WO2021160138A1 - 抗表皮生长因子受体的抗原结合蛋白及其应用 - Google Patents

抗表皮生长因子受体的抗原结合蛋白及其应用 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)
French (fr)
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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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    • 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

提供了一种能够特异性结合细胞膜上的表皮生长因子受体(EGFR)的抗原结合蛋白，其能够结合EGFR的II结构域的氨基酸表位，使复合物内化。还提供了该抗原结合蛋白***的方法和在制备药物中的用途。

Description

抗表皮生长因子受体的抗原结合蛋白及其应用 技术领域
本申请涉及生物医药领域，具体的涉及一种抗表皮生长因子受体的抗原结合蛋白。另外，本申请还涉及该抗体的制备方法以及用途。
背景技术
细胞在活性异常的生长因子受体控制下，容易失去正常增殖，从而转变为癌细胞，引发癌症。这种失控可能是由很多因素引起，比如生长因子的过度表达或生长因子受体的过度表达，以及由生长因子调控的生化途径的自发性激活。参与致癌的生长因子受体包括表皮生长因子受体(Epidermal Growth Factor Receptor，EGFR)，来源于血小板的生长因子受体(PDGFR)，类胰岛素生长因子受体(IGFG)，神经生长因子受体(NGFR)和成纤维生长因子受体(FGF)等。EGFR的高度表达可能促进肿瘤细胞的增殖、血管生成、黏附、侵袭和转移、抑制肿瘤细胞的凋亡、导致肿瘤患者存活率低，预后差，疗效差，肿瘤转移可能性大，容易引起肿瘤细胞对各种细胞毒性药物的耐药性。
针对EGFR高表达相关的癌细胞增殖分化，目前已上市的EGFR抑制剂主要为小分子和抗体药物，他们针对靶向EGFR胞外部分的EGF结合位点或胞内部分的酪氨酸激酶活性位点，但是存在毒副作用强、易产生耐药性的问题。因此，亟需开发高效、新型的EGFR抗体。
发明内容
本申请提供了一种分离的抗原结合蛋白，其能够特异性结合细胞膜上的表皮生长因子受体(EGFR)，使复合物内化，从而进入细胞。
一方面，本申请提供了一种分离的抗原结合蛋白，其能够特异性结合细胞膜上的表皮生长因子受体(EGFR)，使复合物内化。
在某些实施方式中，所述分离的抗原结合蛋白能够特异性结合EGFR的II结构域，所述EGFR的II结构域包含SEQ ID NO:13所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白能够特异性结合EGFR的II结构域上至少一个选自下组的氨基酸：Ser196，Ser222，Lys269和Ser282。
在某些实施方式中，所述分离的抗原结合蛋白能够与LZ-8蛋白竞争性结合所述EGFR。
在某些实施方式中，所述分离的抗原结合蛋白能够与参比抗体竞争结合所述EGFR，其 中所述参比抗体包含重链可变区VH和轻链可变区VL，所述参比抗体的VH包含HCDR1、HCDR2和HCDR3，所述参比抗体的VL包含LCDR1、LCDR2和LCDR3，且所述参比抗体的HCDR1包含SEQ ID NO:15、SEQ ID NO:29和SEQ ID NO:43中任一项所示的氨基酸序列，所述参比抗体的HCDR2包含SEQ ID NO:16、SEQ ID NO:30和SEQ ID NO:44中任一项所示的氨基酸序列，所述参比抗体的HCDR3包含SEQ ID NO:17、SEQ ID NO:31和SEQ ID NO:45中任一项所示的氨基酸序列，所述参比抗体的LCDR1包含SEQ ID NO:18、SEQ ID NO:32和SEQ ID NO:46中任一项所示的氨基酸序列，所述参比抗体的LCDR2包含SEQ ID NO:19、SEQ ID NO:33和SEQ ID NO:47中任一项所示的氨基酸序列，所述参比抗体的LCDR3包含SEQ ID NO:20、SEQ ID NO:34和SEQ ID NO:48中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab，Fab’，F(ab) 2、Fv片段、F(ab’) 2、scFv、di-scFv和/或dAb。
在某些实施方式中，所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。例如，所述抗体可以为单克隆抗体。
在某些实施方式中，所述分离的抗原结合蛋白包含重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:68、SEQ ID NO:72和SEQ ID NO:76中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR3，且所述HCDR3包含SEQ ID NO:17、SEQ ID NO:31和SEQ ID NO:45中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR2，且所述HCDR2包含SEQ ID NO:16、SEQ ID NO:30和SEQ ID NO:44中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1，且所述HCDR1包含SEQ ID NO:15、SEQ ID NO:29和SEQ ID NO:43中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1、HCDR2和HCDR3，且所述HCDR1包含SEQ ID NO:15、SEQ ID NO:29和SEQ ID NO:43中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:16、SEQ ID NO:30和SEQ ID NO:44中任一项所示的氨基酸序列，所述HCDR3包含SEQ ID NO:17、SEQ ID NO:31和SEQ ID NO:45中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1、HCDR2和HCDR3，且所述HCDR1、HCDR2和HCDR3选自以下任一组氨基酸序列：
(1)HCDR1：SEQ ID NO:15，HCDR2：SEQ ID NO:16和HCDR3：SEQ ID NO:17；
(2)HCDR1：SEQ ID NO:29，HCDR2：SEQ ID NO:30和HCDR3：SEQ ID NO:31；
和，
(3)HCDR1：SEQ ID NO:43，HCDR2：SEQ ID NO:44和HCDR3：SEQ ID NO:45。
在某些实施方式中，所述分离的抗原结合蛋白包含重链可变区VH，其中所述VH包括框架区H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:21、SEQ ID NO:35和SEQ ID NO:49中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含重链可变区VH，其中所述VH包括框架区H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:22、SEQ ID NO:36和SEQ ID NO:50中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含重链可变区VH，其中所述VH包括框架区H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:23、SEQ ID NO:37和SEQ ID NO:51中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含重链可变区VH，其中所述VH包括框架区H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:24、SEQ ID NO:38和SEQ ID NO:52中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1、H-FR2、H-FR3和H-FR4，其中所述H-FR1包含SEQ ID NO:21、SEQ ID NO:35和SEQ ID NO:49中任一项所示的氨基酸序列，所述H-FR2包含SEQ ID NO:22、SEQ ID NO:36和SEQ ID NO:50中任一项所示的氨基酸序列，所述H-FR3包含SEQ ID NO:23、SEQ ID NO:37和SEQ ID NO:51中任一项所示的氨基酸序列且所述H-FR4包含SEQ ID NO:24、SEQ ID NO:38和SEQ ID NO:52中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1、H-FR2、H-FR3和H-FR4，且所述H-FR1、H-FR2、H-FR3和H-FR4选自以下任一组氨基酸序列：
(1)F-FR1：SEQ ID NO:21，H-FR2：SEQ ID NO:22，H-FR3：SEQ ID NO:23和H-FR4：SEQ ID NO:24；
(2)H-FR1：SEQ ID NO:35，H-FR2：SEQ ID NO:36，H-FR3：SEQ ID NO:37和H-FR4：SEQ ID NO:38；和，
(3)H-FR1：SEQ ID NO:49，H-FR2：SEQ ID NO:50，H-FR3：SEQ ID NO:51和H-FR4：SEQ ID NO:52。
在某些实施方式中，所述分离的抗原结合蛋白包含重链可变区VH，所述重链可变区VH包含SEQ ID NO:68，SEQ ID NO:72和SEQ ID NO:76中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括重链恒定区，且所述重链恒定区源自IgG1。
在某些实施方式中，所述重链恒定区包含SEQ ID NO:57或SEQ ID NO:58所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体重链，且所述抗体重链包含SEQ ID NO:60、SEQ ID NO:62和SEQ ID NO:64中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含轻链可变区VL中的至少一个CDR，所述VL包含SEQ ID NO:69、SEQ ID NO:73和SEQ ID NO:77中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR3，且所述LCDR3包含SEQ ID NO:20、SEQ ID NO:34和SEQ ID NO:48中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR2，且所述LCDR2包含SEQ ID NO:19、SEQ ID NO:33和SEQ ID NO:47中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR1，且所述LCDR1包含SEQ ID NO:18、SEQ ID NO:32和SEQ ID NO:46中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR1、LCDR2和LCDR3，且所述LCDR1包含SEQ ID NO:18、SEQ ID NO:32和SEQ ID NO:46中任一项所示的氨基酸序列，所述LCDR2包含SEQ ID NO:19、SEQ ID NO:33和SEQ ID NO:47中任一项所示的氨基酸序列，所述LCDR3包含SEQ ID NO:20、SEQ ID NO:34和SEQ ID NO:48中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR1、LCDR2和LCDR3，且所述LCDR1、LCDR2和LCDR3选自以下任一组氨基酸序列：
(1)LCDR1：SEQ ID NO:18，LCDR2：SEQ ID NO:19和LCDR3：SEQ ID NO:20；
(2)LCDR1：SEQ ID NO:32，LCDR2：SEQ ID NO:33和LCDR3：SEQ ID NO:34；
和，
(3)LCDR1：SEQ ID NO:46，LCDR2：SEQ ID NO:47和LCDR3：SEQ ID NO:48。
在某些实施方式中，所述分离的抗原结合蛋白包含轻链可变区VL，其中所述VL包括框架区L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:25、SEQ ID NO:39和SEQ ID NO:53中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含轻链可变区VL，其中所述VL包含框架区L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:26、SEQ ID NO:40和SEQ ID NO:54中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含轻链可变区VL，其中所述VL包含框架区L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:27、SEQ ID NO:41和SEQ ID NO:55中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含轻链可变区VL，其中所述VL包含框架区L-FR4，所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4包含SEQ ID NO:28、SEQ ID NO:42和SEQ ID NO:56中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR1、L-FR2、L-FR3和L-FR4，其中所述L-FR1包含SEQ ID NO:25、SEQ ID NO:39和SEQ ID NO:53中任一项所示的氨基酸序列，所述L-FR2包含SEQ ID NO:26、SEQ ID NO:40和SEQ ID NO:54中任一项所示的氨基酸序列，所述L-FR3包含SEQ ID NO:27、SEQ ID NO:41和SEQ ID NO:55中任一项所示的氨基酸序列且所述L-FR4包含SEQ ID NO:28、SEQ ID NO:42和SEQ ID NO:56中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR1、L-FR2、L-FR3和L-FR4，且所述L-FR1、L-FR2、L-FR3和L-FR4选自以下任一组氨基酸序列：
(1)F-FR1：SEQ ID NO:39，L-FR2：SEQ ID NO:40，L-FR3：SEQ ID NO:41和L-FR4：SEQ ID NO:42；
(2)L-FR1：SEQ ID NO:53，L-FR2：SEQ ID NO:54，L-FR3：SEQ ID NO:55和L-FR4：SEQ ID NO:56；和，
(3)L-FR1：SEQ ID NO:25，L-FR2：SEQ ID NO:26，L-FR3：SEQ ID NO:27和L-FR4：SEQ ID NO:28。
在某些实施方式中，所述分离的抗原结合蛋白包含轻链可变区VL，且所述轻链可变区VL包含SEQ ID NO:69，SEQ ID NO:73和SEQ ID NO:77中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括轻链恒定区，且所述轻链恒定区包含SEQ ID NO:59所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体轻链，且所述抗体轻链包含SEQ ID NO:61、SEQ ID NO:63和SEQ ID NO:65中任一项所示的氨基酸序列。
一方面，本申请提供了多肽，其包含所述分离的抗原结合蛋白。
一方面，本申请提供了免疫缀合物，其包含所述分离的抗原结合蛋白或所述多肽，以及小分子化合物。
在某些实施方式中，所述小分子化合物包含细胞毒性剂和/或标记物。
在某些实施方式中，所述小分子化合物与所述分离的抗原结合蛋白的上氨基酸残基的伯胺基团偶联。
在某些实施方式中，所述免疫缀合物能够使细胞内化。
一方面，本申请提供了分离的一种或多种核酸分子，其编码所述分离的抗原结合蛋白。
在某些实施方式中，所述的核酸分子包含SEQ ID NO：1，SEQID NO：5、SEQ ID NO：9、SEQ ID NO：2，SEQID 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和SEQ ID NO:75中任一项所示的核苷酸序列。
一方面，本申请提供了载体，其包含所述核酸分子。
一方面，本申请提供了细胞，其包含所述核酸分子或根据所述载体。
一方面，本申请提供了制备所述分离的抗原结合蛋白的方法，所述方法包括在使得所述分离的抗原结合蛋白表达的条件下，培养所述细胞。
一方面，本申请提供了药物组合物，其包含所述分离的抗原结合蛋白、所述多肽、所述免疫缀合物、所述核酸分子、所述载体和/或所述细胞，以及任选地药学上可接受的载体。
一方面，本申请提供了所述分离的抗原结合蛋白、所述多肽、所述免疫缀合物、所述核酸分子、所述载体、所述细胞和/或所述药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗与EGFR活性有关的疾病。
在某些实施方式中，所述与EGFR活性有关的疾病包括肿瘤。
在某些实施方式中，所述肿瘤包括与EGFR异常表达相关的肿瘤。在某些实施方式中，所述与EGFR异常表达相关的肿瘤包括与EGFR过表达相关的肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤。
在某些实施方式中，所述肿瘤包括神经胶质细胞瘤、乳腺癌、肺癌、卵巢癌、头颈癌、***、食管癌、***癌、肝癌、结肠癌和/或胃癌。
一方面，本申请提供了预防、缓解或治疗与EGFR活性有关的疾病的方法，所述方法包括向有需要的受试者施用所述分离的抗原结合蛋白、所述多肽、所述免疫缀合物、所述核酸分子、所述载体、所述细胞和/或所述药物组合物。
在某些实施方式中，所述与EGFR活性有关的疾病包括肿瘤。
在某些实施方式中，所述肿瘤包括与EGFR异常表达相关的肿瘤。在某些实施方式中， 所述与EGFR异常表达相关的肿瘤包括与EGFR过表达相关的肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤。
在某些实施方式中，所述肿瘤包括神经胶质细胞瘤、乳腺癌、肺癌、卵巢癌、头颈癌、***、食管癌、***癌、肝癌、结肠癌和/或胃癌。
一方面，本申请提供了所述分离的抗原结合蛋白、所述多肽、所述免疫缀合物、所述核酸分子、所述载体、所述细胞和/或所述药物组合物，用于预防、缓解或治疗与EGFR活性有关的疾病。
在某些实施方式中，所述与EGFR活性有关的疾病包括肿瘤。
在某些实施方式中，所述肿瘤包括与EGFR异常表达相关的肿瘤。在某些实施方式中，所述与EGFR异常表达相关的肿瘤包括与EGFR过表达相关的肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤。
在某些实施方式中，所述肿瘤包括神经胶质细胞瘤、乳腺癌、肺癌、卵巢癌、头颈癌、***、食管癌、***癌、肝癌、结肠癌和/或胃癌。
一方面，本申请提供了一种用于检测EGFR的存在和/或含量的方法，其包括施用所述分离的抗原结合蛋白或所述多肽。
一方面，本申请提供了试剂盒，其包含所述分离的抗原结合蛋白、所述多肽、所述免疫缀合物、所述核酸分子、所述载体、所述细胞和/或所述药物组合物。
一方面，本申请提供了一种使细胞内化的方法，其包括施用所述分离的抗原结合蛋白、所述多肽、所述免疫缀合物、所述核酸分子、所述载体、所述细胞和/或所述药物组合物。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是转染5天后的细胞上清液蛋白电泳以及蛋白免疫印迹(泳道1：还原条件， 泳道2：非还原条件，泳道P：人源IgG1作为阳性对照)。
图2显示的是EGFR胞外域和LZ8等温量热滴定实验(ITC)结果。
图3显示的是使用SDS-PAGE分离EGFR胞外域与LZ8复合体。
图4显示的是利用化学交联偶联质谱法确定EGFR与LZ8的相互作用界面，参与互作的氨基酸残基用球形表示。
图5显示的是EGFR胞外域与LZ8对接最优形态，关键氨基酸残基柱状标注D20，K41分别与S222，K269，S282，S196作用。
图6显示的是Alexa Fluor 568 NHS Ester结构式。
图7显示的是1Z4-AF568偶联物的纯化。
图8显示的是1Z4-AF568偶联物内化，左边图中椭圆形为细胞核，偶联物形成的囊泡结构如右边所示。
图9显示的是流式细胞仪分析1Z4与EGFR阳性、阴性细胞的结合，其中92.8和99.2对应的细胞为MD-MB-453，9660和17420对应的细胞为MD-MB-468。
图10显示的是本申请所述抗原结合蛋白1Z4与西妥昔单抗和mAb806的内化强度对比分析，椭圆形状为细胞核。
图11显示的是本申请所述抗原结合蛋白1Z4与NIH3T3 EGFR野生型细胞株作用的结果，椭圆形状为细胞核。
图12显示的是本申请所述抗原结合蛋白1Z4与NIH3T3 EGFR突变细胞株作用的结果，椭圆形状为细胞核。
图13显示的是本申请所述抗原结合蛋白1Z4抗体内化进入细胞的结果，椭圆形状为细胞核。
图14显示的是本申请所述抗原结合蛋白6F2抗体内化进入细胞的结果，椭圆形状为细胞核。
图15显示的是本申请所述抗原结合蛋白4B1抗体内化进入细胞的结果，椭圆形状为细胞核。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，本领域技术人员可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“表皮生长因子受体(EGFR)”也可称为“HER-1”或“Erb-B1”，属于ErbB(ErbB 1-4)家族的一员。EGFR由细胞外的配体结合区、疏水跨膜结构域和细胞内的激酶区3个部分组成。EGFR的胞内结构包含1个酪氨酸激酶域(tyrosine kinase domain)和具有多个自身磷酸化位点的羧基末端尾，属于受体酪氨酸激酶家族(receptor tyrosine kinase，RTKs)；EGFR的细胞外区域由配体结合位点和2个富含半胱氨酸的区域所构成。EGFR的胞外结构域可进一步分为I、II(氨基酸165-310)、III和IV四个结构域，EGFR能结合具有激动功能的多种配体，主要有表皮生长因子(epidermal growth factor，EGF)、转化生长因子(transforming growth factor a，TGFa)，EGFR的配体还有AREG、epigen(EPGN)、BTC(betacellulin)、eregregulin(EREG)和/或HBEGF(肝素结合EGF)。EGFR通过酪氨酸激酶介导的信号转导途径调控多个细胞过程，包括但不限于活化控制细胞增殖、分化、细胞存活、细胞凋亡、血管生成、有丝***发生，和转移的信号转导途径。
术语“EGFR”涵盖任何脊椎动物来源的任何天然EGFR或经修饰的EGFR，包括其片段以及相关多肽，相关多肽包括但不限于等位基因变体、剪接变体、衍生变体、取代变体、缺失变体和/或***变体(包括N末端蛋氨酸的添加)、融合多肽和种间同源物，所述任何脊椎动物来源包括哺乳动物，诸如灵长类(例如，人或猴)和啮齿类(例如，小鼠或大鼠)。所述术语涵盖“全长”、未加工的EGFR、由细胞中的加工所产生的任何形式的EGFR，以及其功能性片段和功能性变体(例如可变RNA转录物、截断型、多态性等)。EGFR可作为跨膜蛋白或作为可溶性蛋白存在。所述术语还涵盖天然存在的EGFR的变体，例如剪接变体或等位基因变体。EGFR可以有四种剪结变体。EGFR序列是本领域已知的。示例性的人EGFR蛋白的氨基酸序列可在UniProt登录号P00533-1、P00533-2、P00533-3和/或P00533-4下找到。
在本申请中，术语“EGFR的II结构域”，通常是指EGFR的胞外结构域II。EGFR的II结构域通常富含胱氨酸，其氨基酸序列通常包含EGFR的第165至第310位氨基酸。在本申请中，EGFR的II结构域的氨基酸序列可以包含EGFR的第196至第287位氨基酸。在本申请中，EGFR的II结构域的氨基酸序列可以包含SEQ ID NO:13所示的氨基酸序列。
在本申请中，术语“LZ-8”、“LZ8”也称为Ling Zhi-8或灵芝蛋白8，通常是指一种来源于灵芝的免疫调节蛋白。LZ-8还包含其等位基因变体、剪接变体、衍生变体、取代变体、缺失变体和/或***变体(包括N末端蛋氨酸的添加)、融合多肽和种间同源物。在本申请中，所述LZ-8可以指来源于Ganoderma lucidum的免疫调节蛋白。在本申请中，所述LZ-8可包含SEQ ID NO:14所示的氨基酸序列。
在本申请中，术语“竞争性结合”通常是指第一分子以充分相似于第二分子的结合的方式结合至表位，以使得与第二分子不存在下第一分子的结合相比，在第二分子的存在下，第一分子与其同源表位的结合的结果可检测地减少。例如，本申请的抗原结合蛋白以充分相似于LZ-8的结合方式结合至EGFR，与LZ-8不存在的本申请的抗原结合蛋白相比，在LZ-8的存在下，本申请的抗原结合蛋白与EGFR的结合的结果可检测的减少；或者，与本申请的抗原结合蛋白不存在的LZ-8相比，在本申请的抗原结合蛋白的存在下，LZ-8与EGFR的结合的结果可检测的减少。第一分子能够干扰第二分子结合靶标的程度，可以使用竞争结合试验来确定，例如，FACS试验、ELISA或BIACORE试验。一般来讲，当第二分子过量存在时，其会将第一分子与共同表位的特异性结合抑制至少40％、45％、50％、55％、60％、65％、70％或75％。在一些情况下，结合被抑制至少80％、85％、90％、95％或97％或更多。
在本申请中，术语“抗原结合蛋白”通常是指包含结合抗原部分的蛋白质，以及任选地允许结合抗原的部分采用促进抗原结合蛋白与抗原结合的构象的支架或骨架部分。抗原结合蛋白可典型地包含抗体轻链可变区(VL)、抗体重链可变区(VH)或上述两者，及其功能性片段。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗原结合蛋白的实例包括但不限于抗体、抗原结合片段、免疫缀合物、多特异性抗体(例如双特异性抗体)、抗体片段、抗体衍生物、抗体类似物或融合蛋白等，只要它们显示出所需的抗原结合活性即可。
在本申请中，术语“抗体”通常是指对指定蛋白质或肽或其片段有反应性的免疫球蛋白。抗体可以是来自任何类的抗体，包括但不限于IgG、IgA、IgM、IgD和IgE，及来自任何亚类(例如IgG1、IgG2、IgG3、和IgG4)的抗体。抗体可具有选自例如IgG1、IgG2、IgG3、或IgG4的重链恒定区。抗体还可具有选自例如kappa(κ)或lambda(λ)的轻链。本申请的抗体可衍生自任何物种。
在本申请中，术语“抗原结合片段”通常是指抗体分子的某部分，该部分包含氨基酸残基，该氨基酸残基与抗原相互作用并赋予抗体对于抗原的特异性和亲和力。抗原结合片段的实例可包括但不限于Fab，Fab’，F(ab) 2，Fv片段，F(ab’) 2，scFv，di-scFv和/或dAb。在本申请中，术语“Fab”通常是指含有重链可变结构域和轻链可变结构域的片段，并且还含有轻链的恒定结构域和重链的第一恒定结构域(CH1)；术语“Fab’”通常是指在重链CH1结构域的羧基端添加少量残基(包括一个或多个来自抗体铰链区的半胱氨酸)而不同于Fab的片段；术语“F(ab') 2”通常是指Fab’的二聚体，包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。术语“Fv”通常是指含有完整抗原识别与结合位点的最小抗体片段。在某些情形 中，该片段可以由一个重链可变区和一个轻链可变区以紧密非共价结合的二聚体组成；术语“dsFv”通常是指二硫键稳定的Fv片段，其单个轻链可变区与单个重链可变区之间的键是二硫键。术语“dAb片段”通常是指由VH结构域组成的抗体片段。在本申请中，术语“scFv”通常是指抗体的一个重链可变结构域和一个轻链可变结构域通过柔性肽连接子共价连接配对形成的单价分子；此类scFv分子可具有一般结构：NH 2-VL-连接子-VH-COOH或NH 2-VH-连接子-VL-COOH。
在本申请中，术语“可变区”或“可变结构域”通常是指参与抗体与抗原的结合的抗体重链或轻链的结构域。在本申请中，术语“可变”通常是指，抗体的可变结构域的序列的某些部分变化强烈，形成各种特定抗体对其特定抗原的结合和特异性。变异性并非均匀地分布在抗体的整个可变区中。它集中在轻链可变区和重链可变区中的三个区段，被称为互补决定区(CDR)或高变区(HVR)，分别为LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3。可变域中更高度保守的部分被称为框架区(FR)。天然重链和轻链的可变结构域各自包含四个FR区(H-FR1，H-FR2，H-FR3，H-FR4，L-FR1，L-FR2，L-FR3，L-FR4)，大部分采用β-折叠构型，通过三个CDR结构环区连接。每条链中的CDR通过FR区紧密靠近在一起，并与来自另一条链的CDR一起形成抗体的抗原结合位点。
在本领域中，可以通过多种方法来编码抗体的可变区或划分抗体的CDR，例如基于序列可变性的Kabat编号方案和定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))，基于结构环区域位置的Chothia编号方案和定义规则(参见，A1-Lazikani等人，JMol Biol 273:927-48,1997)，efranc等人的基于种系V基因的氨基酸序列比对的IMGT编号方案和定义规则，还有Honneger’s编号方案(AHo’s)，Martin编号方案，Gelfand编号方案等，可参见Mathieu Dondelinger等人，Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition,Front.Immunol.,16October 2018.
在本申请中，术语“单克隆抗体”通常是指从一群基本上同质的抗体获得的抗体，即构成群体的各个抗体相同，除了可能以极小量存在的可能的天然存在突变和/或翻译后修饰(例如异构化、酰胺化)外。单克隆抗体是高度特异性的，针对单一抗原性位点。
在本申请中，术语“嵌合抗体”通常是指其中可变区源自一个物种，而恒定区源自另一个物种的抗体。通常，可变区源自实验动物诸如啮齿动物的抗体(“亲本抗体”)，且恒定区源自人类抗体，使得所得嵌合抗体与亲本(例如小鼠来源)抗体相比，在人类个体中引发不良免疫反应的可能性降低。
在本申请中，术语“人源化抗体”通常是指非人抗体(例如小鼠抗体)的CDR区以外的部分或全部有的氨基酸被源自人免疫球蛋白的相应的氨基酸置换的抗体。在CDR区中，氨基酸的添加、缺失、***、置换或修饰也可以是允许的，只要它们仍保留抗体结合特定抗原的能力。人源化抗体可任选地包含人类免疫球蛋白恒定区的至少一部分。“人源化抗体”保留类似于原始抗体的抗原特异性。非人(例如鼠)抗体的“人源化”形式可以最低限度地包含衍生自非人免疫球蛋白的序列的嵌合抗体。在某些情形中，可以将人免疫球蛋白(受体抗体)中的CDR区残基用具有所期望性质、亲和力和/或能力的非人物种(供体抗体)(诸如小鼠，大鼠，家兔或非人灵长类动物)的CDR区残基替换。在某些情形中，可以将人免疫球蛋白的FR区残基用相应的非人残基替换。此外，人源化抗体可包含在受体抗体中或在供体抗体中没有的氨基酸修饰。
在本申请中，术语“全人源抗体”通常是指所有部分(包括抗体的可变区和恒定区)均由人类来源的基因所编码的抗体。本领域获得全人源抗体的方法可以有噬菌体展示技术、转基因小鼠技术、核糖体展示技术和RNA-多肽技术等。
在本申请中，术语“结合”、“特异性结合”或“对…特异性的”通常是指可测量且可再现的相互作用，诸如抗原和抗体之间的结合，其可以确定在存在分子(包括生物学分子)的异质群体的情况中靶物的存在。例如，抗体通过其抗原结合域与表位结合，并且该结合需要抗原结合域和表位之间的一些互补性。例如，特异性结合靶物(其可以是表位)的抗体是以比其结合其它靶物更大的亲和力、亲合力、更容易和/或以更大的持续时间结合此靶物的抗体。当抗体相比于其将结合随机的、不相关的表位而言更容易通过其抗原结合域与表位结合时，抗体被称为“特异性结合”该抗原。
在本申请中，术语“多肽”或“蛋白质”可互换地使用，通常是指氨基酸残基的聚合物。该术语也适用于其中一个或多个氨基酸残基是相应的天然存在的氨基酸的类似物或模拟物的氨基酸聚合物、以及天然存在的氨基酸聚合物。该术语也可包括修饰的氨基酸聚合物，例如，通过添加糖残基以形成糖蛋白或被磷酸化修饰。多肽和蛋白质可由天然存在的和非重组的细胞或由遗传工程改造的或重组的细胞产生，并且可包含具有天然蛋白质的氨基酸序列的分子、或具有天然序列的一个或多个氨基酸的缺失、添加和/或取代的分子。术语“多肽”和“蛋白质”特别包括本申请所述的抗原结合蛋白的一个或多个氨基酸的缺失、添加和/或取代的序列。
在本申请中，术语“分离的”通常是指大体上不含其天然存在的环境中通常伴随或与之相互作用的组分的生物材料(例如病毒、核酸或蛋白质)。所述分离的生物材料任选地包含 在其天然环境(例如，核酸或蛋白质)中所述生物材料未发现具有的另外的材料。在本申请中，当涉及蛋白质时，“分离”通常是指所述的分子从发现该分子天然存在的整个生物体中分离和分开，或基本不存在其它相同类型的生物大分子。当涉及核酸分子时，它与天然与其结合的序列完全或部分分离，或该核酸具有与其结合的异源序列，或该核算从染色体分离。
在本申请中，术语“免疫缀合物”通常是指抗原结合蛋白与其它活性剂连接形成的物质，其他活性剂可以是小分子活性剂，例如化疗剂、毒素、免疫治疗剂、成像探针或光谱探针。
在本申请中，术语“核酸”分子通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。
在本申请中，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，其将***的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA***细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。
在本申请中，术语“细胞”通常是指可以包含或已经含有包括本申请所述的核酸分子的质粒或载体，或者能够表达本申请所述的抗原结合蛋白的个体细胞、细胞系或细胞培养物。所述细胞可以包括单个宿主细胞的子代。由于天然的、意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述的抗体或其抗原结合片段即可。所述细胞可以通过使用本申请所述的载体体外转染细胞而得到。所述细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。在某些情形中，所述细胞可以是哺乳动物细胞。例如，所述哺乳动物细胞可以是CHO-K1细胞。
在本申请中，术语“药物组合物”通常是指这样的制剂，其以允许活性成分的生物学活性有效的形式存在，并且不包含对将施用所述组合物的对象具有不可接受的毒性的另外的成分。
在本申请中，术语“治疗”通常是指期望改变所治疗个体的天然病程，且可为实现防治或在临床病变过程中进行的临床介入。合乎需要的治疗效果包括但不限于防止疾病发生或复发性、减轻症状、减弱疾病的任何直接或间接病理学后果、防止转移、降低疾病进展速率、改善或缓解疾病状态以及缓和或改善预后。在一些情形中，抗体(例如，抗PD-1抗体)可用 来延迟疾病发展或减缓疾病进展。
在本申请中，术语“施用”通常是指向受试者(例如，患者)给予一定剂量的化合物(例如，抗癌治疗剂)或药物组合物(例如，包含抗癌治疗剂的药物组合物)的方法。施用可通过任何合适的方式进行，包括肠胃外、肺内和鼻内，以及(如果局部治疗需要)损伤内施用。胃肠外输注包括例如肌肉内、静脉内、动脉内、腹膜内或皮下施用。
在本申请中，术语“肿瘤”通常是指所有赘生性细胞生长和增殖(无论恶性还是良性)以及所有癌前和癌性细胞和组织。在本申请中，所述肿瘤可以为细胞和组织的EGFR异常表达的肿瘤。在本申请中，所述肿瘤可以为细胞和组织的EGFR过表达的肿瘤。肿瘤可包括实体瘤和/或非实体瘤(例如，血液瘤、淋巴瘤)。
与EGFR活性有关的疾病包括肿瘤细胞增殖、病理性新血管形成(其促进实体瘤生长)、眼中的新血管形成(糖尿病性视网膜病、年龄诱发的黄斑变性等等)和炎症(银屑病、类风湿性关节炎等等)。临床研究表明，许多类型的肿瘤如神经胶质细胞瘤、乳腺癌、肺癌、卵巢癌、头颈部鳞癌、***、食管癌、***癌、肝癌、结肠癌、胃癌等有高水平的EGFR表达。“EGFR异常表达”通常是指，与相同组织类型的正常细胞相比，某一细胞的表面上具有可测量程度的更高或更低水平的EGFR。“EGFR过表达”通常是指，与相同组织类型的正常细胞相比，某一细胞的表面上具有可测量程度的更高水平的EGFR。该异常表达(例如，过表达)可以由基因扩增或增加的转录或翻译而导致。在本申请中，EGFR异常表达(例如，过表达)可能是EGFR发生突变导致，EGFR配体(例如EGF和TGFα)异常表达(例如，过表达)导致EGFR信号转导***过度激活，或EGFR自身异常表达(例如，过表达)。异常表达(例如，过表达)可以指蛋白质和核酸水平的异常表达(例如，过表达)(由于增加的转录，转录后加工，翻译，翻译后加工，改变的稳定性和改变的蛋白质降解)，以及蛋白质运输模式改变导致的局部异常表达(例如，核定位增加)和增强的功能活性，例如，如增加底物的酶水解。EGFR表达(或，过表达)可以在诊断或预后测定中，通过本领域已知的技术评估存在于细胞表面上或细胞裂解物中的EGFR水平来测定：例如，免疫组织化学测定、免疫荧光测定、免疫酶测定、ELISA、流式细胞术、放射免疫测定法、蛋白质印迹、配体结合和/或激酶活性等。此外，可以通过测量细胞中编码EGFR的核酸分子的水平评估是否过表达，例如通过原位杂交中的荧光、DNA印迹法、或PCR技术。将正常细胞中的EGFR水平与受细胞增殖病症(例如肿瘤)影响的细胞的水平比较，以确定EGFR是否异常表达。异常表达可以是与正常细胞或比较细胞EGFR表达水平具有约50％、60％、70％、80％、90％或更多的差异。过表达可以是高于正常细胞或比较细胞EGFR表达水平的约50％、60％、70％、80％、90％ 或更多。
在本申请中，术语“内化”通常是指细胞外的物质(例如，蛋白质、核酸或小分子)穿过细胞膜(例如，质膜、内体膜和内质网膜)的过程。所述内化包括依赖能量(即，主动)的转运机制(例如吞噬、内吞、胞饮、配体内化和抗体内化)和不依赖能量(即，被动)的转运机制(例如扩散)。在本申请中，内化可以指受体介导的内吞作用，蛋白质或多肽通过结合细胞膜表面的受体，被细胞吸收。
在本申请中，术语“在……之间”通常是指某种氨基酸片段的C端与第一氨基酸片段的N端直接或间接连接，并且其N端与第二氨基酸片段的C端直接或间接连接。在轻链中，例如，所述L-FR2的N末端与所述LCDR1的C末端直接或间接相连，且所述L-FR2的C末端与所述LCDR2的N末端直接或间接相连。又例如，所述L-FR3的N末端与所述LCDR2的C末端直接或间接相连，且所述L-FR3的C末端与所述LCDR3的N末端直接或间接相连。在重链中，例如，所述H-FR2的N末端与所述HCDR1的C末端直接或间接相连，且所述H-FR2的C末端与所述HCDR2的N末端直接或间接相连。又例如，所述H-FR3的N末端与所述HCDR2的C末端直接或间接相连，且所述H-FR3的C末端与所述HCDR3的N末端直接或间接相连。在本申请中，“第一氨基酸片段”和“第二氨基酸片段”可以为相同或不同的任意一段氨基酸片段。
在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
抗原结合蛋白
一方面，本申请提供一种分离的抗原结合蛋白，其能够特异性结合细胞膜上的表皮生长因子受体(EGFR)，使细胞内化。
在本申请中，所述分离的抗原结合蛋白能够特异性结合EGFR的II结构域，所述EGFR的II结构域可包含SEQ ID NO:13所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白能够特异性结合EGFR的II结构域上至少一个选自下组的氨基酸：Ser196，Ser222，Lys269和Ser282。
在本申请中，所述分离的抗原结合蛋白能够特异性结合EGFR的II结构域上的至少一个氨基酸，所述氨基酸选自下组：Ser196，Ser222，Lys269和Ser282。在本申请中，所述分离的抗原结合蛋白能够特异性结合EGFR的II结构域上的至少二个氨基酸，所述氨基酸选自下组：Ser196，Ser222，Lys269和Ser282。在本申请中，所述分离的抗原结合蛋白能够特异性结合EGFR的II结构域上的三个氨基酸，所述氨基酸选自下组：Ser196，Ser222，Lys269和Ser282。在本申请中，所述分离的抗原结合蛋白能够特异性结合EGFR的II结构域上的至少四个氨基酸，所述氨基酸选自下组：Ser196，Ser222，Lys269和Ser282。
在本申请中，所述分离的抗原结合蛋白能够与LZ-8蛋白竞争性结合所述EGFR。所述LZ-8蛋白可包含SEQ ID NO:14所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白能够与参比抗体竞争结合所述EGFR，其中所述参比抗体包含重链可变区VH和轻链可变区VL，所述参比抗体的VH包含HCDR1、HCDR2和HCDR3，所述参比抗体的VL包含LCDR1、LCDR2和LCDR3，且所述参比抗体的HCDR1包含SEQ ID NO:15、SEQ ID NO:29和SEQ ID NO:43中任一项所示的氨基酸序列，所述参比抗体的HCDR2包含SEQ ID NO:16、SEQ ID NO:30和SEQ ID NO:44中任一项所示的氨基酸序列，所述参比抗体的HCDR3包含SEQ ID NO:17、SEQ ID NO:31和SEQ ID NO:45中任一项所示的氨基酸序列，所述参比抗体的LCDR1包含SEQ ID NO:18、SEQ ID NO:32和SEQ ID NO:46中任一项所示的氨基酸序列，所述参比抗体的LCDR2包含SEQ ID NO:19、SEQ ID NO:33和SEQ ID NO:47中任一项所示的氨基酸序列，所述参比抗体的LCDR3包含SEQ ID NO:20、SEQ ID NO:34和SEQ ID NO:48中任一项所示的氨基酸序列。
在本申请中，所述参比抗体可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3的氨基酸序列选自以下任一组：(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和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和LCDR3：SEQ ID NO:34；和，(3)HCDR1：SEQ ID NO:43，HCDR2：SEQ ID NO:44，HCDR3：SEQ ID NO:45，SEQ ID NO:46，LCDR2：SEQ ID NO:47和LCDR3：SEQ ID NO:48。
在本申请中，所述分离的抗原结合蛋白可包含重链可变区VH中的CDR3，所述VH可包含SEQ ID NO:68、SEQ ID NO:72和SEQ ID NO:76中任一项所示的氨基酸序列
在本申请中，所述分离的抗原结合蛋白可包含HCDR3，且所述HCDR3可包含SEQ ID  NO:17、SEQ ID NO:31和SEQ ID NO:45中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链可变区VH中的CDR2，所述VH可包含SEQ ID NO:68、SEQ ID NO:72和SEQ ID NO:76中任一项所示的氨基酸序列
在本申请中，所述分离的抗原结合蛋白可包含HCDR2，且所述HCDR2可包含SEQ ID NO:16、SEQ ID NO:30和SEQ ID NO:44中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链可变区VH中的CDR1，所述VH可包含SEQ ID NO:68、SEQ ID NO:72和SEQ ID NO:76中任一项所示的氨基酸序列
在本申请中，所述分离的抗原结合蛋白可包含HCDR1，且所述HCDR1可包含SEQ ID NO:15、SEQ ID NO:29和SEQ ID NO:43中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，且所述HCDR1可包含SEQ ID NO:15、SEQ ID NO:29和SEQ ID NO:43中任一项所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:16、SEQ ID NO:30和SEQ ID NO:44中任一项所示的氨基酸序列，所述HCDR3可包含SEQ ID NO:17、SEQ ID NO:31和SEQ ID NO:45中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，且所述HCDR1可包含SEQ ID NO:15所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:16所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:17所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，且所述HCDR1可包含SEQ ID NO:29所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:30所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:31所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，且所述HCDR1可包含SEQ ID NO:43所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:44所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:45所示的氨基酸序列。
在本申请中，所述分离的结合蛋白可包含框架区H-FR1，且所述H-FR1可包含SEQ ID NO:21、SEQ ID NO:35和SEQ ID NO:49中任一项所示的氨基酸序列。
在本申请中，所述分离的结合蛋白可包含框架区H-FR2，且所述H-FR2可包含SEQ ID NO:22、SEQ ID NO:36和SEQ ID NO:50中任一项所示的氨基酸序列。
在本申请中，所述分离的结合蛋白可包含框架区H-FR3，且所述H-FR3可包含SEQ ID NO:23、SEQ ID NO:37和SEQ ID NO:51中任一项所示的氨基酸序列。
在本申请中，所述分离的结合蛋白可包含框架区H-FR4，且所述H-FR4可包含SEQ ID  NO:24、SEQ ID NO:38和SEQ ID NO:52中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含H-FR1、H-FR2、H-FR3和H-FR4，且所述H-FR1可包含SEQ ID NO:21、SEQ ID NO:35和SEQ ID NO:49中任一项所示的氨基酸序列，所述H-FR2可包含SEQ ID NO:22、SEQ ID NO:36和SEQ ID NO:50中任一项所示的氨基酸序列，所述H-FR3可包含SEQ ID NO:23、SEQ ID NO:37和SEQ ID NO:51中任一项所示的氨基酸序列，所述H-FR4可包含SEQ ID NO:24、SEQ ID NO:38和SEQ ID NO:52中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含H-FR1、H-FR2、H-FR3和H-FR4，且所述H-FR1可包含SEQ ID NO：21所示的氨基酸序列，所述H-FR2可包含SEQ ID NO：22所示的氨基酸序列，所述H-FR3可包含SEQ ID NO：23所示的氨基酸序列，所述H-FR4可包含SEQ ID NO：24所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含H-FR1、H-FR2、H-FR3和H-FR4，且所述H-FR1可包含SEQ ID NO：35所示的氨基酸序列，所述H-FR2可包含SEQ ID NO：36所示的氨基酸序列，所述H-FR3可包含SEQ ID NO：37所示的氨基酸序列，所述H-FR4可包含SEQ ID NO：38所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含H-FR1、H-FR2、H-FR3和H-FR4，且所述H-FR1可包含SEQ ID NO：49所示的氨基酸序列，所述H-FR2可包含SEQ ID NO：50所示的氨基酸序列，所述H-FR3可包含SEQ ID NO：51所示的氨基酸序列，所述H-FR4可包含SEQ ID NO：52所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链可变区VH，且所述VH可包含SEQ ID NO:68、SEQ ID NO:72和SEQ ID NO:76中任一项所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链可变区VH，且所述VH可包含SEQ ID NO：68所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链可变区VH，且所述VH可包含SEQ ID NO：72所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链可变区VH，且所述VH可包含SEQ ID NO：76所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包括重链恒定区，且所述重链恒定可源自IgG1。
在本申请中，所述重链恒定区可包含SEQ ID NO:57或SEQ ID NO:58所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链，且所述抗体重链可包含SEQ ID NO:60、SEQ ID NO:62和SEQ ID NO:64中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链，且所述抗体重链可包含SEQ ID NO:3、SEQ ID NO:7和SEQ ID NO:11中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含轻链可变区VL中的CDR3，所述VL可包含SEQ ID NO:69、SEQ ID NO:73和SEQ ID NO:77中任一项所示的氨基酸序列
在本申请中，所述分离的抗原结合蛋白可包含LCDR3，且所述LCDR3可包含SEQ ID NO:20、SEQ ID NO:34和SEQ ID NO:48中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含轻链可变区VL中的CDR2，所述VL可包含SEQ ID NO:69、SEQ ID NO:73和SEQ ID NO:77中任一项所示的氨基酸序列
在本申请中，所述分离的抗原结合蛋白可包含LCDR2，且所述LCDR2可包含SEQ ID NO:19、SEQ ID NO:33和SEQ ID NO:47中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含轻链可变区VL中的CDR1，所述VL可包含SEQ ID NO:69、SEQ ID NO:73和SEQ ID NO:77中任一项所示的氨基酸序列
在本申请中，所述分离的抗原结合蛋白可包含LCDR1，且所述LCDR1可包含SEQ ID NO:18、SEQ ID NO:32和SEQ ID NO:46中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含LCDR1、LCDR2和LCDR3，且所述LCDR1包含SEQ ID NO:18、SEQ ID NO:32和SEQ ID NO:46中任一项所示的氨基酸序列，所述LCDR2包含SEQ ID NO:19、SEQ ID NO:33和SEQ ID NO:47中任一项所示的氨基酸序列，所述LCDR3包含SEQ ID NO:20、SEQ ID NO:34和SEQ ID NO:48中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含LCDR1、LCDR2和LCDR3，且所述LCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:19所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:20所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含LCDR1、LCDR2和LCDR3，且所述LCDR1可包含SEQ ID NO:32所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:33所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:34所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含LCDR1、LCDR2和LCDR3，且所述LCDR1可包含SEQ ID NO:46所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:47所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:48所示的氨基酸序列。
在本申请中，所述分离的结合蛋白可包含框架区L-FR1，且所述L-FR1可包含SEQ ID NO:25、SEQ ID NO:39和SEQ ID NO:53中任一项所示的氨基酸序列。
在本申请中，所述分离的结合蛋白可包含框架区L-FR2，且所述L-FR2可包含SEQ ID NO:26、SEQ ID NO:40和SEQ ID NO:54中任一项所示的氨基酸序列。
在本申请中，所述分离的结合蛋白可包含框架区L-FR3，且所述L-FR3可包含SEQ ID NO:27、SEQ ID NO:41和SEQ ID NO:55中任一项所示的氨基酸序列。
在本申请中，所述分离的结合蛋白可包含框架区L-FR4，且所述L-FR4可包含SEQ ID NO:28、SEQ ID NO:42和SEQ ID NO:56中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含L-FR1、L-FR2、L-FR3和L-FR4，且所述所述L-FR1包含SEQ ID NO:25、SEQ ID NO:39和SEQ ID NO:53中任一项所示的氨基酸序列，所述L-FR2包含SEQ ID NO:26、SEQ ID NO:40和SEQ ID NO:54中任一项所示的氨基酸序列，所述L-FR3包含SEQ ID NO:27、SEQ ID NO:41和SEQ ID NO:55中任一项所示的氨基酸序列且所述L-FR4包含SEQ ID NO:28、SEQ ID NO:42和SEQ ID NO:56中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含L-FR1、L-FR2、L-FR3和L-FR4，且所述L-FR1可包含SEQ ID NO：39所示的氨基酸序列，所述L-FR2可包含SEQ ID NO：40所示的氨基酸序列，所述L-FR3可包含SEQ ID NO：41所示的氨基酸序列，所述L-FR4可包含SEQ ID NO：42所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含L-FR1、L-FR2、L-FR3和L-FR4，且所述L-FR1可包含SEQ ID NO：53所示的氨基酸序列，所述L-FR2可包含SEQ ID NO：54所示的氨基酸序列，所述L-FR3可包含SEQ ID NO：55所示的氨基酸序列，所述L-FR4可包含SEQ ID NO：56所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含L-FR1、L-FR2、L-FR3和L-FR4，且所述L-FR1可包含SEQ ID NO：25所示的氨基酸序列，所述L-FR2可包含SEQ ID NO：26所示的氨基酸序列，所述L-FR3可包含SEQ ID NO：27所示的氨基酸序列，所述L-FR4可包含SEQ ID NO：28所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含轻链可变区VL，且所述VL可包含SEQ ID NO:69、SEQ ID NO:73和SEQ ID NO:77中任一项所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含轻链可变区VL，且所述VL可包含SEQ ID NO：69所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含轻链可变区VL，且所述VL可包含SEQ ID NO：73所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含轻链可变区VL，且所述VL可包含SEQ ID NO：77所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包括轻链恒定区，且轻链恒定区可包含SEQ ID NO:59所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体轻链，且所述抗体轻链可包含SEQ ID NO:61、SEQ ID NO:63和SEQ ID NO:65中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体轻链，且所述抗体轻链可包含SEQ ID NO:4、SEQ ID NO:8和SEQ ID NO:12中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1可包含SEQ ID NO:15、SEQ ID NO:29和SEQ ID NO:43中任一项所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:16、SEQ ID NO:30和SEQ ID NO:44中任一项所示的氨基酸序列，所述HCDR3可包含SEQ ID NO:17、SEQ ID NO:31和SEQ ID NO:45中任一项所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:18、SEQ ID NO:32和SEQ ID NO:46中任一项所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:19、SEQ ID NO:33和SEQ ID NO:47中任一项所示的氨基酸序列，所述LCDR3可包含SEQ ID NO:20、SEQ ID NO:34和SEQ ID NO:48中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1可包含SEQ ID NO:15所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:16所示的氨基酸序列，所述HCDR3可包含SEQ ID NO:17所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:19所示的氨基酸序列，所述LCDR3可包含SEQ ID NO:20所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1可包含SEQ ID NO:29所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:30所示的氨基酸序列，所述HCDR3可包含SEQ ID NO:31所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:32所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:33所示的氨基酸序列，所述LCDR3可包含SEQ ID NO:34中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1可包含SEQ ID NO:43所示的氨基酸序列，所述HCDR2可包含 SEQ ID NO:44所示的氨基酸序列，所述HCDR3可包含SEQ ID NO:45所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:46所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:47所示的氨基酸序列，所述LCDR3可包含SEQ ID NO:48所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:68、SEQ ID NO:72和SEQ ID NO:76中任一项所示的氨基酸序列，所述VL可包含SEQ ID NO:69、SEQ ID NO:73和SEQ ID NO:77中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:68所示的氨基酸序列，所述VL可包含SEQ ID NO:69所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:72所示的氨基酸序列，所述VL可包含SEQ ID NO:73所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:76所示的氨基酸序列，所述VL可包含SEQ ID NO:77所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链和轻链，所述重链可包含SEQ ID NO:60、SEQ ID NO:62和SEQ ID NO:64中任一项所示的氨基酸序列，所述轻链可包含SEQ ID NO:61、SEQ ID NO:63和SEQ ID NO:65中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链和轻链，所述重链可包含SEQ ID NO:60所示的氨基酸序列，所述轻链可包含SEQ ID NO:61所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链和轻链，所述重链可包含SEQ ID NO:62所示的氨基酸序列，所述轻链可包含SEQ ID NO:63所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链和轻链，所述重链可包含SEQ ID NO:64所示的氨基酸序列，所述轻链可包含SEQ ID NO:65所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链和轻链，所述重链可包含SEQ ID NO:3、SEQ ID NO:7和SEQ ID NO:11中任一项所示的氨基酸序列，所述轻链可包含SEQ ID NO:4、SEQ ID NO:8和SEQ ID NO:12中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链和轻链，所述重链可包含SEQ ID NO:3所示的氨基酸序列，所述轻链可包含SEQ ID NO:4所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链和轻链，所述重链可包含SEQ ID NO:7所示的氨基酸序列，所述轻链可包含SEQ ID NO:8所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链和轻链，所述重链可包含SEQ ID NO:11所示的氨基酸序列，所述轻链可包含SEQ ID NO:12所示的氨基酸序列。
核酸、载体、宿主细胞和制备方法
在另一个方面，本申请还提供了分离的一种或多种核酸分子，所述一种或多种核酸分子可编码本申请所述的抗原结合蛋白。例如，所述一种或多种核酸分子中的每一个核酸分子可以编码完整的所述抗原结合蛋白，也可以编码其中的一部分(例如，HCDR1-3、LCDR1-3、VL、VH、轻链或重链中的一种或多种)。
本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。
在另一个方面，本申请提供了一种或多种载体，其包含本申请所述的一种或多种核酸分子。每种载体中可包含一种或多种所述核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。例如，所述载体为表达载体。
在另一个方面，本申请提供了宿主细胞，所述宿主细胞可包含本申请所述的一种或多种核酸分子和/或本申请所述的一种或多种载体。在某些实施方式中，每种或每个宿主细胞可包含一个或一种本申请所述的核酸分子或载体。在某些实施方式中，每种或每个宿主细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体
在另一个方面，本申请提供了制备所述抗原结合片段的方法。所述方法可包括，在使得所述的抗体或其抗原结合片段表达的条件下，培养所述本申请所述的宿主细胞。例如，可通过使用适当的培养基、适当的温度和培养时间等，这些方法是本领域普通技术人员所了解的。
药物组合物、方法、用途
另一方面，本申请提供了一种免疫缀合物，其可以包含所述分离的抗原结合蛋白或所述多肽，以及小分子化合物。免疫缀合物通常是指采用特定的连接子将抗体和小分子细胞毒药物连接起来，其主要组成成分可以包括抗体、连接子和小分子细胞毒药物。在本申请中，，所述小分子化合物可以包含细胞毒性剂和/或标记物。在本申请中，所述小分子化合物可以与所述分离的抗原结合蛋白的上氨基酸残基的伯胺基团偶联。在本申请中，所述免疫缀合物能够 使细胞内化。
另一方面，本申请提供了一种试剂盒，其可以包含本申请所述的抗原结合蛋白，嵌合抗原受体，基因修饰的细胞，免疫缀合物，和/或本申请所述的药物组合物。其可在单一常用容器中包括本申请所述的抗原结合蛋白，嵌合抗原受体，基因修饰的细胞，和/或免疫缀合物，也可任选地与一种或多种治疗剂组合，任选地一起配制于药物组合物中。
另一方面，本申请提供了一种给药装置，它可以用来施用本申请所述的抗原结合蛋白或其药物组合物。
在另一个方面，本申请提供了一种药物组合物，其可包含本申请所述的抗原结合蛋白，所述多肽，所述的核酸分子，所述的载体，所述的宿主细胞，以及任选地药学上可接受的载体。所述药学上可接受的佐剂在所采用的剂量和浓度下对接受者无毒性，本申请中的药物组合物还可含有多于一种活性化合物，通常为不会不利地影响彼此的具有互补活性的那些活性化合物。
所述抗原结合蛋白，所述多肽，所述的核酸分子，所述的载体，所述的宿主细胞，所述免疫缀合物和/或所述药物组合物可以用于治疗与EGFR活性有关的疾病。例如了以用于***，或抑制肿瘤生长。例如，本申请的药物组合物可以抑制或延缓疾病的发展或进展，可以减小肿瘤大小(甚至基本消除肿瘤)，和/或可以减轻和/或稳定疾病状态。
本申请所述的药物组合物可以包含预防和/或治疗有效量的所述抗原结合蛋白，所述多肽，所述的核酸分子，所述的载体，所述的宿主细胞，所述免疫缀合物和/或所述药物组合物。所述预防和/或治疗有效量是能够预防和/或治疗(至少部分治疗)患有或具有发展风险的受试者中的疾病或病症和/或其任何并发症而所需的剂量。
另一方面，本申请提供了所述抗原结合蛋白，所述多肽，所述的核酸分子，所述的载体，所述的宿主细胞，所述免疫缀合物和/或所述药物组合物在制备药物中的用途。
所述药物用于治疗与EGFR活性有关的疾病。在某些实施方式中，所述肿瘤包括EGFR异常表达(例如，过表达)的肿瘤。在某些实施方式中，所述与EGFR活性有关的疾病包括肿瘤。在本申请中，所述肿瘤可以包括与EGFR异常表达(例如，过表达)相关的肿瘤。在本申请中，所述肿瘤可以包括实体瘤和/或非实体瘤。例如，所述肿瘤为实体瘤。在本申请中，所述肿瘤可以包括神经胶质细胞瘤、乳腺癌、肺癌、卵巢癌、头颈癌、***、食管癌、***癌、肝癌、结肠癌和/或胃癌。
一方面，本申请提供了一种使细胞内化的方法，其包括施用所述分离的抗原结合蛋白、所述多肽、所述免疫缀合物、所述核酸分子、所述载体、所述细胞和/或所述药物组合物。例 如，所述方法可以是离体或体外方法。例如，所述方法可以是非治疗目的的方法。
另一方面，本申请提供了一种用于检测EGFR蛋白的存在和/或含量的方法，其包括施用所述分离的抗原结合蛋白和/或所述的多肽。例如，所述方法可以是离体或体外方法。例如，所述方法可以是非治疗目的的方法。
本申请还提供了抗原结合蛋白在诊断患有肿瘤或癌症的受试者的方法中的用途，所述方法包括：通过使样品与本申请的抗原结合蛋白接触并检测结合的抗体的存在来确定获自受试者的样品中EGFR的存在或表达水平。
另一方面，本申请还提供了以下实施方案：
1.一种鼠源抗表皮生长因子的单克隆抗体，其特征在于，所述抗体可结合于细胞膜上的EGFR并引起抗原抗体复合物剧烈内化进入肿瘤细胞。
2.如实施方案1所述单克隆抗体，可特异与EGFR的II结构域(氨基酸Ser196-Cys287)结合，引起抗原抗体复合物内化。
3.如实施方案2所述单克隆抗体，至少可特异性结合EGFR的II结构域上如下氨基酸：Ser196，Ser222，Lys269，Ser282。
4.如实施方案2所述单克隆抗体，它包含重链可变区和轻链可变区，其特征在于，所述重链可变区具有SEQ ID NO：3,SEQ ID NO：7,SEQ ID NO：11所示的氨基酸序列，所述轻链可变区具有SEQ ID NO：4，SEQ ID NO：8，SEQ ID NO：12所示的氨基酸序列。
5.根据实施方案4所述的重链可变区，其特征在于，该重链可变区含有SEQ ID NO：1，SEQ ID NO：5，SEQ ID NO：9所示的核苷酸序列。
6.根据实施方案4所述的轻链可变区，其特征在于，该轻链可变区含有SEQ ID NO：2，SEQ ID NO：6，SEQ ID NO：10所示的核苷酸序列。
7.如实施方案2所述单克隆抗体，可与LZ-8竞争性结合于EGFR。
8.如实施方案2所述单克隆抗体，可由经人EGFR蛋白免疫小鼠脾细胞与骨髓瘤细胞融合所获得的杂交瘤细胞进行制备。
9.如实施方案8所述的杂交瘤细胞，其特征在于，可稳定分泌如实施方案2所述的单克隆抗体。
10.如实施方案9所述的杂交瘤细胞，可通过与LZ-8竞争性结合EGFR进行筛选获得。
11.如实施方案10所述的杂交瘤细胞的筛选方式，包括但不限于通过LZ-8的竞争性ELISA筛选。
12.如实施方案2所述单克隆抗体，可与小分子化合物偶联形成抗体-小分子化合物偶联 物。
13.如实施方案12所述抗体-小分子化合物偶联物，其偶联方式包括但不限于此抗体上氨基酸残基的伯胺基团。
14.如实施方案12所述抗体-小分子化合物偶联物，可内化进入肿瘤细胞。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请发明的各个技术方案，而不用于限制本申请发明的范围。
实施例
实施例1 抗原结合蛋白的制备和筛选
准备材料如表1。
表1 实验材料
抗原名称 提供方 数量 目的
人EGFR蛋白 GenScirpt 3mg，(>85％purity,>0.5mg/ml) 免疫/筛选
LZ8 本实验室 1mg，(>85％purity,>0.5mg/ml) 筛选(竞争)
采用人EGFR蛋白对Balb/C，C57BL/6小鼠进行免疫。具体方法如下：
1.动物免疫：EGFR抗原以完全弗氏佐剂乳化，采用皮下或腹腔注射方法免疫小鼠，五组动物(Balb/C小鼠)将按照表2所示进行免疫。免疫后取尾血以ELISA法梯度稀释测定血清效价，选取抗体效价最高的小鼠进行细胞融合。
表2 免疫进程表
流程 时间 免疫途径 免疫剂量
免疫前采血 T＝-4天    
初疫 T＝0天 s.c.注射 每只动物50μgA蛋白
第一次加免 T＝14天s i.p.注射 每只动物25μgA蛋白
测试血清1 T＝21天s    
第二次加免 T＝28天s s.c.注射 每只动物25μgA蛋白
测试血清2 T＝35天s    
终免 T＝56±7天 i.p.注射 每只动物25μgA蛋白
融合 T＝终免+4天    
2.细胞融合和筛选
骨髓瘤细胞采用BALB/c来源的sp2/0，融合时处于对数生长期；取已免疫小鼠脾脏，制成淋巴细胞单细胞悬液；小鼠脾淋巴细胞与骨髓瘤细胞以1:5-1:10混合，滴加37℃的50％PEG(PH 8.0)1ml，加入不完全培养基及其余终止液，离心弃去上清后加入HAT培养基悬浮混 匀，MC定容到50ml，分装到3.5cm培养皿中，放于湿盒中，置于37℃，5％CO 2恒温培养箱中进行培养。
3.筛选和竞争性筛选
细胞融合7-10天内挑选细胞克隆，针对人EGFR蛋白对所有母克隆进行ELISA测试，挑取OD450阳性值较高的120个单克隆孔。将上述单克隆进行一步第一轮亚克隆，进行有限稀释，并针对LZ8和抗体做竞争性ELISA筛选，进而得到12个亚克隆可以阻断LZ8与EGFR结合。将上述亚克隆进行第二轮亚克隆，并再次进行针对LZ8和EGFR抗体竞争性ELISA筛选，最后根据12个亚克隆的阳性值以及上清中抗体的含量分析，得到3个阳性值较高的稳定的单克隆，对应融合板细胞株为UMAB1Z4，UMAB4B1，UMAB6F2。
4.细胞上清单抗的制备与纯化
将上述3个细胞株用含15％血清的DMEM培养基培养于10cm培养皿中，培养至4×10 7小时，800rpm离心5min，弃去上清并将细胞转移到2L瓶中，加入无血清培养基，使细胞密度约为3×10 5个/ml。继续培养1～2周，当细胞死亡率达到60％-70％时，收取细胞悬液6000rpm高速离心20min，取上清，亲和柱层析法进行上清纯化，根据抗体压型选择相应柱料，单抗UMAB1Z4，UMAB4B1，UMAB6F2亚型为IgG1，采用protein G进行纯化。纯化后的单抗浓度测定、分装(100ml/管，浓度为1mg/ml，保存在4-8℃)。
实施例2 抗原结合蛋白的序列确定
对上述细胞株UMAB1Z4(后简称为1Z4)，UMAB4B1(后简称为4B1)，UMAB6F2(后简称为6F2)的抗EGFR单克隆细胞株进行测序。从获得的杂交瘤细胞中提取总RNA，用鼠源抗体可变区通用引物，逆转录成cDNA，进行PCR扩增，获得的轻链和重链可变区委托Genscript公司进行测序。
SEQ ID NO：67和SEQ ID NO：68分别是本申请获得的高活性高单克隆抗体1Z4的重链可变区和轻链可变区的核苷酸编码序列；SEQID NO：69和SEQ ID NO：70分别是1Z4的重链可变区和轻链可变区的氨基酸序列。SEQ ID NO：71和SEQ ID NO：72分别是单克隆抗体4B1的重链可变区和轻链可变区的核苷酸编码序列；SEQID NO：73和SEQ ID NO：74分别是4B1的重链可变区和轻链可变区的氨基酸序列。SEQ ID NO：75和SEQ ID NO：76分别是单克隆抗体6F2的重链可变区和轻链可变区的核苷酸编码序列；SEQID NO：77和SEQ ID NO：78分别是6F2的重链可变区和轻链可变区的氨基酸序列。
实施例3 抗原结合蛋白表达载体的纯化
选用实施例2中1Z4的抗体做进步一的分析，通过PCR扩增，将1Z4抗体重链和轻链可变区分别***到表达载体pcDNA3.1(+)EcoRI和HindIII位点中，构建成鼠源抗EGFR单克隆抗体基因的表达载体。
本实施例采用HiTrapTm MabSelect SuRe蛋白柱技术，纯化表达在Expi293F TM细胞的抗体，以获得浓度在0.5mg/ml，纯度在90％以上的抗体。具体步骤如下：
Expi293F TM细胞在无血清Expi293 TM表达培养基(Thermo Fisher Science)中培养。随后将细胞保存在Erlenmeyer培养瓶中，放置于37℃，8％二氧化碳的培养箱中。当细胞浓度达到2.0×10 6个/mL后，将DNA与ExpiFectamine TM293按1:2.7加入到转染的细胞培养瓶中。
将编码抗体1Z4重链和轻链的重组质粒共同转染到悬浮培养的Expi293F细胞中。转染约17h后，将ExpiFectamine TM293转染增强剂1和ExpiFectamine TM293转染增强剂2加入培养瓶中。转染后第5天测定细胞密度和细胞活力，收集约1mL细胞培养上清液用于抗体表达评价。取第6天收获的细胞培养上清进行纯化。
将第6天收集细胞培养上清液进行抗体纯化。用过滤后的上清液以3ml/min加载到HiTrapTM MabSelect Sure 5ml(GE，目录号11-0034-95)上。用4柱体积的PBS缓冲液(pH7.2)洗脱，用50mM柠檬酸(pH3.0)洗脱。在洗脱过程中，每1mL组分立即用124mol/L 1M Tris-HCl缓冲液(pH9.0)中和。将部分峰混合在一起，然后将缓冲液交换到PBS缓冲液(pH7.2)。用SDS-PAGE和Western blot分析其分子量、蛋白产量和纯度(图1)。样品浓度用A280法测定，消光系数为1.505。
实施例4 LZ8与EGFR结合位点测定
(1)EGFR胞外域和LZ8的化学交联
为定位LZ8与EGFR结合的表位，将EGFR胞外区与单克隆抗体在磷酸缓冲液中孵化，并采用DSS/BS3交联剂按照比例(1:4)室温交联1h之后，SDS-PAGE凝胶分离交联物并储存在10％醋酸溶液中。
(2)液质串联质谱分析
蛋白中的半胱氨酸残基分别用Tris-(carboxyethyl)phosphine hydrochloride(TCEP)试剂和chloroacetamide(CAA)试剂进行还原和烷基化，分别在56℃反应60分钟以及在室温下反应45分钟。随后用胰蛋白酶(Thermo Fisher Scientific)在37℃条件下酶切过夜，经过C18柱脱盐后上样到液质联用串联质谱中；
肽段经过纳升流液相***Nano1200，流速为300nL/min，洗脱梯度为:5to 60％B(60min),60–70％B(20min),and 70％B(10min)。其中A液为0.1％formic acid in water，B液为95％Acetonitrile,0.1％formic acid in water；
质谱数据采集在Orbitrap Fusion Lumos串联质谱上进行(Thermo Fisher Scientific)，一级谱采集的质荷比的范围是400–2000，分辨率为70,000(m/z 400)。二级谱的数据采集方式为数据依赖型，对一级谱中信号最强的前10个肽段进一步进行二级谱的数据采集，分辨率为35,000(m/z 400)，最后通过SIM-XL对交联肽段进行鉴定。
(3)等温量热滴定(ITC)实验
等温量热滴定实验用MicroCal VP-ITC(GE Healthcare)仪器进行。将400ìM LZ8依次滴入到含有20ìM EGFR胞外域的样品池中，每滴3ml，总共19滴，反应的缓冲液为20mm磷酸缓冲液，150mm NaCl(pH 8.0)，温度为20℃。将获得的ITC数据用MicroCal Origin软件进行分析，求出
Figure PCTCN2021076393-appb-000001
K，n以及解离常数值。
结果表明，EGFR的胞外域与LZ8形成复合体时的摩尔比为1:2，这暗示EGFR的胞外域以单体的形式与LZ8的二聚体互作。结果如图2所示。
随后将经过交联形成的EGFR胞外域与LZ8复合体用SDS-PAGE分离后用胰蛋白酶处理，结果如图3所示，然后进行了串联质谱的分析。
串联质谱总共检测到26条交联肽段，其中有3条具有高置信度的交联肽段是由EGFR的胞外域与LZ8之间的交联形成的，能够反映EGFR与LZ8互作的界面信息。随后将EGFR与LZ8之间发生交联的位点分别标记在LZ8和EGFR胞外域的三维结构上(图4)。从图4中可以看出，EGFR胞外域与LZ8的作用界面包括丝氨酸169位，丝氨酸222位以及赖氨酸269位，这三个位点与EGFR的胞外域的二聚体化有关。LZ8蛋白上参与互作的氨基酸为41位赖氨酸，这个残基在LZ8蛋白的环BC上的。此外，利用“零长度”的交联剂NHS/EDC对EGFR/LZ8复合体进行化学交联，进一步验证利用BS3/DSS获得的交联结果。结果显示，在LZ8的第20位的天冬氨酸和EGFR的第282位丝氨酸之间形成的交联肽段，而这两个氨基酸残基恰好在上面鉴定到的EGFR/LZ8作用界面的附近，进一步验证了交联结果的可靠性。
结果显示，LZ8与EGFR结合表位位于EGFR的II结构域(氨基酸Ser196-Cys287)。
实施例5 结合计算机模拟推测LZ8与EGFR结合关键位点
根据LZ8的结构(PDB：3F3H.pdb)，在Discovery Studio 4.0中进行prepare ligand,EGFR的结构在PDB bank中下载，PDB ID:4kro.pdb，在Discovery Studio 4.0中进行去水，加氢再进行prepare protein,然后将配体与EGFR靶点进行CDOCKER对接，参数设置如下：Top  Hits＝10,RandomConformations＝10,Orientations to Refine＝10,Force field＝CHARMm,Use Full Potential＝False,其他参数选择默认设置。
经过对接后，确定LZ8和EGFR在氨基酸Ser196-Cys287形成结合界面，其中4个氨基酸残基位点(Ser196，Ser222，Lys269，Ser282)与EGFR有较强的键合作用力，表明其为抗原结合蛋白与EGFR作用的关键氨基酸残基。实验结果如图5所示。
实施例6 抗原结合蛋白1Z4可携载小分子化合物内化进入肿瘤细胞
本实施例将Alexa Fluor 568NHS Ester(AF568)偶联到1Z4抗体，以检测1Z4抗体在小分子化合物修饰后的内化能力。AF568是分子量为791.8的小分子化合物，其结构如图6所示。AF568可与蛋白质氨基酸残基中伯胺基团(R-NH 2)发生反应，从而偶联形成复合物。
本实施例首先将10mg的1Z4抗体溶于1mL的0.1M碳酸氢钠缓冲液中，5mg AF568溶解在0.5mL DMSO中，然后在涡旋搅拌1Z4抗体溶液的同时，缓慢加入100ìL AF568 DMSO溶液，并于室温下搅拌1小时，使其充分反应。反应结束后，使用Sephadex TM G-25凝胶过滤层析柱进行纯化，如图7所示，收集第一个峰的产物即为1Z4-AF568偶联物，第二个峰为过量未偶联的AF568。
将40nM 1Z4-AF568偶联物在4℃与HeLa细胞孵育30分钟后使其与细胞表面受体结合，然后转移到374℃细胞培养箱中继续孵育1小时。采用OMX超高分辨率显微镜观察1Z4-AF568偶联物内化情况。结果表明，1Z4-AF568偶联物可以囊泡形式内化进入肿瘤细胞中，囊泡直径1微米左右(图8)。
实施例7 本申请所述抗原结合蛋白可结合细胞膜上的EGFR并引发高强度内化
7.1抗原结合蛋白1Z4、6F2和4B1可结合细胞膜上的EGFR并引发高强度内化
分别将小分子化合物Alexa Fluor 568NHS Ester修饰后的40nM 1Z4、6F2和4B1抗体(修饰方法同实施例6)在4℃与HeLa细胞孵育30分钟，然后在37℃继续孵育1小时使其内化。采用OMX成像***观察1Z4、6F2和4B1抗体的内化情况。图13、图14和图15的结果表明1Z4、6F2和4B1抗体均成功内化进入细胞中。
7.2抗原结合蛋白1Z4可结合细胞膜上的EGFR并引发高强度内化
以EGFR单克隆抗体西妥昔单抗(Cetuximab)为对照，采用流式细胞术检测了1Z4单克隆抗体与EGFR阳性细胞系MD-MB-468和EGFR阴性细胞系MD-MB-453细胞表面EGFR的结合情况。将Alexa Fluor 647NHS Ester修饰的1Z4(修饰方法同实施例6)在4℃分别与两种细胞孵育30分钟，再用预冷PBS溶液洗去未结合抗体后进行流式检测。结果表明1Z4 单克隆抗体可特异性的结合于EGFR阳性细胞系MD-MB-468，而不与EGFR阴性细胞系MD-MB-453结合(图9)。
以西妥昔单抗和mAb806为对照，采用高内涵成像***分析了1Z4抗体的内化强度。分别将小分子化合物Alexa Fluor 568NHS Ester修饰后的40nM西妥昔单抗、mAb806和1Z4抗体(修饰方法同实施例6)在4℃与HeLa细胞孵育30分钟，然后在37℃继续孵育1小时使其内化。结果表明1Z4的内化强度远远高于西妥昔单抗和mAb806(图10)。
实施例8 抗原结合蛋白1Z4与EGFR结合位点的验证
将Alexa Fluor 488偶联到1Z4单克隆抗体(修饰方法同实施例6)，以比较1Z4单克隆抗体与突变EGFR(S196A/S222A/K269A/S282A)和野生型EGFR的结合能力。其中突变EGFR突变位点为LZ8与EGFR结合的关键位点。
本实施例选取NIH3T3 EGFR(S196A/S222A/K269A/S282A)突变细胞株和NIH3T3 EGFR wildtype细胞株开展相关研究。取mAb1Z4-Alexa Fluor 488与上述贴壁培养的细胞分别在冰上孵育30min使其与细胞表面受体结合，然后用多聚甲醛固定，并用Hoechst 33342浸染细胞核。采用高内涵***分析平均荧光强度来体现在细胞水平上mAb1Z4与EGFR及其突变体的结合能力差别。
图11和图12的结果表明，1Z4与野生型EGFR的结合能力明显优于突变型EGFR，这说明S196A、S222A、K269A和S282A是影响mAb1Z4与EGFR结合的关键位点。此外已有研究证明S196A、S222A、K269A和S282A位于LZ8与EGFR的结合界面，是LZ8结合EGFR的关键位点，而mAb1Z4是通过与LZ8竞争性结合EGFR筛选获得的。综上所述，1Z4与EGFR结合的位点与LZ8相同，S196A、S222A、K269A和S282A均为两者的关键结合位点。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方式的范围内。

Claims (68)

  1. 一种分离的抗原结合蛋白，其能够特异性结合细胞膜上的表皮生长因子受体(EGFR)，使复合物内化。
  2. 根据权利要求1所述的分离的抗原结合蛋白，其能够特异性结合EGFR的II结构域，所述EGFR的II结构域包含SEQ ID NO:13所示的氨基酸序列。
  3. 根据权利要求1或2所述的分离的抗原结合蛋白，其能够特异性结合EGFR的II结构域上至少一个选自下组的氨基酸：Ser196，Ser222，Lys269和Ser282。
  4. 根据权利要求1-3中任一项所述的分离的抗原结合蛋白，其能够与LZ-8蛋白竞争性结合所述EGFR。
  5. 根据权利要求1-4中任一项所述的分离的抗原结合蛋白，其能够与参比抗体竞争结合所述EGFR，其中所述参比抗体包含重链可变区VH和轻链可变区VL，所述参比抗体的VH包含HCDR1、HCDR2和HCDR3，所述参比抗体的VL包含LCDR1、LCDR2和LCDR3，且所述参比抗体的HCDR1包含SEQ ID NO:15、SEQ ID NO:29和SEQ ID NO:43中任一项所示的氨基酸序列，所述参比抗体的HCDR2包含SEQ ID NO:16、SEQ ID NO:30和SEQ ID NO:44中任一项所示的氨基酸序列，所述参比抗体的HCDR3包含SEQ ID NO:17、SEQ ID NO:31和SEQ ID NO:45中任一项所示的氨基酸序列，所述参比抗体的LCDR1包含SEQ ID NO:18、SEQ ID NO:32和SEQ ID NO:46中任一项所示的氨基酸序列，所述参比抗体的LCDR2包含SEQ ID NO:19、SEQ ID NO:33和SEQ ID NO:47中任一项所示的氨基酸序列，所述参比抗体的LCDR3包含SEQ ID NO:20、SEQ ID NO:34和SEQ ID NO:48中任一项所示的氨基酸序列。
  6. 根据权利要求1-5中任一项所述的分离的抗原结合蛋白，其包括抗体或其抗原结合片段。
  7. 根据权利要求6所述的分离的抗原结合蛋白，其中所述抗原结合片段包括Fab，Fab’，F(ab) 2、Fv片段、F(ab’) 2、scFv、di-scFv、VHH和/或dAb。
  8. 根据权利要求1-7中任一项所述的分离的抗原结合蛋白，其中所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
  9. 根据权利要求1-8中任一项所述的分离的抗原结合蛋白，其包含重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:68、SEQ ID NO:72和SEQ ID NO:76中任一项所示的氨基酸序列。
  10. 根据权利要求1-9中任一项所述的分离的抗原结合蛋白，其包含HCDR3，且所述HCDR3包含SEQ ID NO:17、SEQ ID NO:31和SEQ ID NO:45中任一项所示的氨基酸序列。
  11. 根据权利要求1-10中任一项所述的分离的抗原结合蛋白，其包含HCDR2，且所述HCDR2包含SEQ ID NO:16、SEQ ID NO:30和SEQ ID NO:44中任一项所示的氨基酸序列。
  12. 根据权利要求1-11中任一项所述的分离的抗原结合蛋白，其包含HCDR1，且所述HCDR1包含SEQ ID NO:15、SEQ ID NO:29和SEQ ID NO:43中任一项所示的氨基酸序列。
  13. 根据权利要求1-12中任一项所述的分离的抗原结合蛋白，其包含HCDR1、HCDR2和HCDR3，且所述HCDR1包含SEQ ID NO:15、SEQ ID NO:29和SEQ ID NO:43中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:16、SEQ ID NO:30和SEQ ID NO:44中任一项所示的氨基酸序列，所述HCDR3包含SEQ ID NO:17、SEQ ID NO:31和SEQ ID NO:45中任一项所示的氨基酸序列。
  14. 根据权利要求1-13中任一项所述的分离的抗原结合蛋白，其包含HCDR1、HCDR2和HCDR3，且所述HCDR1、HCDR2和HCDR3选自以下任一组氨基酸序列：
    (1)HCDR1：SEQ ID NO:15，HCDR2：SEQ ID NO:16和HCDR3：SEQ ID NO:17；(2)HCDR1：SEQ ID NO:29，HCDR2：SEQ ID NO:30和HCDR3：SEQ ID NO:31；和，
    (3)HCDR1：SEQ ID NO:43，HCDR2：SEQ ID NO:44和HCDR3：SEQ ID NO:45。
  15. 根据权利要求1-14中任一项所述的分离的抗原结合蛋白，其包含重链可变区VH，其中所述VH包括框架区H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:21、SEQ ID NO:35和SEQ ID NO:49中任一项所示的氨基酸序列。
  16. 根据权利要求1-15中任一项所述的分离的抗原结合蛋白，其包含重链可变区VH，其中所述VH包括框架区H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:22、SEQ ID NO:36和SEQ ID NO:50中任一项所示的氨基酸序列。
  17. 根据权利要求1-16中任一项所述的分离的抗原结合蛋白，其包含重链可变区VH，其中所述VH包括框架区H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:23、SEQ ID NO:37和SEQ ID NO:51中任一项所示的氨基酸序列。
  18. 根据权利要求1-17中任一项所述的分离的抗原结合蛋白，其包含重链可变区VH，其中所述VH包括框架区H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所 述H-FR4包含SEQ ID NO:24、SEQ ID NO:38和SEQ ID NO:52中任一项所示的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的分离的抗原结合蛋白，其包含H-FR1、H-FR2、H-FR3和H-FR4，其中所述H-FR1包含SEQ ID NO:21、SEQ ID NO:35和SEQ ID NO:49中任一项所示的氨基酸序列，所述H-FR2包含SEQ ID NO:22、SEQ ID NO:36和SEQ ID NO:50中任一项所示的氨基酸序列，所述H-FR3包含SEQ ID NO:23、SEQ ID NO:37和SEQ ID NO:51中任一项所示的氨基酸序列且所述H-FR4包含SEQ ID NO:24、SEQ ID NO:38和SEQ ID NO:52中任一项所示的氨基酸序列。
  20. 根据权利要求1-19中任一项所述的分离的抗原结合蛋白，其包含H-FR1、H-FR2、H-FR3和H-FR4，且所述H-FR1、H-FR2、H-FR3和H-FR4选自以下任一组氨基酸序列：
    (1)F-FR1：SEQ ID NO:21，H-FR2：SEQ ID NO:22，H-FR3：SEQ ID NO:23和H-FR4：SEQ ID NO:24；
    (2)H-FR1：SEQ ID NO:35，H-FR2：SEQ ID NO:36，H-FR3：SEQ ID NO:37和H-FR4：SEQ ID NO:38；和，
    (3)H-FR1：SEQ ID NO:49，H-FR2：SEQ ID NO:50，H-FR3：SEQ ID NO:51和H-FR4：SEQ ID NO:52。
  21. 根据权利要求1-20中任一项所述的分离的抗原结合蛋白，其包含重链可变区VH，所述重链可变区VH包含SEQ ID NO:68、SEQ ID NO:72和SEQ ID NO:76中任一项所示的氨基酸序列。
  22. 根据权利要求1-21中任一项所述的分离的抗原结合蛋白，其包括重链恒定区，且所述重链恒定区源自IgG1。
  23. 根据权利要求1-22中任一项所述的分离的抗原结合蛋白，其中所述重链恒定区包含SEQ ID NO:57或SEQ ID NO:58所示的氨基酸序列。
  24. 根据权利要求1-23中任一项所述的分离的抗原结合蛋白，其包含抗体重链，且所述抗体重链包含SEQ ID NO:60、SEQ ID NO:62和SEQ ID NO:64中任一项所示的氨基酸序列。
  25. 根据权利要求1-24中任一项所述的分离的抗原结合蛋白，其包含轻链可变区VL中的至少一个CDR，所述VL包含SEQ ID NO:69、SEQ ID NO:73和SEQ ID NO:77中任一项所示的氨基酸序列。
  26. 根据权利要求1-25中任一项所述的分离的抗原结合蛋白，其包含LCDR3，且所述 LCDR3包含SEQ ID NO:20、SEQ ID NO:34和SEQ ID NO:48中任一项所示的氨基酸序列。
  27. 根据权利要求1-26中任一项所述的分离的抗原结合蛋白，其包含LCDR2，且所述LCDR2包含SEQ ID NO:19、SEQ ID NO:33和SEQ ID NO:47中任一项所示的氨基酸序列。
  28. 根据权利要求1-27中任一项所述的分离的抗原结合蛋白，其包含LCDR1，且所述LCDR1包含SEQ ID NO:18、SEQ ID NO:32和SEQ ID NO:46中任一项所示的氨基酸序列。
  29. 根据权利要求1-28中任一项所述的分离的抗原结合蛋白，其包含LCDR1、LCDR2和LCDR3，且所述LCDR1包含SEQ ID NO:18、SEQ ID NO:32和SEQ ID NO:46中任一项所示的氨基酸序列，所述LCDR2包含SEQ ID NO:19、SEQ ID NO:33和SEQ ID NO:47中任一项所示的氨基酸序列，所述LCDR3包含SEQ ID NO:20、SEQ ID NO:34和SEQ ID NO:48中任一项所示的氨基酸序列。
  30. 根据权利要求1-29中任一项所述的分离的抗原结合蛋白，其包含LCDR1、LCDR2和LCDR3，且所述LCDR1、LCDR2和LCDR3选自以下任一组氨基酸序列：
    (1)LCDR1：SEQ ID NO:18，LCDR2：SEQ ID NO:19和LCDR3：SEQ ID NO:20；
    (2)LCDR1：SEQ ID NO:32，LCDR2：SEQ ID NO:33和LCDR3：SEQ ID NO:34；和，
    (3)LCDR1：SEQ ID NO:46，LCDR2：SEQ ID NO:47和LCDR3：SEQ ID NO:48。
  31. 根据权利要求1-30中任一项所述的分离的抗原结合蛋白，其包含轻链可变区VL，其中所述VL包括框架区L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:25、SEQ ID NO:39和SEQ ID NO:53中任一项所示的氨基酸序列。
  32. 根据权利要求1-31中任一项所述的分离的抗原结合蛋白，其包含轻链可变区VL，其中所述VL包含框架区L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:26、SEQ ID NO:40和SEQ ID NO:54中任一项所示的氨基酸序列。
  33. 根据权利要求1-32中任一项所述的分离的抗原结合蛋白，其包含轻链可变区VL，其中所述VL包含框架区L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:27、SEQ ID NO:41和SEQ ID NO:55中任一项所示的氨基酸序列。
  34. 根据权利要求1-33中任一项所述的分离的抗原结合蛋白，其包含轻链可变区VL，其中所述VL包含框架区L-FR4，所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4包含SEQ ID NO:28、SEQ ID NO:42和SEQ ID NO:56中任一项所示的氨基酸序列。
  35. 根据权利要求1-34中任一项所述的分离的抗原结合蛋白，其包含L-FR1、L-FR2、L-FR3和L-FR4，其中所述L-FR1包含SEQ ID NO:25、SEQ ID NO:39和SEQ ID NO:53中任一项所示的氨基酸序列，所述L-FR2包含SEQ ID NO:26、SEQ ID NO:40和SEQ ID NO:54中任一项所示的氨基酸序列，所述L-FR3包含SEQ ID NO:27、SEQ ID NO:41和SEQ ID NO:55中任一项所示的氨基酸序列且所述L-FR4包含SEQ ID NO:28、SEQ ID NO:42和SEQ ID NO:56中任一项所示的氨基酸序列。
  36. 根据权利要求1-35中任一项所述的分离的抗原结合蛋白，其包含L-FR1、L-FR2、L-FR3和L-FR4，且所述L-FR1、L-FR2、L-FR3和L-FR4选自以下任一组氨基酸序列：
    (1)F-FR1：SEQ ID NO:39，L-FR2：SEQ ID NO:40，L-FR3：SEQ ID NO:41和L-FR4：SEQ ID NO:42；
    (2)L-FR1：SEQ ID NO:53，L-FR2：SEQ ID NO:54，L-FR3：SEQ ID NO:55和L-FR4：SEQ ID NO:56；和，
    (3)L-FR1：SEQ ID NO:25，L-FR2：SEQ ID NO:26，L-FR3：SEQ ID NO:27和L-FR4：SEQ ID NO:28。
  37. 根据权利要求1-36中任一项所述的分离的抗原结合蛋白，其包含轻链可变区VL，且所述轻链可变区VL包含SEQ ID NO:69、SEQ ID NO:73和SEQ ID NO:77中任一项所示的氨基酸序列。
  38. 根据权利要求1-37中任一项所述的分离的抗原结合蛋白，其包括轻链恒定区，且所述轻链恒定区包含SEQ ID NO:59所示的氨基酸序列。
  39. 根据权利要求1-38中任一项所述的分离的抗原结合蛋白，其包含抗体轻链，且所述抗体轻链包含SEQ ID NO:61、SEQ ID NO:63和SEQ ID NO:65中任一项所示的氨基酸序列。
  40. 多肽，其包含权利要求1-39中任一项所述的分离的抗原结合蛋白。
  41. 免疫缀合物，其包含权利要求1-39中任一项所述的分离的抗原结合蛋白或权利要求40所述的多肽，以及小分子化合物。
  42. 根据权利要求41所述的免疫缀合物，所述小分子化合物包含细胞毒性剂和/或标记物。
  43. 根据权利要求41-42中任一项所述的免疫缀合物，所述小分子化合物与所述分离的抗原结合蛋白的上氨基酸残基的伯胺基团偶联。
  44. 根据权利要求41-43中任一项所述的免疫缀合物，其能够使复合物内化。
  45. 分离的一种或多种核酸分子，其编码权利要求1-39中任一项所述的分离的抗原结合蛋白。
  46. 根据权利要求45所述的核酸分子，其包含SEQ ID NO：1，SEQID NO：5、SEQ ID NO：9、SEQ ID NO：2，SEQID NO：6、SEQ ID NO：10、SEQ ID NO：66，SEQID NO：67、SEQ ID NO：70、SEQ ID NO：71，SEQID NO：74和SEQ ID NO：75中任一项所示的核苷酸序列。
  47. 载体，其包含根据权利要求45-46中任一项所述的核酸分子。
  48. 细胞，其包含根据权利要求45-46中任一项所述的核酸分子或根据权利要求47所述的载体。
  49. 制备权利要求1-39中任一项所述的分离的抗原结合蛋白的方法，所述方法包括在使得权利要求1-39中任一项所述的分离的抗原结合蛋白表达的条件下，培养根据权利要求48所述的细胞。
  50. 药物组合物，其包含权利要求1-39中任一项所述的分离的抗原结合蛋白、权利要求40所述的多肽、权利要求41-44中任一项所述的免疫缀合物、权利要求45-46中任一项所述的核酸分子、权利要求47所述的载体和/或权利要求48所述的细胞，以及任选地药学上可接受的载体。
  51. 权利要求1-39中任一项所述的分离的抗原结合蛋白、权利要求40所述的多肽、权利要求41-44中任一项所述的免疫缀合物、权利要求45-46中任一项所述的核酸分子、权利要求47所述的载体、权利要求48所述的细胞和/或权利要求50所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗与EGFR活性有关的疾病。
  52. 根据权利要求51所述的用途，所述与EGFR活性有关的疾病包括肿瘤。
  53. 根据权利要求52所述的用途，所述肿瘤包括与EGFR异常表达相关的肿瘤。
  54. 根据权利要求52-53中任一项所述的用途，所述肿瘤包括实体瘤。
  55. 根据权利要求52-54中任一项所述的用途，所述肿瘤包括神经胶质细胞瘤、乳腺癌、肺癌、卵巢癌、头颈癌、***、食管癌、***癌、肝癌、结肠癌和/或胃癌。
  56. 预防、缓解或治疗与EGFR活性有关的疾病的方法，所述方法包括向有需要的受试者施用权利要求1-39中任一项所述的分离的抗原结合蛋白、权利要求40所述的多肽、权利要求41-44中任一项所述的免疫缀合物、权利要求45-46中任一项所述的核酸分子、权 利要求47所述的载体、权利要求48所述的细胞和/或权利要求50所述的药物组合物。
  57. 根据权利要求56所述的方法，所述与EGFR活性有关的疾病包括肿瘤。
  58. 根据权利要求57所述的方法，所述肿瘤包括与EGFR异常表达相关的肿瘤。
  59. 根据权利要求57-58中任一项所述的方法，所述肿瘤包括实体瘤。
  60. 根据权利要求57-59中任一项所述的方法，所述肿瘤包括神经胶质细胞瘤、乳腺癌、肺癌、卵巢癌、头颈癌、***、食管癌、***癌、肝癌、结肠癌和/或胃癌。
  61. 权利要求1-39中任一项所述的分离的抗原结合蛋白、权利要求40所述的多肽、权利要求41-44中任一项所述的免疫缀合物、权利要求45-46中任一项所述的核酸分子、权利要求47所述的载体、权利要求48所述的细胞和/或权利要求50所述的药物组合物，用于预防、缓解或治疗与EGFR活性有关的疾病。
  62. 根据权利要求61所述的分离的抗原结合蛋白、多肽、免疫缀合物、核酸分子、载体、细胞和/或药物组合物，所述与EGFR活性有关的疾病包括肿瘤。
  63. 根据权利要求62所述的分离的抗原结合蛋白、多肽、免疫缀合物、核酸分子、载体、细胞和/或药物组合物，所述肿瘤包括与EGFR异常表达相关的肿瘤。
  64. 根据权利要求62-63中任一项所述的分离的抗原结合蛋白、多肽、免疫缀合物、核酸分子、载体、细胞和/或药物组合物，所述肿瘤包括实体瘤。
  65. 根据权利要求62-64中任一项所述的分离的抗原结合蛋白、多肽、免疫缀合物、核酸分子、载体、细胞和/或药物组合物，所述肿瘤包括神经胶质细胞瘤、乳腺癌、肺癌、卵巢癌、头颈癌、***、食管癌、***癌、肝癌、结肠癌和/或胃癌。
  66. 一种用于检测EGFR的存在和/或含量的方法，其包括施用权利要求1-39中任一项所述的分离的抗原结合蛋白或权利要求40所述的多肽。
  67. 试剂盒，其包含权利要求1-39中任一项所述的分离的抗原结合蛋白、权利要求40所述的多肽、权利要求41-44中任一项所述的免疫缀合物、权利要求45-46中任一项所述的核酸分子、权利要求47所述的载体、权利要求48所述的细胞和/或权利要求50所述的药物组合物。
  68. 一种使复合物内化的方法，其包括施用权利要求1-39中任一项所述的分离的抗原结合蛋白、权利要求40所述的多肽、权利要求41-44中任一项所述的免疫缀合物、权利要求45-46中任一项所述的核酸分子、权利要求47所述的载体、权利要求48所述的细胞和/或权利要求50所述的药物组合物。
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