • the isolated antigen binding protein can specifically bind to at least two amino acids on the II domain of EGFR, and the amino acids are selected from the group consisting of Ser196, Ser222, Lys269 and Ser282.
  • the isolated antigen binding protein can specifically bind to three amino acids on the II domain of EGFR, and the amino acids are selected from the group consisting of Ser196, Ser222, Lys269 and Ser282.
  • the isolated antigen binding protein can specifically bind to at least four amino acids on the II domain of EGFR, and the amino acids are selected from the group consisting of Ser196, Ser222, Lys269 and Ser282.
  • the isolated antigen binding protein can competitively bind to the EGFR with the LZ-8 protein.
  • the LZ-8 protein may include the amino acid sequence shown in SEQ ID NO: 14.
  • the isolated antigen binding protein can compete with a reference antibody for binding to the EGFR, wherein the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL, and the reference antibody VH includes HCDR1, HCDR2, and HCDR3, the VL of the reference antibody includes LCDR1, LCDR2, and LCDR3, and the HCDR1 of the reference antibody includes any of SEQ ID NO: 15, SEQ ID NO: 29, and SEQ ID NO: 43
  • the amino acid sequence shown in one item, the HCDR2 of the reference antibody comprises the amino acid sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 30 and SEQ ID NO: 44, and the HCDR3 of the reference antibody Contains the amino acid sequence shown in any one of SEQ ID NO: 17, SEQ ID NO: 31 and SEQ ID NO: 45, and the LCDR1 of the reference antibody includes SEQ ID NO: 18, SEQ ID NO: 32 and SEQ ID
  • the reference antibody may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the amino acid sequence of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 is selected from any of the following groups: (1 ) HCDR1: SEQ ID NO: 15, HCDR2: SEQ ID NO: 16, HCDR3: SEQ ID NO: 17, LCDR1: SEQ ID NO: 18, LCDR2: SEQ ID NO: 19 and LCDR3: SEQ ID NO: 20; ( 2) HCDR1: SEQ ID NO: 29, HCDR2: SEQ ID NO: 30, HCDR3: SEQ ID NO: 31, LCDR1: SEQ ID NO: 32, LCDR2: SEQ ID NO: 33 and LCDR3: SEQ ID NO: 34; And, (3) HCDR1: SEQ ID NO: 43, HCDR2: SEQ ID NO: 44, HCDR3: SEQ ID NO: 45, SEQ ID NO: 46, LCDR2:
  • the isolated antigen binding protein may include CDR3 in the heavy chain variable region VH, and the VH may include any one of SEQ ID NO: 68, SEQ ID NO: 72, and SEQ ID NO: 76 Amino acid sequence shown
  • the isolated antigen binding protein may include HCDR3, and the HCDR3 may include the amino acid sequence shown in any one of SEQ ID NO: 17, SEQ ID NO: 31, and SEQ ID NO: 45.
  • the isolated antigen binding protein may include CDR2 in the heavy chain variable region VH, and the VH may include any one of SEQ ID NO: 68, SEQ ID NO: 72, and SEQ ID NO: 76 Amino acid sequence shown
  • the isolated antigen binding protein may include HCDR2, and the HCDR2 may include the amino acid sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 30, and SEQ ID NO: 44.
  • the isolated antigen binding protein may include CDR1 in the heavy chain variable region VH, and the VH may include any one of SEQ ID NO: 68, SEQ ID NO: 72, and SEQ ID NO: 76 Amino acid sequence shown
  • the isolated antigen binding protein may include HCDR1, and the HCDR1 may include the amino acid sequence shown in any one of SEQ ID NO: 15, SEQ ID NO: 29, and SEQ ID NO: 43.
  • the isolated antigen binding protein may include HCDR1, HCDR2, and HCDR3, and the HCDR1 may include any one of SEQ ID NO: 15, SEQ ID NO: 29, and SEQ ID NO: 43.
  • An amino acid sequence the HCDR2 may include the amino acid sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 30 and SEQ ID NO: 44, and the HCDR3 may include SEQ ID NO: 17, SEQ ID NO: The amino acid sequence shown in any one of 31 and SEQ ID NO: 45.
  • the isolated antigen binding protein may include HCDR1, HCDR2, and HCDR3, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 15, and the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 16. Amino acid sequence, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 17.
  • the isolated antigen binding protein may include HCDR1, HCDR2, and HCDR3, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 29, and the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 30. Amino acid sequence, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 31.
  • the isolated antigen binding protein may include HCDR1, HCDR2, and HCDR3, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 43, and the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 44 Amino acid sequence, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:45.
  • the isolated binding protein may include the framework region H-FR1, and the H-FR1 may include any one of SEQ ID NO: 21, SEQ ID NO: 35, and SEQ ID NO: 49 The amino acid sequence.
  • the isolated binding protein may include the framework region H-FR2, and the H-FR2 may include any one of SEQ ID NO: 22, SEQ ID NO: 36, and SEQ ID NO: 50.
  • the amino acid sequence may include the framework region H-FR2, and the H-FR2 may include any one of SEQ ID NO: 22, SEQ ID NO: 36, and SEQ ID NO: 50. The amino acid sequence.
  • the isolated binding protein may include the framework region H-FR3, and the H-FR3 may include any one of SEQ ID NO: 23, SEQ ID NO: 37, and SEQ ID NO: 51.
  • the amino acid sequence may include the framework region H-FR3, and the H-FR3 may include any one of SEQ ID NO: 23, SEQ ID NO: 37, and SEQ ID NO: 51. The amino acid sequence.
  • the isolated binding protein may include the framework region H-FR4, and the H-FR4 may include any one of SEQ ID NO: 24, SEQ ID NO: 38, and SEQ ID NO: 52.
  • the amino acid sequence may include the amino acid sequence.
  • the isolated antigen binding protein may include H-FR1, H-FR2, H-FR3, and H-FR4, and the H-FR1 may include SEQ ID NO: 21, SEQ ID NO: 35 and The amino acid sequence shown in any one of SEQ ID NO: 49, the H-FR2 may include the amino acid sequence shown in any one of SEQ ID NO: 22, SEQ ID NO: 36, and SEQ ID NO: 50, so The H-FR3 may include the amino acid sequence shown in any one of SEQ ID NO: 23, SEQ ID NO: 37 and SEQ ID NO: 51, and the H-FR4 may include SEQ ID NO: 24, SEQ ID NO: The amino acid sequence shown in any one of 38 and SEQ ID NO: 52.
  • the isolated antigen binding protein may include H-FR1, H-FR2, H-FR3, and H-FR4, and the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 21, so
  • the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 22
  • the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 23
  • the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 24 Amino acid sequence.
  • the isolated antigen binding protein may include H-FR1, H-FR2, H-FR3, and H-FR4, and the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 35, so
  • the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 36
  • the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 37
  • the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 38 Amino acid sequence.
  • the isolated antigen binding protein may include H-FR1, H-FR2, H-FR3, and H-FR4, and the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 49, so The H-FR2 may include the amino acid sequence shown in SEQ ID NO: 50, the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 51, and the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 52 Amino acid sequence.
  • the isolated antigen binding protein may include a heavy chain variable region VH, and the VH may include any one of SEQ ID NO: 68, SEQ ID NO: 72, and SEQ ID NO: 76 The amino acid sequence.
  • the isolated antigen binding protein may include the heavy chain variable region VH, and the VH may include the amino acid sequence shown in SEQ ID NO: 68.
  • the isolated antigen binding protein may include a heavy chain variable region VH, and the VH may include the amino acid sequence shown in SEQ ID NO: 72.
  • the isolated antigen binding protein may include the heavy chain variable region VH, and the VH may include the amino acid sequence shown in SEQ ID NO: 76.
  • the isolated antigen binding protein may include a heavy chain constant region, and the heavy chain constant may be derived from IgG1.
  • the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO: 57 or SEQ ID NO: 58.
  • the isolated antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include any one of SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64. Amino acid sequence.
  • the isolated antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include any one of SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 11. Amino acid sequence.
  • the isolated antigen binding protein may include CDR3 in the light chain variable region VL, and the VL may include any one of SEQ ID NO: 69, SEQ ID NO: 73 and SEQ ID NO: 77 Amino acid sequence shown
  • the isolated antigen binding protein may include LCDR3, and the LCDR3 may include the amino acid sequence shown in any one of SEQ ID NO: 20, SEQ ID NO: 34, and SEQ ID NO: 48.
  • the isolated antigen binding protein may include CDR2 in the light chain variable region VL, and the VL may include any one of SEQ ID NO: 69, SEQ ID NO: 73 and SEQ ID NO: 77 Amino acid sequence shown
  • the isolated antigen binding protein may include LCDR2, and the LCDR2 may include the amino acid sequence shown in any one of SEQ ID NO: 19, SEQ ID NO: 33, and SEQ ID NO: 47.
  • the isolated antigen binding protein may include CDR1 in the light chain variable region VL, and the VL may include any one of SEQ ID NO: 69, SEQ ID NO: 73 and SEQ ID NO: 77 Amino acid sequence shown
  • the isolated antigen binding protein may include LCDR1, and the LCDR1 may include the amino acid sequence shown in any one of SEQ ID NO: 18, SEQ ID NO: 32, and SEQ ID NO: 46.
  • the isolated antigen binding protein may include LCDR1, LCDR2, and LCDR3, and the LCDR1 includes the amino acid shown in any one of SEQ ID NO: 18, SEQ ID NO: 32, and SEQ ID NO: 46
  • the LCDR2 includes the amino acid sequence shown in any one of SEQ ID NO: 19, SEQ ID NO: 33 and SEQ ID NO: 47
  • the LCDR3 includes SEQ ID NO: 20, SEQ ID NO: 34 and SEQ ID NO: the amino acid sequence shown in any one of 48.
  • the isolated antigen binding protein may include LCDR1, LCDR2, and LCDR3, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 18, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 19. Amino acid sequence, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO:20.
  • the isolated antigen binding protein may include LCDR1, LCDR2, and LCDR3, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 32, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 33 Amino acid sequence, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 34.
  • the isolated antigen binding protein may include LCDR1, LCDR2, and LCDR3, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 46, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 47 Amino acid sequence, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 48.
  • the isolated binding protein may include the framework region L-FR1, and the L-FR1 may include any one of SEQ ID NO: 25, SEQ ID NO: 39, and SEQ ID NO: 53 The amino acid sequence.
  • the isolated binding protein may include the framework region L-FR2, and the L-FR2 may include any one of SEQ ID NO: 26, SEQ ID NO: 40, and SEQ ID NO: 54 The amino acid sequence.
  • the isolated binding protein may include the framework region L-FR3, and the L-FR3 may include any one of SEQ ID NO: 27, SEQ ID NO: 41, and SEQ ID NO: 55 The amino acid sequence.
  • the isolated binding protein may include the framework region L-FR4, and the L-FR4 may include any one of SEQ ID NO: 28, SEQ ID NO: 42 and SEQ ID NO: 56 The amino acid sequence.
  • the isolated antigen binding protein may include L-FR1, L-FR2, L-FR3, and L-FR4, and the L-FR1 includes SEQ ID NO: 25, SEQ ID NO: 39 And the amino acid sequence shown in any one of SEQ ID NO: 53, the L-FR2 includes the amino acid sequence shown in any one of SEQ ID NO: 26, SEQ ID NO: 40 and SEQ ID NO: 54, so The L-FR3 includes the amino acid sequence shown in any one of SEQ ID NO: 27, SEQ ID NO: 41, and SEQ ID NO: 55, and the L-FR4 includes SEQ ID NO: 28, SEQ ID NO: 42 and The amino acid sequence shown in any one of SEQ ID NO: 56.
  • the isolated antigen binding protein may include L-FR1, L-FR2, L-FR3, and L-FR4, and the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 39, so The L-FR2 may include the amino acid sequence shown in SEQ ID NO: 40, the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 41, and the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 42 Amino acid sequence.
  • the isolated antigen binding protein may include L-FR1, L-FR2, L-FR3, and L-FR4, and the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 53, so The L-FR2 may include the amino acid sequence shown in SEQ ID NO: 54, the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 55, and the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 56 Amino acid sequence.
  • the isolated antigen binding protein may include L-FR1, L-FR2, L-FR3, and L-FR4, and the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 25, so
  • the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 26
  • the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 27
  • the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 28. Amino acid sequence.
  • the isolated antigen binding protein may include the light chain variable region VL, and the VL may include any one of SEQ ID NO: 69, SEQ ID NO: 73, and SEQ ID NO: 77 The amino acid sequence.
  • the isolated antigen binding protein may include the light chain variable region VL, and the VL may include the amino acid sequence shown in SEQ ID NO: 69.
  • the isolated antigen binding protein may include the light chain variable region VL, and the VL may include the amino acid sequence shown in SEQ ID NO: 73.
  • the isolated antigen binding protein may include the light chain variable region VL, and the VL may include the amino acid sequence shown in SEQ ID NO: 77.
  • the isolated antigen binding protein may include a light chain constant region, and the light chain constant region may include the amino acid sequence shown in SEQ ID NO:59.
  • the isolated antigen binding protein may include an antibody light chain, and the antibody light chain may include any one of SEQ ID NO: 61, SEQ ID NO: 63, and SEQ ID NO: 65. Amino acid sequence.
  • the isolated antigen binding protein may include an antibody light chain, and the antibody light chain may include any one of SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 12. Amino acid sequence.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and the HCDR1 may include SEQ ID NO: 15, SEQ ID NO: 29 and SEQ ID NO: 43.
  • the amino acid sequence shown in any item the HCDR2 may include the amino acid sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 30 and SEQ ID NO: 44, and the HCDR3 may include SEQ ID NO: 17.
  • the LCDR1 may include any one of SEQ ID NO: 18, SEQ ID NO: 32 and SEQ ID NO: 46
  • the LCDR2 may include the amino acid sequence shown in any one of SEQ ID NO: 19, SEQ ID NO: 33 and SEQ ID NO: 47
  • the LCDR3 may include SEQ ID NO: 20, SEQ ID The amino acid sequence shown in any one of NO:34 and SEQ ID NO:48.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 15, and the HCDR2 may include SEQ ID
  • the amino acid sequence shown in NO: 16 the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 17
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 18
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO:
  • the amino acid sequence shown in 19, the LCDR3 may include the amino acid sequence shown in SEQ ID NO:20.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 29, and the HCDR2 may include SEQ ID
  • the amino acid sequence shown in NO: 30, the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 31, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 32, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO:
  • the amino acid sequence shown in 33, the LCDR3 may include the amino acid sequence shown in any one of SEQ ID NO: 34.
  • the isolated antigen binding protein may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 43, and the HCDR2 may include SEQ ID
  • the amino acid sequence shown in NO: 44, the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 45, the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 46, and the LCDR2 may include SEQ ID NO: 47.
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 48.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL
  • the VH may include SEQ ID NO: 68, SEQ ID NO: 72, and SEQ ID NO: 76
  • the amino acid sequence shown in any one of the VL may include the amino acid sequence shown in any one of SEQ ID NO: 69, SEQ ID NO: 73 and SEQ ID NO: 77.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 68, and the VL may include SEQ ID NO: 69 amino acid sequence.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 72, and the VL may include SEQ ID NO: The amino acid sequence shown in 73.
  • the isolated antigen binding protein may include a heavy chain variable region VH and a light chain variable region VL, the VH may include the amino acid sequence shown in SEQ ID NO: 76, and the VL may include SEQ ID NO: The amino acid sequence shown in 77.
  • the isolated antigen binding protein may include a heavy chain and a light chain, and the heavy chain may include any one of SEQ ID NO: 60, SEQ ID NO: 62, and SEQ ID NO: 64.
  • An amino acid sequence, the light chain may include the amino acid sequence shown in any one of SEQ ID NO: 61, SEQ ID NO: 63, and SEQ ID NO: 65.
  • the isolated antigen binding protein may include a heavy chain and a light chain
  • the heavy chain may include the amino acid sequence shown in SEQ ID NO: 60
  • the light chain may include the amino acid sequence shown in SEQ ID NO: 61 The amino acid sequence.
  • the isolated antigen binding protein may include a heavy chain and a light chain
  • the heavy chain may include the amino acid sequence shown in SEQ ID NO: 62
  • the light chain may include the amino acid sequence shown in SEQ ID NO: 63 The amino acid sequence.
  • the isolated antigen binding protein may include a heavy chain and a light chain
  • the heavy chain may include the amino acid sequence shown in SEQ ID NO: 64
  • the light chain may include the amino acid sequence shown in SEQ ID NO: 65 The amino acid sequence.
  • the isolated antigen binding protein may include a heavy chain and a light chain, and the heavy chain may include any one of SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 11.
  • An amino acid sequence, the light chain may include the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 12.
  • the isolated antigen binding protein may include a heavy chain and a light chain
  • the heavy chain may include the amino acid sequence shown in SEQ ID NO: 3
  • the light chain may include the amino acid sequence shown in SEQ ID NO: 4 The amino acid sequence.
  • the isolated antigen binding protein may include a heavy chain and a light chain
  • the heavy chain may include the amino acid sequence shown in SEQ ID NO: 7
  • the light chain may include the amino acid sequence shown in SEQ ID NO: 8 The amino acid sequence.
  • the isolated antigen binding protein may include a heavy chain and a light chain
  • the heavy chain may include the amino acid sequence shown in SEQ ID NO: 11
  • the light chain may include the amino acid sequence shown in SEQ ID NO: 12 The amino acid sequence.
  • the present application also provides isolated one or more nucleic acid molecules, which can encode the antigen binding protein described in the present application.
  • each nucleic acid molecule in the one or more nucleic acid molecules may encode the entire antigen binding protein, or may encode part of it (for example, HCDR1-3, LCDR1-3, VL, VH, light chain Or one or more of the heavy chain).
  • the nucleic acid molecules described in this application may be isolated. For example, it can be produced or synthesized by the following methods: (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified , For example, fractionation by restriction enzyme digestion and gel electrophoresis, or (iv) synthesized, for example, by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
  • this application provides one or more vectors, which comprise one or more nucleic acid molecules described in this application.
  • Each vector may contain one or more of the nucleic acid molecules.
  • the vector may also contain other genes, such as a marker gene that allows the vector to be selected in a suitable host cell and under suitable conditions.
  • the vector may also contain expression control elements that allow the coding region to be correctly expressed in a suitable host.
  • the vector is an expression vector.
  • the application provides a host cell, which may comprise one or more nucleic acid molecules described in this application and/or one or more vectors described in this application.
  • each or each host cell may contain one or one of the nucleic acid molecules or vectors described in this application.
  • each or each host cell may contain multiple (e.g., 2 or more) or multiple (e.g., 2 or more) nucleic acid molecules or vectors described in the present application
  • the application provides a method for preparing the antigen-binding fragment.
  • the method may include culturing the host cell described in the present application under conditions such that the antibody or antigen-binding fragment thereof is expressed. For example, it is possible to use an appropriate medium, an appropriate temperature, a culture time, etc., and these methods are understood by those of ordinary skill in the art.
  • the present application provides an immunoconjugate, which may include the isolated antigen binding protein or the polypeptide, and a small molecule compound.
  • Immunoconjugates usually refer to the use of specific linkers to connect antibodies and small molecule cytotoxic drugs. Its main components can include antibodies, linkers and small molecule cytotoxic drugs.
  • the small molecule compound may contain a cytotoxic agent and/or a marker.
  • the small molecule compound can be coupled to the primary amine group of the upper amino acid residue of the isolated antigen binding protein.
  • the immunoconjugate is capable of internalizing cells.
  • this application provides a kit, which may contain the antigen binding protein, chimeric antigen receptor, genetically modified cell, immunoconjugate, and/or the drug described in this application combination. It can include the antigen binding protein, chimeric antigen receptor, genetically modified cell, and/or immunoconjugate described in the present application in a single common container, and can optionally be combined with one or more therapeutic agents , Optionally formulated together in a pharmaceutical composition.
  • this application provides a drug delivery device, which can be used to administer the antigen binding protein described in this application or a pharmaceutical composition thereof.
  • the application provides a pharmaceutical composition, which may comprise the antigen binding protein, the polypeptide, the nucleic acid molecule, the vector, the host cell, and any Select a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable adjuvant is non-toxic to the recipient at the dose and concentration used.
  • the pharmaceutical composition in this application may also contain more than one active compound, usually those that do not adversely affect each other. Those active compounds with complementary activities.
  • the antigen binding protein, the polypeptide, the nucleic acid molecule, the carrier, the host cell, the immunoconjugate and/or the pharmaceutical composition can be used for the treatment of EGFR activity related disease.
  • it can be used to treat tumors or inhibit tumor growth.
  • the pharmaceutical composition of the present application can inhibit or delay the development or progression of the disease, can reduce the tumor size (or even substantially eliminate the tumor), and/or can reduce and/or stabilize the disease state.
  • the pharmaceutical composition described in the present application may comprise a preventive and/or therapeutically effective amount of the antigen binding protein, the polypeptide, the nucleic acid molecule, the carrier, the host cell, and the immunoconjugate And/or the pharmaceutical composition.
  • the prophylactic and/or therapeutically effective amount is a dose required to prevent and/or treat (at least partially treat) a disease or disorder and/or any complications thereof in a subject suffering from or at risk of development.
  • the present application provides the antigen binding protein, the polypeptide, the nucleic acid molecule, the carrier, the host cell, the immunoconjugate and/or the pharmaceutical composition in Use in the preparation of medicines.
  • the tumor includes a tumor in which EGFR is abnormally expressed (e.g., overexpressed).
  • the disease associated with EGFR activity includes tumors.
  • the tumor may include tumors related to abnormal expression (for example, overexpression) of EGFR.
  • the tumor may include solid tumors and/or non-solid tumors.
  • the tumor is a solid tumor.
  • the tumor may include glioma, breast cancer, lung cancer, ovarian cancer, head and neck cancer, cervical cancer, esophageal cancer, prostate cancer, liver cancer, colon cancer and/or gastric cancer.
  • the application provides a method for internalizing a cell, which comprises administering the isolated antigen binding protein, the polypeptide, the immunoconjugate, the nucleic acid molecule, the vector, the cell And/or the pharmaceutical composition.
  • the method can be an ex vivo or in vitro method.
  • the method may be a method for non-therapeutic purposes.
  • the present application provides a method for detecting the presence and/or content of EGFR protein, which comprises administering the isolated antigen binding protein and/or the polypeptide.
  • the method can be an ex vivo or in vitro method.
  • the method may be a method for non-therapeutic purposes.
  • the application also provides the use of an antigen binding protein in a method for diagnosing a subject suffering from a tumor or cancer, the method comprising: contacting a sample with the antigen binding protein of the application and detecting the presence of bound antibody The presence or expression level of EGFR in a sample obtained from a subject.
  • a mouse-derived anti-epidermal growth factor monoclonal antibody characterized in that the antibody can bind to EGFR on the cell membrane and cause the antigen-antibody complex to vigorously internalize into tumor cells.
  • the monoclonal antibody described in embodiment 1 can specifically bind to the II domain (amino acid Ser196-Cys287) of EGFR, causing the internalization of the antigen-antibody complex.
  • the monoclonal antibody according to embodiment 2 which can at least specifically bind to the following amino acids on the II domain of EGFR: Ser196, Ser222, Lys269, Ser282.
  • the monoclonal antibody according to embodiment 2 which comprises a heavy chain variable region and a light chain variable region, characterized in that the heavy chain variable region has SEQ ID NO: 3, SEQ ID NO: 7, The amino acid sequence shown in SEQ ID NO: 11, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12.
  • the heavy chain variable region according to embodiment 4 characterized in that the heavy chain variable region contains the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9 .
  • the light chain variable region according to embodiment 4 characterized in that the light chain variable region contains the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10 .
  • the monoclonal antibody according to embodiment 2 can be prepared from hybridoma cells obtained by fusion of mouse spleen cells immunized with human EGFR protein and myeloma cells.
  • the hybridoma cell according to embodiment 8 characterized in that it can stably secrete the monoclonal antibody according to embodiment 2.
  • hybridoma cells according to embodiment 9 which can be obtained by screening for competitive binding with EGFR with LZ-8.
  • the monoclonal antibody as described in embodiment 2 which can be conjugated with a small molecule compound to form an antibody-small molecule compound conjugate.
  • the antibody-small molecule compound conjugate according to embodiment 12, the coupling method includes but is not limited to the primary amine group of the amino acid residue on the antibody.
  • the antibody-small molecule compound conjugate according to embodiment 12 can be internalized into tumor cells.
  • the preparation materials are shown in Table 1.
  • mice were immunized with human EGFR protein.
  • the specific method is as follows:
  • mice are immunized by subcutaneous or intraperitoneal injection. Five groups of animals (Balb/C mice) will be immunized as shown in Table 2. After immunization, tail blood was collected and the serum titer was determined by ELISA method. The mouse with the highest antibody titer was selected for cell fusion.
  • the myeloma cells were sp2/0 derived from BALB/c, which were in the logarithmic growth phase when fused; the spleens of immunized mice were taken to prepare a single cell suspension of lymphocytes; the ratio of mouse spleen lymphocytes to myeloma cells was 1:5 Mix at -1:10, add 1ml of 50% PEG (PH 8.0) at 37°C dropwise, add incomplete medium and the rest of the stop solution, centrifuge to discard the supernatant, add HAT medium to suspend and mix, and dilute MC to 50ml. Dispense them into 3.5cm petri dishes, place them in a humid box, and place them in a 37°C, 5% CO 2 constant temperature incubator for culture.
  • Cell clones were selected within 7-10 days of cell fusion, and ELISA tests were performed on all parent clones against human EGFR protein, and 120 monoclonal wells with higher OD450 positive values were selected.
  • the above single clones were subjected to the first round of subcloning in one step, subjected to limiting dilution, and subjected to competitive ELISA screening against LZ8 and antibodies, and 12 subclones were obtained that could block the binding of LZ8 to EGFR.
  • the above subclones were subjected to the second round of subcloning, and again subjected to competitive ELISA screening against LZ8 and EGFR antibodies.
  • the above 3 cell lines were cultured in a 10 cm culture dish with 15% serum-containing DMEM medium, cultured to 4 ⁇ 10 7 hours, centrifuged at 800 rpm for 5 minutes, discarded the supernatant and transferred the cells to a 2L flask, and added serum-free Medium so that the cell density is about 3 ⁇ 10 5 cells/ml. Continue to culture for 1 to 2 weeks. When the cell death rate reaches 60%-70%, collect the cell suspension and centrifuge at 6000 rpm for 20 minutes, take the supernatant, and purify the supernatant by affinity column chromatography. Select the corresponding column according to the antibody pressure type.
  • monoclonal antibodies UMAB1Z4, UMAB4B1, UMAB6F2 subtypes are IgG1, and protein G is used for purification.
  • concentration of purified monoclonal antibody was determined and divided into aliquots (100ml/tube, concentration of 1mg/ml, stored at 4-8°C).
  • the anti-EGFR monoclonal cell lines of the aforementioned cell lines UMAB1Z4 (hereinafter referred to as 1Z4), UMAB4B1 (hereinafter referred to as 4B1), and UMAB6F2 (hereinafter referred to as 6F2) were sequenced.
  • Total RNA was extracted from the obtained hybridoma cells, reverse transcribed into cDNA with universal primers for the variable region of murine antibody, and then amplified by PCR.
  • the variable regions of the light and heavy chains obtained were entrusted to Genscript for sequencing.
  • SEQ ID NO: 67 and SEQ ID NO: 68 are the nucleotide coding sequences of the heavy chain variable region and the light chain variable region of the highly active and highly active monoclonal antibody 1Z4 obtained in this application;
  • SEQ ID NO: 69 and SEQ ID NO: 70 is the amino acid sequence of the heavy chain variable region and the light chain variable region of 1Z4, respectively.
  • SEQ ID NO: 71 and SEQ ID NO: 72 are the nucleotide coding sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 4B1, respectively;
  • SEQ ID NO: 73 and SEQ ID NO: 74 are respectively of 4B1 The amino acid sequence of the variable region of the heavy chain and the variable region of the light chain.
  • SEQ ID NO: 75 and SEQ ID NO: 76 are the nucleotide coding sequences of the heavy chain variable region and the light chain variable region of monoclonal antibody 6F2, respectively; SEQ ID NO: 77 and SEQ ID NO: 78 are for 6F2, respectively The amino acid sequence of the variable region of the heavy chain and the variable region of the light chain.
  • the 1Z4 antibody in Example 2 was selected for further analysis, and the heavy chain and light chain variable regions of the 1Z4 antibody were inserted into the expression vector pcDNA3.1(+)EcoRI and HindIII sites by PCR amplification to construct Murine anti-EGFR monoclonal antibody gene expression vector.
  • HiTrapTm MabSelect SuRe protein column technology was used to purify antibodies expressed in Expi293F TM cells to obtain antibodies with a concentration of 0.5 mg/ml and a purity of more than 90%. Specific steps are as follows:
  • Expi293F TM cells were cultured in serum-free Expi293 TM expression medium (Thermo Fisher Science). The cells were then stored in Erlenmeyer culture flasks and placed in an incubator at 37°C and 8% carbon dioxide. When the cell concentration reached 2.0 ⁇ 10 6 cells/mL, DNA and ExpiFectamine TM 293 were added to the transfected cell culture flask at a ratio of 1:2.7.
  • the recombinant plasmids encoding the heavy and light chains of antibody 1Z4 were co-transfected into Expi293F cells in suspension culture. About 17 hours after transfection, add ExpiFectamine TM 293 Transfection Enhancer 1 and ExpiFectamine TM 293 Transfection Enhancer 2 into the culture flask. On the 5th day after transfection, the cell density and cell viability were measured, and about 1 mL of cell culture supernatant was collected for evaluation of antibody expression. Take the cell culture supernatant harvested on the 6th day for purification.
  • the cell culture supernatant was collected on day 6 for antibody purification.
  • the filtered supernatant was loaded onto HiTrapTM MabSelect Sure 5ml (GE, catalog number 11-0034-95) at 3ml/min. It was eluted with 4 column volumes of PBS buffer (pH 7.2), and eluted with 50 mM citric acid (pH 3.0). During the elution process, each 1mL component was immediately neutralized with 124mol/L 1M Tris-HCl buffer (pH9.0). Part of the peaks were mixed together, and then the buffer was exchanged to PBS buffer (pH 7.2). The molecular weight, protein yield and purity were analyzed by SDS-PAGE and Western blot ( Figure 1). The concentration of the sample was determined by the A280 method, and the extinction coefficient was 1.505.
  • the extracellular region of EGFR and the monoclonal antibody were incubated in phosphate buffer, and cross-linked with DSS/BS3 cross-linking agent according to the ratio (1:4) at room temperature for 1 hour, then SDS-PAGE The gel separates the cross-linked product and stores it in a 10% acetic acid solution.
  • Cysteine residues in the protein were reduced and alkylated with Tris-(carboxyethyl)phosphine hydrochloride (TCEP) reagent and chloroacetamide (CAA) reagent, respectively, and reacted at 56°C for 60 minutes and at room temperature for 45 minutes. Then it was digested with trypsin (Thermo Fisher Scientific) overnight at 37°C, desalted on a C18 column, and loaded into the LC/MS tandem mass spectrometer;
  • TCEP Tris-(carboxyethyl)phosphine hydrochloride
  • CAA chloroacetamide
  • the peptides pass through the Nano1200, the flow rate is 300nL/min, and the elution gradient is: 5 to 60% B (60 min), 60-70% B (20 min), and 70% B (10 min).
  • a liquid is 0.1% formal acid in water
  • B liquid is 95% Acetonitrile, 0.1% formal acid in water;
  • Mass spectrometry data collection is performed on Orbitrap Fusion Lumos tandem mass spectrometry (Thermo Fisher Scientific).
  • the mass-to-charge ratio range of the first-order spectrum collection is 400-2000, and the resolution is 70,000 (m/z 400).
  • the data acquisition method of the secondary spectrum is data-dependent.
  • the first 10 peptides with the strongest signal in the primary spectrum are further collected for secondary spectrum data with a resolution of 35,000 (m/z 400), and finally through SIM- XL identifies cross-linked peptides.
  • Isothermal calorimetric titration experiments were performed with MicroCal VP-ITC (GE Healthcare) instrument. Drop 400 ⁇ M LZ8 into the sample pool containing 20 ⁇ M EGFR extracellular domain, 3ml per drop, 19 drops in total.
  • the reaction buffer is 20mm phosphate buffer, 150mm NaCl (pH 8.0), and the temperature is 20°C. Analyze the obtained ITC data with MicroCal Origin software to find K, n and dissociation constant values.
  • the amino acid involved in the interaction on the LZ8 protein is lysine 41, and this residue is on the ring BC of the LZ8 protein.
  • the "zero-length" cross-linking agent NHS/EDC was used to chemically cross-link the EGFR/LZ8 complex to further verify the cross-linking results obtained with BS3/DSS.
  • the results showed that the cross-linked peptide formed between aspartic acid at position 20 of LZ8 and serine at position 282 of EGFR, and these two amino acid residues are just near the EGFR/LZ8 interaction interface identified above , Which further verified the reliability of the cross-linking results.
  • Example 5 Combining computer simulation to speculate on the key sites of LZ8 binding to EGFR
  • Antigen binding protein 1Z4 can carry small molecule compounds to internalize into tumor cells
  • Alexa Fluor 568NHS Ester (AF568) was coupled to 1Z4 antibody to detect the internalization ability of 1Z4 antibody after modification of small molecule compounds.
  • AF568 is a small molecule compound with a molecular weight of 791.8, and its structure is shown in Figure 6.
  • AF568 can react with the primary amine group (R-NH 2 ) in the amino acid residue of the protein to couple to form a complex.
  • the 40nM 1Z4-AF568 conjugate was incubated with HeLa cells at 4°C for 30 minutes, and then allowed to bind to cell surface receptors, and then transferred to a 374°C cell incubator for further incubation for 1 hour.
  • the internalization of the 1Z4-AF568 conjugate was observed with an OMX ultra-high resolution microscope. The results showed that the 1Z4-AF568 conjugate can be internalized into tumor cells in the form of vesicles with a diameter of about 1 micron ( Figure 8).
  • Example 7 The antigen binding protein of the present application can bind to EGFR on the cell membrane and trigger high-intensity internalization
  • Antigen binding proteins 1Z4, 6F2 and 4B1 can bind to EGFR on the cell membrane and trigger high-intensity internalization
  • the 40 nM 1Z4, 6F2 and 4B1 antibodies modified by the small molecule compound Alexa Fluor 568 NHS Ester (modification method is the same as in Example 6) were incubated with HeLa cells at 4°C for 30 minutes, and then incubated at 37°C for 1 hour to make them internalized.
  • the OMX imaging system was used to observe the internalization of 1Z4, 6F2 and 4B1 antibodies.
  • the results of Figure 13, Figure 14 and Figure 15 indicate that the 1Z4, 6F2 and 4B1 antibodies were successfully internalized into the cells.
  • Antigen binding protein 1Z4 can bind to EGFR on the cell membrane and trigger high-intensity internalization
  • the internalization intensity of 1Z4 antibody was analyzed using a high-content imaging system. Incubate the 40nM cetuximab, mAb806, and 1Z4 antibody (modification method is the same as in Example 6) with HeLa cells at 4°C for 30 minutes, and then continue to incubate at 37°C for 1 hour. Its internalization. The results showed that the internalization intensity of 1Z4 was much higher than that of cetuximab and mAb806 ( Figure 10).
  • the Alexa Fluor 488 was coupled to the 1Z4 monoclonal antibody (the modification method is the same as in Example 6) to compare the binding ability of the 1Z4 monoclonal antibody with mutant EGFR (S196A/S222A/K269A/S282A) and wild-type EGFR.
  • the mutated EGFR mutation site is the key site for the binding of LZ8 to EGFR.
  • NIH3T3 EGFR (S196A/S222A/K269A/S282A) mutant cell lines and NIH3T3 EGFR wildtype cell lines were selected for related research.
  • a high-content system was used to analyze the average fluorescence intensity to reflect the difference in binding ability between mAb1Z4 and EGFR and its mutants at the cellular level.

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Abstract

L'invention concerne une protéine de liaison à l'antigène capable de se lier spécifiquement à un récepteur du facteur de croissance épidermique (EGFR) sur une membrane cellulaire, ladite protéine pouvant se lier à un épitope d'acide aminé du domaine II de l'EGFR de façon à internaliser un complexe. L'invention concerne en outre une méthode de traitement de tumeurs à l'aide de la protéine de liaison à l'antigène et une utilisation de celle-ci dans la préparation d'un médicament.
PCT/CN2021/076393 2020-02-10 2021-02-09 Protéine de liaison à l'antigène de l'anti-récepteur du facteur de croissance épidermique et son application WO2021160138A1 (fr)

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WO2023172981A1 (fr) * 2022-03-09 2023-09-14 Erasca, Inc. Anticorps anti-egfr et leurs utilisations

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