WO2019114793A1 - 一种egfr抗体及其制备方法和应用 - Google Patents

一种egfr抗体及其制备方法和应用 Download PDF

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WO2019114793A1
WO2019114793A1 PCT/CN2018/120906 CN2018120906W WO2019114793A1 WO 2019114793 A1 WO2019114793 A1 WO 2019114793A1 CN 2018120906 W CN2018120906 W CN 2018120906W WO 2019114793 A1 WO2019114793 A1 WO 2019114793A1
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seq
amino acid
sequence
sequence listing
acid sequence
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PCT/CN2018/120906
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French (fr)
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张莹
刘思琪
杨翠青
刘礼乐
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凯惠科技发展(上海)有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the invention relates to the field of biomedicine, in particular to an EGFR antibody and a preparation method and application thereof.
  • EGFR Human epidermal growth factor receptor
  • Erb-B1 Human epidermal growth factor receptor
  • Aminokinase activity (Modjtahedi et al., Br. J. Cancer 73: 228-235, 1996; Herbst and Shin, Cancer 94: 1593-1611, 2002).
  • the full length sequence of EGFR is numbered P00533 in the SwissProt database.
  • EGFR regulates a variety of cellular physiological processes through tyrosine kinase-mediated signal transduction pathways, including cell proliferation and differentiation, cell survival and apoptosis, angiogenesis, and cell mitosis and cell migration (Atalay et al., Ann .Oncology 14: 1346-1363, 2003; Tsao and Herbst, Signal 4: 4-9, 2003; Herbst and Shin, Cancer 94: 1593-1611, 2002; Modjtahedi et al., Br. J. Cancer 73: 228-235 , 1996).
  • Ligands for EGFR include EGF, TGFA/TGF-alpha, amphiregulin, epigen/EPGN, BTC/betacellulin, epiregulin/EREG and HBEGF/heparin-binding EGF.
  • Receptor-ligand binding triggers EGFR formation of homologous or heterodimers, allowing autophosphorylation of the intracellular domain, further activating complex downstream signaling cascades, including the following signaling pathways: RAS- RAF-MEK-ERK signaling pathway, phosphatidylinositol 3-kinase (PI3K) signaling pathway, PLCgamma-PKC signaling pathway and STATs modules signaling pathway.
  • PI3K phosphatidylinositol 3-kinase
  • Overexpression of EGFR has been found in a variety of tumors, including bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, prostate cancer, and kidney cancer (Atalay et al., Ann . Oncology 14: 1346-1363, 2003; Herbst and Shin, Cancer 94: 1593-1611, 2002; and Modjtahedi et al., Br. J. Cancer 73: 228-235, 1996). In many cases, overexpression of EGFR is associated with poor prognosis in patients. (Herbst and Shin, Cancer 94: 1593-1611, 2002; Modjtahedi et al., Br. J. Cancer 73: 228-235, 1996). EGFR is also expressed in normal tissues and is expressed in higher amounts in epithelial tissues of the skin, liver and gastrointestinal tract, but at a much lower level than tumor tissues (Herbst and Shin, Cancer 94: 1593-1611, 2002).
  • the effective rate (ORR) of treatment of colorectal cancer with Erbitux and irinotecan is 23%, and the effective rate of treatment of head and neck cancer with fluoropyrimidine and other chemotherapy drugs is 13%-30%. Due to the chimeric antibody of human and mouse, Erbitux produced antibody responses in 3.7% of patients in clinical trials.
  • panitumumab (Vectibix, panitumumab, Amgen), a fully humanized monoclonal antibody prepared using transgenic mouse technology, without a murine protein sequence.
  • the antibody targets the epidermal growth factor receptor (EGFR) and was approved by the FDA in September 2006. It is used in combination with fluoropyrimidine, oxaliplatin and irinotecan or after chemotherapy to treat EGFR-positive metastatic junctions. Rectal cancer. In 2006, the FDA approved its monotherapy for chemotherapy-resistant metastatic colorectal cancer (mCRC). However, panitumumab is an IgG2 subtype antibody.
  • antibody drugs having a higher biological activity against the epidermal growth factor receptor particularly antibody drugs which are effective against KRAS mutants, such as bispecific antibody drugs, antibody-conjugated drugs, and inlays.
  • Antigen receptor T cell immunotherapy and the like to further improve the therapeutic effect.
  • the technical problem to be solved by the present invention is to overcome the defects of the current lack of EGFR antibody, and to provide an EGFR antibody with high affinity and specificity, a preparation method and application thereof.
  • the EGFR antibody has high affinity with the EGFR protein and has a cytotoxic effect on EGFR-positive cells, and thus can be used for the preparation of drugs for treating tumors and the like.
  • the invention adopts hybridoma technology, comprehensively uses a plurality of immunization methods, and screens by using effective cell level functional experiments, and develops a group of EGFR lead antibodies with high affinity, high biological activity and high diversity.
  • the amino acid sequence of the heavy chain variable region of the EGFR antibody and the light chain variable region of the EGFR antibody is then sequenced by molecular biological methods.
  • the antibody or antigen-binding region obtained by the hybridoma technique of the present invention comprises an antibody light chain variable region and a heavy chain variable region, and each variable region comprises three domains of CDR1, CDR2 and CDR3.
  • Methods and techniques for identifying CDRs within an antibody molecule's heavy chain variable region and light chain variable region, and antibody variable region amino acid sequences are well known in the art and can be used to identify specific antibodies disclosed herein.
  • CDRs within the amino acid sequence of the variable region are well known in the art and can be used to identify specific antibodies disclosed herein.
  • Boundary example conventions for CDRs that can be used for identification include, for example, Kabat definition, Chothia definition, and AbM definition.
  • the Kabat definition is based on sequence diversity
  • the Chothia definition is based on the position of the structural loop region
  • the AbM definition is a compromise between the Kabat definition and the Chothia method.
  • Other public databases can also be used to identify CDR sequences in antibodies.
  • the numbering of CDRs in the present invention is based on the definition of Chothia.
  • the invention provides an EGFR antibody comprising a complementarity determining region (CDR): one or more of a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and/or a light chain CDR1, a light chain CDR2 of an EGFR antibody And one or more of the light chain CDR3, wherein the amino acid sequence of the heavy chain CDR1 is SEQ ID No. 2, SEQ ID No. 10, SEQ ID No. 18, SEQ ID No. 26 in the sequence listing, SEQ ID No. 34, SEQ ID No. 42, SEQ ID No. 50, SEQ ID No. 58, SEQ ID No. 66, SEQ ID No. 74, SEQ ID No. 82 or SEQ ID No.
  • CDR complementarity determining region
  • the amino acid sequence of the heavy chain CDR2 is SEQ ID No. 3, SEQ ID No. 11, SEQ ID No. 19, SEQ ID No. 27, SEQ ID No. 35, SEQ ID No. 43, SEQ ID in the sequence listing. No. 51, SEQ ID No. 59, SEQ ID No. 67, SEQ ID No. 75, SEQ ID No. 83 or SEQ ID No. 91;
  • the amino acid sequence of the heavy chain CDR3 is as SEQ ID in the Sequence Listing No. 4, SEQ ID No. 12, SEQ ID No. 20, SEQ ID No. 28, SEQ ID No. 36, SEQ ID No. 44, SEQ ID No. 52, SEQ ID No. 60, SEQ ID No. 68, SEQ ID No.
  • the amino acid sequence of the CDR1 of the chain is SEQ ID No. 6, SEQ ID No. 14, SEQ ID No. 22, SEQ ID No. 30, SEQ ID No. 38, SEQ ID No. 46, SEQ ID No. 54 in the Sequence Listing. , SEQ ID No. 62, SEQ ID No. 70, SEQ ID No. 78, SEQ ID No. 86 or SEQ ID No. 94; the amino acid sequence of the light chain CDR2 is SEQ ID No. 7 in the sequence listing. SEQ ID No. 15, SEQ ID No. 23, SEQ ID No. 31, SEQ ID No. 39, SEQ ID No. 47, SEQ ID No. 55, SEQ ID No.
  • amino acid sequence of the light chain CDR3 is SEQ ID No. 8, SEQ ID No. 16, SEQ ID No. 24, SEQ in the sequence listing. ID No. 32, SEQ ID No. 40, SEQ ID No. 48, SEQ ID No. 56, SEQ ID No. 64, SEQ ID No. 72, SEQ ID No. 80, SEQ ID No. 88 or SEQ ID No. As shown in .96;
  • the amino acid sequence of the heavy chain CDR1 is as SEQ ID No. 2, SEQ ID No. 10, SEQ ID No. 18, SEQ ID No. 26, SEQ ID No. 34, SEQ ID No. 42 in the sequence listing. At least 80% sequence homology of the amino acid sequence represented by SEQ ID No. 50, SEQ ID No. 58, SEQ ID No. 66, SEQ ID No. 74, SEQ ID No. 82, and SEQ ID No. 90
  • the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 3, SEQ ID No. 11, SEQ ID No. 19, SEQ ID No. 27, SEQ ID No. 35, SEQ in the sequence listing. At least 80% of the amino acid sequences represented by ID No.
  • amino acid sequence of the sequence homology is shown; the amino acid sequence of the heavy chain CDR3 is as SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 20, SEQ ID No. 28, SEQ ID No in the sequence listing. .36.
  • the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 6, SEQ ID No. 14, SEQ ID No. 22, SEQ ID No. 30, SEQ ID No. 38, SEQ ID No in the sequence listing. 46.
  • the amino acid sequence represented by SEQ ID No. 54, SEQ ID No. 62, SEQ ID No. 70, SEQ ID No. 78, SEQ ID No. 86, and SEQ ID No. 94 has at least 80% sequence identity.
  • the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 7, SEQ ID No. 15, SEQ ID No. 23, SEQ ID No. 31, SEQ ID No. 39 in the sequence listing.
  • SEQ ID No. 55 has at least 80 % amino acid sequence of sequence homology; amino acid sequence of the light chain CDR3 and SEQ ID No. 8, SEQ ID No. 16, SEQ ID No. 24, SEQ ID No. 32, SEQ in the sequence listing Amino acids represented by ID No. 40, SEQ ID No. 48, SEQ ID No. 56, SEQ ID No. 64, SEQ ID No. 72, SEQ ID No. 80, SEQ ID No. 88, SEQ ID No. 96 Ammonia with at least 80% sequence homology Acid sequence shown in FIG.
  • the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID No. 2 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 3 of the Sequence Listing
  • the heavy chain CDR3 The amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 10 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR2 is SEQ ID No in the Sequence Listing.
  • the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 12 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR1 is as shown in SEQ ID No.
  • the heavy chain The amino acid sequence of CDR2 is shown in SEQ ID No. 19 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 20 of the sequence; the amino acid sequence of the heavy chain CDR1 is as shown in the sequence listing. As shown in ID No. 26, the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 27 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 28 of the Sequence Listing; The amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 34 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR2 is as follows.
  • the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 36 of the Sequence Listing; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 42 of the Sequence Listing.
  • the amino acid sequence of the heavy chain CDR2 is set forth in SEQ ID No. 43 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR3 is set forth in SEQ ID No. 44 of the Sequence Listing; the amino acid sequence of the heavy chain CDR1 As shown in SEQ ID No. 50 of the Sequence Listing, the amino acid sequence of the heavy chain CDR2 is set forth in SEQ ID No. 51 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR3 is SEQ ID No. 52 in the Sequence Listing.
  • the amino acid sequence of the heavy chain CDR1 is set forth in SEQ ID No. 58 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR2 is set forth in SEQ ID No. 59 of the Sequence Listing
  • the heavy chain CDR3 The amino acid sequence is shown in SEQ ID No. 60 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR1 is set forth in SEQ ID No. 66 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR2 is SEQ ID No. in the Sequence Listing.
  • the amino acid sequence of the heavy chain CDR3 is as shown in SEQ ID No. 68 of the Sequence Listing
  • the heavy chain CDR1 The amino acid sequence is shown in SEQ ID No.
  • the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID No. 75 in the Sequence Listing
  • the amino acid sequence of the heavy chain CDR3 is SEQ ID No in the Sequence Listing.
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 82 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 83 of the Sequence Listing
  • the heavy chain is shown.
  • the amino acid sequence of CDR3 is shown in SEQ ID No. 84 of the Sequence Listing;
  • the amino acid sequence of the heavy chain CDR1 is set forth in SEQ ID No.
  • the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 6 of the Sequence Listing
  • the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 7 of the Sequence Listing
  • the amino acid sequence of the light chain CDR3 is as The amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 14 of the Sequence Listing
  • the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 15 of the Sequence Listing.
  • the amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 16 of the Sequence Listing
  • the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No.
  • amino acid sequence of the light chain CDR2 As shown in SEQ ID No. 23 of the Sequence Listing, and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 24 of the Sequence Listing; the amino acid sequence of the light chain CDR1 is SEQ ID No. 30 in the Sequence Listing. As shown, the amino acid sequence of the light chain CDR2 is set forth in SEQ ID No. 31 of the Sequence Listing, and the amino acid sequence of the light chain CDR3 is set forth in SEQ ID No. 32 of the Sequence Listing; The amino acid sequence is shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the light chain CDR2 is SEQ I in the Sequence Listing. D No.
  • amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 40 of the Sequence Listing; the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 46 of the Sequence Listing.
  • the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 47 of the Sequence Listing, and the amino acid sequence of the light chain CDR3 is shown as SEQ ID No. 48 in the Sequence Listing; the amino acid sequence of the light chain CDR1 is as in the sequence listing.
  • amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 55 of the Sequence Listing
  • amino acid sequence of the light chain CDR3 is as shown in SEQ ID No.
  • the amino acid sequence of the light chain CDR1 is set forth in SEQ ID No. 62 of the Sequence Listing
  • the amino acid sequence of the light chain CDR2 is set forth in SEQ ID No. 63 of the Sequence Listing
  • the amino acid sequence of the light chain CDR3 is as The amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 70 of the Sequence Listing
  • the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 71 of the Sequence Listing.
  • the amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 72 of the Sequence Listing;
  • the amino acid sequence of the light chain CDR1 The sequence is shown in SEQ ID No.
  • the amino acid sequence of the light chain CDR2 is set forth in SEQ ID No. 79 of the Sequence Listing, and the amino acid sequence of the light chain CDR3 is SEQ ID No. in the Sequence Listing. 80; the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 86 of the Sequence Listing, the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No. 87 of the Sequence Listing, and the light chain CDR3
  • the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 94 of the Sequence Listing, and the amino acid sequence of the light chain CDR2 is SEQ ID No in the Sequence Listing. Shown in .95, and the amino acid sequence of the light chain CDR3 is set forth in SEQ ID No. 96 of the Sequence Listing.
  • the EGFR antibody comprises a EGFR antibody heavy chain variable region comprising the CDR and/or a light chain variable region of an EGFR antibody, the amino acid sequence of the heavy chain variable region being SEQ ID No in the Sequence Listing .1, SEQ ID No. 9, SEQ ID No. 17, SEQ ID No. 25, SEQ ID No. 33, SEQ ID No. 41, SEQ ID No. 49, SEQ ID No. 57, SEQ ID No. 65 SEQ ID No. 73, SEQ ID No. 81 or SEQ ID No. 89; the amino acid sequence of the light chain variable region is SEQ ID No. 5, SEQ ID No. 13, SEQ ID No in the sequence listing. .21, SEQ ID No. 29, SEQ ID No. 37, SEQ ID No. 45, SEQ ID No. 53, SEQ ID No. 61, SEQ ID No. 69, SEQ ID No. 77, SEQ ID No. 85 Or as shown in SEQ ID No. 93.
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 1 of the Sequence Listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 5 of the Sequence Listing;
  • the amino acid sequence of the heavy chain variable region is set forth in SEQ ID No. 9 of the Sequence Listing, and the amino acid sequence of the light chain variable region is set forth in SEQ ID No. 13 of the Sequence Listing;
  • the amino acid sequence of the variable region is shown in SEQ ID No. 17 of the Sequence Listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 21 of the Sequence Listing; the amino acid sequence of the heavy chain variable region As shown in SEQ ID No.
  • amino acid sequence of the light chain variable region is shown as SEQ ID No. 29 in the Sequence Listing; the amino acid sequence of the heavy chain variable region is as shown in the Sequence Listing ID No. 33, and the amino acid sequence of the light chain variable region is set forth in SEQ ID No. 37 of the Sequence Listing; the amino acid sequence of the heavy chain variable region is set forth in SEQ ID No. 41 of the Sequence Listing. And the amino acid sequence of the light chain variable region is set forth in SEQ ID No. 45 of the Sequence Listing; the amino acid sequence of the heavy chain variable region As shown in SEQ ID No. 49 of the Sequence Listing, and the amino acid sequence of the light chain variable region is set forth in SEQ ID No.
  • amino acid sequence of the heavy chain variable region is as shown in the Sequence Listing ID No. 57, and the amino acid sequence of the light chain variable region is set forth in SEQ ID No. 61 of the Sequence Listing; the amino acid sequence of the heavy chain variable region is set forth in SEQ ID No. 65 of the Sequence Listing.
  • the amino acid sequence of the light chain variable region is set forth in SEQ ID No. 69 of the Sequence Listing; the amino acid sequence of the heavy chain variable region is set forth in SEQ ID No. 73 of the Sequence Listing, and The amino acid sequence of the light chain variable region is set forth in SEQ ID No. 77 of the Sequence Listing; the amino acid sequence of the heavy chain variable region is set forth in SEQ ID No.
  • amino acid sequence is shown in SEQ ID No. 85 of the Sequence Listing; or the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 89 of the Sequence Listing, and the amino acid sequence of the light chain variable region As shown in SEQ ID No. 93 in the sequence listing.
  • the invention provides a panel of antibodies or antigen-binding fragments that react with EGFR:
  • the invention provides a panel of antibodies or antigen-binding fragments that react with EGFR:
  • a heavy chain variable region having an amino acid sequence number of 1 and a gene sequence number of 97, comprising a heavy chain CDR1, CDR2, and CDR3, wherein the amino acid sequence numbers are 2, 3, and 4, respectively;
  • a light chain variable region having an amino acid sequence number of 5 and a gene sequence number of 98, comprising a light chain CDR1, CDR2, and CDR3, wherein the amino acid sequence numbers are 6, 7, and 8, respectively;
  • a light chain variable region having an amino acid sequence number of 13 and a gene sequence number of 100, comprising a light chain CDR1, CDR2, and CDR3, the amino acid sequence numbers of which are 14, 15, and 16, respectively;
  • a heavy chain variable region having an amino acid sequence number of 17 and a gene sequence number of 101, comprising a heavy chain CDR1, CDR2, and CDR3, the amino acid sequence numbers of which are 18, 19, and 20, respectively;
  • a light chain variable region having an amino acid sequence number of 21 and a gene sequence number of 102, comprising a light chain CDR1, CDR2, and CDR3, wherein the amino acid sequence numbers are 22, 23, and 24, respectively;
  • a heavy chain variable region having an amino acid sequence number of 25 and a gene sequence number of 103, comprising a heavy chain CDR1, CDR2, and CDR3, the amino acid sequence numbers of which are 26, 27, and 28, respectively;
  • a heavy chain variable region having an amino acid sequence number of 33 and a gene sequence number of 105, comprising a heavy chain CDR1, CDR2, and CDR3, the amino acid sequence numbers of which are 34, 35, and 36, respectively;
  • a light chain variable region having an amino acid sequence number of 37 and a gene sequence number of 106, comprising a heavy chain CDR1, CDR2, and CDR3, wherein the amino acid sequence numbers are 38, 39, and 40, respectively;
  • a heavy chain variable region having an amino acid sequence number of 41 and a gene sequence number of 107, comprising a heavy chain CDR1, CDR2, and CDR3, the amino acid sequence numbers of which are 42, 43, 44, respectively;
  • a light chain variable region having an amino acid sequence number of 45 and a gene sequence number of 108, comprising a heavy chain CDR1, CDR2, and CDR3, wherein the amino acid sequence numbers are 46, 47, and 48, respectively;
  • a heavy chain variable region having an amino acid sequence number of 49 and a gene sequence number of 109, comprising a heavy chain CDR1, a CDR2 CDR3 having amino acid sequence numbers of 50, 51, and 52, respectively;
  • a light chain variable region having an amino acid sequence number of 53 and a gene sequence number of 110, comprising a heavy chain CDR1, CDR2, and CDR3, the amino acid sequence numbers of which are 54, 54, 56, respectively;
  • a heavy chain variable region having an amino acid sequence number of 57 and a gene sequence number of 111, comprising a heavy chain CDR1, CDR2, and CDR3, wherein the amino acid sequence numbers are 58, 59, and 60, respectively;
  • a light chain variable region having an amino acid sequence number of 61 and a gene sequence number of 112, comprising a heavy chain CDR1, CDR2, and CDR3, the amino acid sequence numbers of which are 62, 63, and 64, respectively;
  • a heavy chain variable region having an amino acid sequence number of 65 and a gene sequence number of 113, comprising a heavy chain CDR1, CDR2, and CDR3, the amino acid sequence numbers of which are 66, 67, and 68, respectively;
  • a light chain variable region having an amino acid sequence number of 69 and a gene sequence number of 114, comprising a heavy chain CDR1, CDR2, and CDR3, the amino acid sequence numbers of which are 70, 71, and 72, respectively;
  • a heavy chain variable region having an amino acid sequence number of 73 and a gene sequence number of 115, comprising a heavy chain CDR1, CDR2, and CDR3, the amino acid sequence numbers of which are 74, 75, and 76, respectively;
  • a light chain variable region having an amino acid sequence number of 77 and a gene sequence number of 116, comprising a heavy chain CDR1, CDR2, and CDR3, the amino acid sequence numbers of which are 78, 79, and 80, respectively;
  • a heavy chain variable region having an amino acid sequence number of 81 and a gene sequence number of 117, comprising a heavy chain CDR1, CDR2, and CDR3, wherein the amino acid sequence numbers are 82, 83, and 84, respectively;
  • a light chain variable region having an amino acid sequence number of 85 and a gene sequence number of 118, comprising a heavy chain CDR1, CDR2, and CDR3, the amino acid sequence numbers of which are 86, 87, and 88, respectively;
  • a heavy chain variable region having an amino acid sequence number of 89 and a gene sequence number of 119, comprising a heavy chain CDR1, CDR2, and CDR3, wherein the amino acid sequence numbers are 90, 91, and 92, respectively;
  • a light chain variable region having an amino acid sequence number of 93 and a gene sequence number of 120, comprising a heavy chain CDR1, CDR2, and CDR3, the amino acid sequence numbers of which are 94, 95, and 96, respectively.
  • the EGFR antibody further comprises a framework region comprising a heavy chain framework region and/or a light chain framework region; preferably, the heavy chain framework region is a human or murine antibody heavy chain framework region, And/or, the light chain framework region is a human or murine antibody light chain framework region; more preferably, the heavy chain framework region is a human antibody heavy chain framework region, and the light chain framework region is a human antibody light chain framework region.
  • the EGFR antibody further comprises an antibody heavy chain constant region and/or an antibody light chain constant region, wherein the antibody heavy chain constant region is preferably a human or mouse-derived antibody heavy chain constant region, the antibody
  • the light chain constant region is preferably a human or mouse derived antibody light chain constant region.
  • the EGFR antibody is preferably a monoclonal antibody of EGFR, an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody, a single domain antibody or a single region antibody.
  • the monoclonal antibodies can be developed by a variety of pathways and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc.
  • the mainstream is the preparation of monoclonal antibodies from wild-type or transgenic mice by hybridoma technology.
  • the full-length antibody protein is a conventional full-length antibody of the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region.
  • the heavy chain variable region and the light chain variable region and the human heavy chain constant region and the human light chain constant region constitute a full human antibody full length protein.
  • the full length protein of the antibody is IgG1, IgG2, IgG3 or IgG4.
  • the single-chain antibody is a conventional single-chain antibody in the art, which comprises a heavy chain variable region, a light chain variable region, and a short peptide of 15-20 amino acids.
  • the antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment of the art comprising a light chain variable region, a light chain constant region, and an Fd segment of a heavy chain constant region.
  • the antigen-antibody binding domain protein fragments are Fab and F(ab').
  • the single domain antibodies are conventional single domain antibodies in the art, including heavy chain variable regions and heavy chain constant regions.
  • the single region antibodies are conventional single region antibodies of the art which include only heavy chain variable regions.
  • the preparation method of the EGFR antibody is a conventional preparation method in the art.
  • the preparation method is preferably obtained by isolating the expression transformant recombinantly expressing the EGFR antibody or by artificially synthesizing the protein sequence.
  • the method for isolating the expression transformant that recombinantly expresses the EGFR antibody is preferably obtained by cloning a nucleic acid molecule encoding the EGFR antibody and having a point mutation into a recombinant vector, and transforming the resulting recombinant vector into a transformant to obtain The transformant is recombinantly expressed, and the EGFR antibody can be isolated and purified by culturing the resulting recombinant expression transformant.
  • the invention also provides a nucleic acid encoding the EGFR antibody described above.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is SEQ ID No. 97, SEQ ID No. 99, SEQ ID No. 101, SEQ ID No. 103, SEQ ID No in the sequence listing. .105, SEQ ID No. 107, SEQ ID No. 109, SEQ ID No. 111, SEQ ID No. 113, SEQ ID No. 115, SEQ ID No. 117 or SEQ ID No. 119; and/or
  • the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. 98, SEQ ID No. 100, SEQ ID No. 102, SEQ ID No. 104, SEQ ID No. 106, SEQ ID No. 108, SEQ ID No. 110, SEQ ID No. 112, SEQ ID No. 114, SEQ ID No. 116, SEQ ID No. 118 or SEQ ID No. 120.
  • nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 97 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is, for example, the sequence listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is represented by SEQ ID No. 99 in the sequence listing, and the nucleoside of the nucleic acid encoding the light chain variable region is shown in SEQ ID No. 98;
  • the acid sequence is as shown in SEQ ID No. 100 of the Sequence Listing;
  • nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No.
  • the nucleotide sequence of the nucleic acid is as shown in SEQ ID No. 102 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 103 of the Sequence Listing, and the encoding is as described.
  • the nucleotide sequence of the nucleic acid of the light chain variable region is set forth in SEQ ID No. 104 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is set forth in SEQ ID No. 105 of the Sequence Listing.
  • nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 106 of the Sequence Listing; the nucleoside encoding the nucleic acid of the heavy chain variable region The sequence is set forth in SEQ ID No. 107 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is set forth in SEQ ID No. 108 of the Sequence Listing; encoding the heavy chain variable region
  • nucleotide sequence of the nucleic acid is as shown in SEQ ID No. 109 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No.
  • nucleotide sequence of the nucleic acid of the chain variable region is as shown in SEQ ID No. 111 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 112 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is set forth in SEQ ID No. 113 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as SEQ ID in the Sequence Listing No. 114; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No.
  • nucleotide sequence of the nucleic acid encoding the light chain variable region is as SEQ ID No. 116 in the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 117 of the Sequence Listing, and The nucleotide sequence of the nucleic acid of the light chain variable region is as shown in SEQ ID No. 118 of the Sequence Listing; or the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is SEQ ID No in the Sequence Listing
  • the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in 119, as shown in SEQ ID No. 120 of the Sequence Listing.
  • the invention also provides a recombinant expression vector comprising the nucleic acid described above.
  • the recombinant expression vector can be obtained by a conventional method in the art, that is, the nucleic acid molecule of the present invention is ligated to various expression vectors.
  • the expression vector is a variety of vectors conventional in the art as long as it can accommodate the aforementioned nucleic acid molecule.
  • the vector preferably includes: various plasmids, cosmids, phage or viral vectors, and the like.
  • the present invention also provides a recombinant expression transformant comprising the above recombinant expression vector.
  • the preparation method of the recombinant expression transformant is a preparation method conventional in the art, and preferably, the recombinant expression vector is transformed into a host cell.
  • the host cell is a variety of host cells conventional in the art, as long as it satisfies the stable self-replication of the above recombinant expression vector, and the nucleic acid carried can be efficiently expressed.
  • the host cell is an E. coli TG1 or BL21 cell (expressing a single-chain antibody or a Fab antibody), or a CHO-K1 cell (expressing a full-length IgG antibody).
  • the recombinant expression plasmid of the present invention can be obtained by transforming the aforementioned recombinant expression plasmid into a host cell.
  • the conversion method is a conventional transformation method in the art, preferably a chemical conversion method, a heat shock method or an electrotransformation method.
  • the present invention also provides a method for producing an EGFR antibody, which comprises the steps of culturing the above recombinant expression transformant and obtaining an EGFR antibody from the culture.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the EGFR antibody described above and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is a carrier conventional in the art and may be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient.
  • the pharmaceutical excipients are conventional pharmaceutical excipients in the art, preferably including pharmaceutically acceptable excipients, fillers or diluents and the like. More preferably, the pharmaceutical composition comprises from 0.01 to 99.99% of the above EGFR or the above immunoconjugate, and from 0.01 to 99.99% of a pharmaceutically acceptable carrier, the percentage being the mass percentage of the pharmaceutical composition.
  • the administration route of the pharmaceutical composition of the present invention is preferably parenteral administration, injection administration or oral administration.
  • the administration by injection preferably includes intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous injection.
  • the pharmaceutical composition is in various forms conventional in the art, preferably in the form of a solid, semi-solid or liquid, that is, it may be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule or a granule.
  • the pharmaceutical composition may also be administered as an aerosol or crude spray, i.e., nasally; or intrathecally, intramedullaryly or intraventricularly. More preferably, the pharmaceutical composition may also be administered transdermally, transdermally, topically, enterally, intravaginally, sublingually or rectally.
  • the dosage level of the pharmaceutical composition of the present invention can be adjusted depending on the amount of the composition that achieves the desired diagnosis or treatment result.
  • the administration regimen can also be a single injection or multiple injections, or adjusted.
  • the selected dosage level and regimen will depend on the activity and stability (ie, half-life) of the pharmaceutical composition, the formulation, the route of administration, the combination with other drugs or treatments, the disease or condition to be detected and/or treated, Reasonable adjustments are made to various factors such as the health status of the subject to be treated and the prior medical history.
  • the therapeutically effective dose of the pharmaceutical composition of the invention can be estimated initially in cell culture experiments or animal models such as rodents, rabbits, dogs, pigs and/or primates. Animal models can also be used to determine the appropriate range and route of administration. This can then be used to determine the useful dose and route of administration in a human. In general, the determination and adjustment of the effective amount or dose administered and the assessment of when and how such adjustments are made are known to those skilled in the art.
  • the above EGFR antibodies, immunoconjugates and/or additional therapeutic or diagnostic agents described above can each be used as a single agent, in any time frame suitable for performing the intended treatment or diagnosis.
  • these single agents can be administered substantially simultaneously (i.e., as a single formulation or over a few minutes or hours) or sequentially.
  • these single agents can be administered within one year, or within 10, 8, 6, 4, or 2 months, or within 4, 3, 2, or 1 week, or 5, 4, 3, 2, or 1 day.
  • the present invention also provides the use of the above EGFR antibody or the above pharmaceutical composition for the preparation of an antitumor drug.
  • the present invention also provides a method for detecting a cell overexpressing an EGFR protein, the method comprising the steps of: contacting the EGFR antibody with a sample to be tested in vitro, and detecting binding of the EGFR antibody to the sample to be tested Just fine.
  • the present invention also provides a composition for detecting a cell overexpressing an EGFR protein, the composition comprising the above EGFR antibody as an active ingredient.
  • the present invention also provides the use of the above EGFR antibody, or the above pharmaceutical composition, for the preparation of a medicament for preventing or treating a disease associated with EGFR expression or dysfunction; preferably, the EGFR expression or dysfunction is associated with
  • the disease is a tumor, and the tumor is preferably bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, prostate cancer or kidney cancer.
  • the reagents and starting materials used in the present invention are commercially available.
  • the EGFR antibody has high affinity with EGFR protein, and can be combined with human EGFR protein and cynomolgus EGFR protein by ELISA, and does not bind to mouse EGFR protein; FACS detection can identify human EGFR protein in tumor tissue .
  • EGFR-positive cells are cytotoxic and can be used in the preparation of drugs such as tumors.
  • Figure 1 shows the results of FACS screening assay of human EGFR protein transfected CHO-K1 cells.
  • Figure 2 shows the results of FACS screening assay of human EGFR protein transfected 293F cells.
  • Figures 3A and 3B show the serum antibody titer of mice after EGFR protein immunization by ELISA.
  • Figure 4 shows the binding reaction of EGFR antibody to A431 by FACS.
  • Therapeutic monoclonal antibodies can be developed by a variety of techniques and approaches, including hybridoma technology, phage display technology, single lymphocyte gene cloning techniques, and the like. However, the preparation of monoclonal antibodies by hybridoma technology is still the mainstream of current preparation methods for therapeutic monoclonal antibodies.
  • the present invention utilizes optimized hybridoma technology to prepare the desired anti-EGFR antibodies based on current state of the art monoclonal antibody technology.
  • the monoclonal antibody prepared from a conventional animal such as a mouse can clone the antibody heavy chain variable region and the light chain variable region gene by conventional molecular biological methods, and the variable region gene can be grafted to the human antibody constant region gene to form a human.
  • Murine chimeric antibodies US Pat. No. 4,816,567, Cabilly et al
  • the CDR domain of the murine antibody variable region can be grafted onto the human antibody framework, thereby reducing the murine antibody component to less than 5%, greatly increasing the safety of the antibody for use in humans.
  • the antibody obtained by this route is called a humanized antibody and is a major product of the current antibody drug market (U.S. Pat. No. 5, 225, 539 to 55, Winter, and U.S. Pat. Nos. 5, 530, 101; 5, 585, 089; 5, 693, 762 and 6, 180, 370 to Queen et al.
  • the invention utilizes monoclonal antibody technology, comprehensively utilizes various immunization methods, and develops a group of EGFR lead antibodies with high affinity, high biological activity and high diversity by using effective cell level functional experiments. These antibodies show excellent properties and are capable of binding to the extracellular domain of human EGFR.
  • the amino acid sequence of the heavy chain variable region of the EGFR antibody and the light chain variable region of the EGFR antibody is then sequenced by molecular biological methods.
  • the nucleotide sequence containing the amino acid sequence encoding the extracellular region of the human EGFR protein was cloned into the pCpC vector (purchased from Invitrogen, V044-50) carrying the human IgG Fc fragment (hFc) and established as Prepare plasmids by standard molecular biology methods. For specific methods, see Sambrook, J., Fritsch, EF, and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press) ).
  • HEK293 cells purchased from Invitrogen
  • PI transiently transfected
  • FreeStyleTM 293 FreeStyleTM 293
  • the culture supernatant was applied to a Protein A affinity chromatography column (Mabselect Sure, available from GE Healthcare) while monitoring the change in ultraviolet absorption value (A280 nm) with an ultraviolet (UV) detector.
  • A280 nm ultraviolet absorption value
  • the protein A affinity column was washed with PBS phosphate buffer (pH 7.2) until the UV absorbance value returned to the baseline, and then eluted with 0.1 M glycine hydrochloride (pH 2.5) to collect affinity from protein A.
  • the hFc-tagged EGFR protein (EGFR-hFc) eluted on the column was dialyzed against PBS phosphate buffer (pH 7.2) overnight at 4 ° C in a refrigerator. The dialyzed protein was sterile-filtered at 0.22 ⁇ m and stored at -80 ° C to obtain purified immunogen A.
  • the nucleotide sequence encoding the human EGFR full-length amino acid sequence was cloned into the pIRES vector (purchased from Clontech) and a plasmid was prepared.
  • HEK293 cell line and CHO-K1 cell line both purchased from Invitrogen
  • PEI plasmid-transfected
  • the cells were selectively cultured for 2 weeks, subcloned in a 96-well culture plate by limiting dilution, and cultured at 37 ° C, 5% (v/v) CO 2 , and selected for partial monoclonal well amplification after approximately 2 weeks.
  • the amplified clones were screened by flow cytometry using a known EGFR antibody (purchased from Absoluteantibody, #Ab00184-1.1). The cell line with good growth and high fluorescence intensity is selected, and the monoclonal cell line continues to expand and the liquid nitrogen is frozen, that is, the immunogen B is obtained.
  • the specific selection results are shown in Table 1 and Figure 1, and the IgG subtype control is a mouse IgG control.
  • Tables 1 and 2 illustrate that CHO-K1 cell lines positive for EGFR expression have been produced, as well as HEK293F cell lines positive for EGFR expression.
  • the abscissa is the fluorescence intensity of the cells, and the ordinate is the number of cells.
  • the results in Figure 1 indicate that CHO-K1-hEGFR 3G2 is a high level expressing cell line of EGFR.
  • Immunogen A immunization 6-8 weeks old BALB/cAnNCrl mice or SJL/JorllcoCrl mice (purchased from Shanghai Slack Company) were used, and the mice were raised under SPF conditions.
  • the immunogen A protein was emulsified with Freund's complete adjuvant and intraperitoneally injected with 0.25 ml, that is, 50 ⁇ g of immunogen A protein was injected per mouse.
  • the immunogen A protein was emulsified with Freund's incomplete adjuvant and intraperitoneally injected with 0.25 ml, that is, 50 ⁇ g of immunogen A protein was injected per mouse.
  • the first and second immunizations were separated by 2 weeks, and each subsequent immunization interval was 3 weeks.
  • blood was collected 1 week after each immunization, and antibody titer and specificity in serum were measured by FACS. After the second booster immunization, the FACS test serum antibody titer reached 1:1000 or more.
  • each mouse selected was intraperitoneally injected with 100 ⁇ g of purified immunogen A (the mouse in which the original A was immunoreactive) or the immunogen B was immunoreactive against the immunogen B.
  • Rats mice were sacrificed 5 days later, and spleen cells were collected. NH 4 OH was added to a final concentration of 1% (w/w), and the red blood cells doped in the spleen cells were lysed to obtain a spleen cell suspension.
  • the cells were washed three times with DMEM basal medium at 1000 rpm, and then mixed with mouse myeloma cells SP2/0 (purchased from ATCC) at a 5:1 ratio of viable cells, using a high-efficiency electrofusion method (see METHODS IN ENZYMOLOGY). , VOL.220) for cell fusion.
  • the fused cells were diluted into DMEM medium containing 20% fetal calf serum, 1 ⁇ HAT, and the percentage was a mass percentage.
  • 1 ⁇ 10 5 /200 ⁇ l per well was added to a 96-well cell culture plate and placed in a 5% CO 2 , 37 ° C incubator, the percentage being a volume percentage.
  • the cell fusion plates were screened by ELISA and Acumen (microplate assay), and positive clones with OD 450nm >1.0 in ELISA and MFI values >100 in Acumen were amplified into 24-well plates at 10% (in 10%).
  • w/w) HT fetal bovine serum, DMEM (invitrogen) was expanded at 37 ° C, 5% (v / v) CO 2 conditions.
  • the culture medium expanded in a 24-well plate was centrifuged, the supernatant was collected, and the supernatant was subjected to antibody subtype analysis, and the binding activity of EGFR protein and EGFR-positive cells was determined by ELISA and FACS (binding activity).
  • the detection method please refer to Example 3A and Example 3B), respectively, and the anti-mouse antibody-MMAF indirect cytotoxic killing experiment (see Example 4 for indirect cytotoxic killing activity detection method).
  • the hybridoma cells with IgG positive cell killing rate of 50% were selected in the ELISA experiment with OD 450nm >1.0, MACS value >50 in FACS experiment and indirect cytotoxic killing experiment.
  • select eligible hybridoma cells were subcloned in 96-well plates by limiting dilution in DMEM medium containing 10% (w/w) FBS (purchased from invitrogen) at 37 ° C, 5% (v/v) Culture under CO 2 conditions.
  • Initial screening was performed by ELISA and Acumen 10 days after subcloning, and single positive monoclonal clones were selected and expanded into 24-well plates for further culture.
  • the best clones were selected and cultured in DMEM medium containing 10% (w/w) FBS (purchased from invitrogen) at 37 ° C, 5% (v/v) CO 2 conditions.
  • the optimal clone is expanded and cultured, and the hybridoma cells of the present invention are obtained by cryopreservation of liquid nitrogen, and can be used for subsequent antibody production and purification.
  • Hybridoma cells produce antibodies at low concentrations, about 1-10 ⁇ g/ml, with large changes in concentration. Moreover, the various proteins produced in the culture of the medium and the fetal bovine serum components contained in the culture medium have different degrees of interference with many biological activity analysis methods, so that small-scale (1-5 mg) antibody production purification is required.
  • the hybridoma cells obtained in Example 1 were inoculated into a T-75 cell culture flask and domesticated for 3 passages using a production medium (Hybridoma serum free medium, available from Invitrogen). After the growth state is good, inoculate the cell culture spinner. 200 ml of production medium was added to each 2 liter culture spinner bottle, and the inoculated cell density was 1.0 ⁇ 10 5 /ml. The cap was capped and the roller was placed in a bottle shaker in a 37 ° C incubator at 3 rpm. After 14 days of continuous spin culture, the cell culture medium was collected, and the cells were removed by filtration, and filtered through a 0.45 ⁇ m filter to clarify the culture supernatant. The clarified culture supernatant can be purified immediately or frozen at -30 °C.
  • a production medium Hybridoma serum free medium, available from Invitrogen
  • the monoclonal antibody in the culture supernatant (200 mL) of the clarified hybridoma cells was purified using a 2 mL Protein A column (purchased from GE Healthcare).
  • the Protein G column was first equilibrated with equilibration buffer (PBS phosphate buffer, pH 7.4), and then the clarified culture supernatant was applied to a Protein A column at a flow rate of 3 mL/min. After loading, the protein G column was washed with equilibration buffer, and the volume of the equilibration buffer was 4 times the volume of the protein A column bed.
  • equilibration buffer PBS phosphate buffer, pH 7.4
  • the EGFR antibody bound to the Protein A column was eluted with an eluent (0.1 M sodium citrate buffer, pH 3.5), and the elution condition (A280 ultraviolet absorption peak) was monitored with a UV detector.
  • the eluted antibody was collected, neutralized by adding 10% 1.0 M Tris-HCl buffer, and the percentage was a volume percentage, and then immediately dialyzed against PBS phosphate buffer overnight, and the next day, the solution was changed once and the dialysis was continued for 3 hours.
  • the dialyzed EGFR antibody was collected, sterile-filtered with a 0.22 micron filter, and stored aseptically to obtain a purified EGFR antibody.
  • the purified EGFR antibody was subjected to detection analysis of protein concentration (A280/1.4), purity, endotoxicity (Lonza kit), and the results are shown in Table 4. As a result, it was found that the endotoxin concentration of the antibody final product was within 1.0 EU/mg.
  • the nucleotide sequence containing the full-length amino acid sequence encoding human EGFR introduced in step (2) of Example 1 was introduced into a CHO-K1 cell line to obtain a CHO-K1 stable cell line containing human EGFR (herein referred to as CHO-K1).
  • CHO-K1 stable cell line containing human EGFR (herein referred to as CHO-K1).
  • -hEGFR stable cell line) then expanded to 90% confluence in T-75 cell culture flask, exhausted the medium, washed twice with PBS buffer (purchased from Invitrogen), then used The cells were treated and collected by enzyme cell dissociation (Versene solution: purchased from Life Technology).
  • the cells were washed twice with PBS buffer, and after cell counting, the cells were diluted with PBS buffer to 2 ⁇ 10 6 cells per ml, and 2% calf serum blocking solution was added, the percentage being mass percentage, and incubation at room temperature for 15 minutes. It was then washed twice with PBS buffer. The collected cells were suspended in FACS buffer (PBS + 2% FBS, the percentage is mass%) to 3 ⁇ 10 6 cells / mL, and added to a 96-well FACS reaction plate at 100 ⁇ L per well, and Example 2 was added. The resulting purified EGFR antibody test sample was 100 ⁇ l per well and incubated at 4 ° C for 1 hour.
  • FACS buffer PBS + 2% FBS, the percentage is mass
  • the cells were washed twice with FACS buffer, and 100 ⁇ l of a fluorescent (Alexa 488)-labeled secondary antibody (purchased from Invitrogen) per well was added and incubated at 4 ° C for 1 hour.
  • the cells were washed three times with FACS buffer, and 100 ⁇ l of fixative [4% (v/v) paraformaldehyde] suspension cells were added to each well, and after 10 minutes, the cells were washed twice with FACS buffer.
  • the cells were suspended in 100 ⁇ l of FACS buffer, and the results were detected and analyzed by FACS (FACS Calibur, available from BD). The results are shown in Figure 4 and Table 5.
  • Table 5 shows that the antibody to be tested binds to EGFR on the cell surface.
  • the IgG control was murine IgG, and the data in the table is the average fluorescence intensity value of the cell population measured by MFI.
  • RNA isolation The supernatant obtained from the subcloning culture of Example 1 was tested for antigen binding (ie, after the assays and activity assays of Examples 3 to 5), and 5 ⁇ 10 7 hybridoma cells were collected by centrifugation and added.
  • Reverse transcription and PCR 1 ⁇ g of total RNA was taken, 20 ⁇ l of the system was placed, reverse transcriptase was added, and the reaction was carried out at 42 ° C for 60 minutes, and the reaction was terminated at 7 ° C for 10 minutes.
  • Configure 50 ⁇ l PCR system including 1 ⁇ l cDNA, 25 pmol of each primer, 1 ⁇ l DNA polymerase and matching buffer system, 250 ⁇ mol dNTPs; set PCR program, pre-denatured at 95 ° C for 3 minutes, denatured at 95 ° C for 30 seconds, annealed at 55 ° C for 30 seconds, extended 72
  • the PCR product was obtained after 35 cycles of 35 cycles and an additional 5 minutes at 72 °C.
  • the kit for reverse transcription was PrimeScript RT Master Mix, purchased from Takara, Catalog No. RR036; the kit used for PCR included Q5 super-fidelity enzyme, available from NEB, Cat. No. M0492.
  • the ligation reaction was carried out: 50 ng of sample, 50 ng of T vector, 0.5 ⁇ l of ligase, 1 ⁇ l of buffer, 10 ⁇ l of reaction system, and reacted at 16 ° C for half an hour to obtain a ligation product, wherein the ligated kit was T4 DNA ligase, purchased from NEB, article number M0402 5 ⁇ l of the ligation product was added to 100 ⁇ l of competent cells (Ecos 101competent cells, purchased from Yeastern, item number FYE607), ice bathed for 5 minutes, then heat-shocked in a 42 ° C water bath for 1 minute, put back on ice for 1 minute, and then added 650 ⁇ l.
  • competent cells Ecos 101competent cells, purchased from Yeastern, item number FYE607
  • the antibiotic SOC medium was incubated at 37 RPM for 30 minutes on a 37 ° C shaker, and 200 ⁇ l of the solution was applied to an antibiotic-containing LB solid medium and incubated at 37 ° C overnight; the next day, the T-carrier primers M13F and M13R were used.
  • a 30 ⁇ l PCR system was set up, colony PCR was performed, and the colony was pipetted in a PCR reaction system with a pipette tip, and 0.5 ⁇ l was sucked onto another LB solid culture dish containing 100 nM ampicillin to preserve the strain; the PCR reaction was ended.
  • the number in Table 6 is the sequence number in the sequence listing, such as the amino acid sequence of the heavy chain protein variable region of 51B12D10 is SEQ ID No. 1, and the amino acid sequence of CDR1 in the heavy chain protein variable region of 51B12D10 is SEQ ID No. 2.
  • the number in Table 8 is the sequence number in the sequence listing, and the nucleotide sequence of the variable region of the heavy chain protein encoding 51B12D10 is SEQ ID No. 97.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 51B12D10 is from positions 76 to 99 of SEQ ID No. 97 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 51B12D10 is from positions 157 to 171 in SEQ ID No. 97 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 51B12D10 is from positions 295 to 318 of SEQ ID No. 97 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 51B12D10 is from position 70 to position 99 of SEQ ID No. 98 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 51B12D10 is from positions 145 to 165 of SEQ ID No. 98 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 51B12D10 is 262th to 288th in SEQ ID No. 98 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 67A6F9 is from positions 76 to 96 of SEQ ID No. 99 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 67A6F9 is from positions 154 to 171 in SEQ ID No. 99 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 67A6F9 is from positions 295 to 330 of SEQ ID No. 99 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 67A6F9 is from positions 70 to 117 in SEQ ID No. 100 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 67A6F9 is from positions 163 to 183 in SEQ ID No. 100 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 67A6F9 is 280th to 306th in SEQ ID No. 100 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 80E11E12 is from positions 76 to 96 of SEQ ID No. 101 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 80E11E12 is 154th to 171th in SEQ ID No. 101 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 80E11E12 is from positions 295 to 324 of SEQ ID No. 101 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 80E11E12 is from positions 70 to 117 of SEQ ID No. 102 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 80E11E12 is from position 163 to position 183 in SEQ ID No. 102 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 80E11E12 is from positions 280 to 309 in SEQ ID No. 102 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 75F10C9 is from positions 76 to 96 of SEQ ID No. 103 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 75F10C9 is from positions 154 to 171 in SEQ ID No. 103 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 75F10C9 is from positions 295 to 318 of SEQ ID No. 103 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 75F10C9 is from positions 70 to 102 of SEQ ID No. 104 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 75F10C9 is 148th to 168th in SEQ ID No. 104 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 75F10C9 is position 265 to 291 in SEQ ID No. 104 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 80E12B7 is from positions 76 to 96 of SEQ ID No. 105 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 80E12B7 is from positions 154 to 171 in SEQ ID No. 105 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 80E12B7 is from positions 295 to 324 of SEQ ID No. 105 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 80E12B7 is from positions 70 to 99 of SEQ ID No. 106 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 80E12B7 is from positions 145 to 165 in SEQ ID No. 106 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 80E12B7 is from positions 262 to 288 in SEQ ID No. 106 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 74B3D4 is from positions 76 to 96 of SEQ ID No. 107 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 74B3D4 is from positions 154 to 171 in SEQ ID No. 107 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 74B3D4 is 295th to 327th in SEQ ID No. 107 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 74B3D4 is the 70th to 99th positions in SEQ ID No. 108 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 74B3D4 is from positions 145 to 165 of SEQ ID No. 108 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 74B3D4 is position 262 to 288 in SEQ ID No. 109 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 122E4D8 is from position 76 to position 96 in SEQ ID No. 109 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 122E4D8 is 154th to 171th in SEQ ID No. 109 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 122E4D8 is 295th to 318th in SEQ ID No. 109 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 122E4D8 is from positions 70 to 102 of SEQ ID No. 110 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 122E4D8 is 148th to 168th in SEQ ID No. 110 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 122E4D8 is position 265 to 291 in SEQ ID No. 110 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 122E4B6 is from positions 76 to 96 of SEQ ID No. 111 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 122E4B6 is 154th to 171th in SEQ ID No. 111 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 122E4B6 is 295th to 318th in SEQ ID No. 111 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 122E4B6 is from positions 70 to 102 of SEQ ID No. 112 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 122E4B6 is 148th to 168th in SEQ ID No. 112 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 122E4B6 is from position 265 to position 291 in SEQ ID No. 112 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 126C3B4 is from position 76 to position 96 of SEQ ID No. 113 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 126C3B4 is from position 154 to position 171 in SEQ ID No. 113 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 126C3B4 is from positions 295 to 318 of SEQ ID No. 113 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 126C3B4 is from positions 70 to 120 of SEQ ID No. 114 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 126C3B4 is 166th to 186th in SEQ ID No. 114 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 126C3B4 is position 283 to 309 in SEQ ID No. 114 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 128C5G12 is from position 76 to position 96 of SEQ ID No. 115 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 128C5G12 is from position 154 to position 171 in SEQ ID No. 115 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 128C5G12 is 295th to 318th in SEQ ID No. 115 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 128C5G12 is from positions 70 to 117 of SEQ ID No. 116 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 128C5G12 is from position 163 to position 183 in SEQ ID No. 116 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 128C5G12 is position 280 to 306 in SEQ ID No. 116 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 132E5F6 is from position 76 to position 96 of SEQ ID No. 117 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 132E5F6 is from position 154 to position 171 in SEQ ID No. 117 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 132E5F6 is from positions 295 to 324 of SEQ ID No. 117 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 132E5F6 is from positions 70 to 102 of SEQ ID No. 118 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 132E5F6 is 148th to 168th in SEQ ID No. 118 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 132E5F6 is position 265 to 291 in SEQ ID No. 118 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 132E8H6 is from position 76 to position 96 of SEQ ID No. 119 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 132E8H6 is from position 154 to position 171 in SEQ ID No. 119 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 1132E8H6 is from positions 295 to 330 of SEQ ID No. 119 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 132E8H6 is from positions 70 to 102 of SEQ ID No. 120 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 132E8H6 is 148th to 168th in SEQ ID No. 120 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 132E8H6 is position 265 to 288 in SEQ ID No. 120 of the Sequence Listing.

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Abstract

一种EGFR抗体及其制备方法和应用。所述抗体包括互补决定区(CDR)：重链CDR1、重链CDR2和重链CDR3中的一种或多种，和/或，EGFR抗体的轻链CDR1、轻链CDR2和轻链CDR3中的一种或多种。所述的EGFR抗体，与EGFR蛋白具有高度亲和力，对EGFR阳性细胞进行细胞毒杀伤作用，因此能够运用于***等药物的制备中。

Description

一种EGFR抗体及其制备方法和应用
本申请要求申请日为2017年12月13日的中国专利申请CN201711332490.5的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及生物医药领域，具体涉及一种EGFR抗体及其制备方法和应用。
背景技术
人类表皮生长因子受体(EGFR，human epidermal growth factor receptor，也称为her-1或Erb-B1)是一个170kDa的跨膜蛋白受体，由原癌基因c-erbB编码，膜内区具有酪氨酸激酶活性(Modjtahedi et al.，Br.J.Cancer 73:228-235，1996；Herbst and Shin，Cancer94:1593-1611，2002)。EGFR全长序列在SwissProt数据库的编号为P00533。EGFR通过酪氨酸激酶介导的信号转导途径调节多种细胞生理过程，主要包括细胞增殖及分化、细胞存活及凋亡、血管生成、以及细胞的有丝***和细胞转移(Atalay et al.，Ann.Oncology14:1346-1363，2003；Tsao and Herbst，Signal 4:4-9，2003；Herbst and Shin，Cancer 94:1593-1611，2002；Modjtahedi et al.，Br.J.Cancer 73:228-235，1996)。
EGFR的配体包括EGF，TGFA/TGF-alpha，amphiregulin，epigen/EPGN，BTC/betacellulin，epiregulin/EREG和HBEGF/heparin-binding EGF。受体-配体结合后会触发EGFR形成同源或者异源二聚体，从而使胞内区发生自身磷酸化，进一步激活复杂的下游信号级联反应，主要包括下列几条信号通路:RAS-RAF-MEK-ERK信号通路，磷脂酰肌醇3-激酶(PI3K)信号通路，PLCgamma-PKC信号通路和STATs modules信号通路。
在多种肿瘤中发现了EGFR的过度表达，包括膀胱癌、脑癌、头颈癌、胰腺癌、肺癌、乳腺癌、卵巢癌、结肠癌、***癌、和肾癌等(Atalay et al.，Ann.Oncology 14:1346-1363，2003；Herbst and Shin，Cancer 94:1593-1611，2002；and Modjtahedi et al.，Br.J.Cancer 73:228-235，1996)。在很多情况下，EGFR的过度表达与患者的不良预后相关。(Herbst and Shin，Cancer 94:1593-1611，2002；Modjtahedi et al.，Br.J.Cancer 73:228-235，1996)。EGFR也表达于正常组织中，在皮肤、肝脏和胃肠道的上皮组织中表达量较高，但表达水平远低于肿瘤组织(Herbst and Shin，Cancer 94:1593-1611，2002)。
目前市场上已有两个抗表皮生长因子受体的治疗性抗体，一个是人鼠嵌合抗体C225抗体(爱必妥，Erbitux或Cetuximab，ImClone(现在Eli Lilly)公司)，与表皮生长因子受体 有特异亲和性，可阻断EGF和TGFα等配体与表皮生长因子受体的结合，抑制其磷酸化和下游信号传导，从而抑制肿瘤细胞的生长，诱导凋亡，减少基质金属蛋白酶和血管上皮生长因子的产生。2004年美国FDA批准爱必妥治疗结直肠癌，2006年批准其治疗头颈部癌，目前有更多临床试验用于其它肿瘤适应症。在临床上，爱必妥和伊立替康合用治疗结直肠癌的有效率(ORR)为23％，与氟嘧啶等化疗药物合用治疗头颈癌的有效率为13％-30％。由于是人鼠嵌合抗体，爱必妥在临床试验中有3.7％的病人产生了抗体反应。
另一个抗表皮生长因子受体的治疗性抗体是帕尼单抗(Vectibix，panitumumab，Amgen公司)，是采用转基因小鼠技术制备的全人化单克隆抗体，无鼠源蛋白序列。该抗体靶向作用于表皮生长因子受体(EGFR)，2006年9月被FDA批准上市，与氟嘧啶、奥沙利铂和伊立替康合用或在化疗后用于治疗EGFR阳性的转移性结直肠癌。2006年FDA批准其单药治疗化疗耐受的转移性结直肠癌(mCRC)。然而帕尼单抗是IgG2亚型抗体，与IgG1相比，IgG2的CDC活性及ADCC等生物学活性明显减低；另外，IgG2亚型抗体稳定性较差，这可能是临床效果上全人抗体帕尼单抗与嵌合抗体爱必妥相比没有明显优势的主要原因。在临床上治疗结直肠癌的总体生存率(OR)才达到8％，非进展生存期只延长了3.6个月。
目前大量的临床数据显示爱必妥单抗和帕尼单抗只对EGFR表达的KRAS野生型(KRAS wild type)有疗效，而对KRAS突变体没有肿瘤生长抑制活性，因此，美国临床肿瘤协会发表的指导原则中明确指出抗EGFR单抗药物只适用于KRAS野生型的结肠癌病人(Allegra CJ，Jessup JM，Somerield MR，Hamilton SR，Hammond EH，Hayes DF，et al.American Soceitey of Clinical Oncology provisional clinical opinion：testing for KRAS gene mutations in patients with metastatic colorectal carcinoma to predict response to anti-epidermal growth factor monoclonal antibody therapy.J.Clin Oncol.2009；27：2091-2096；Bardelli A，Siena S.Molecular mechanisms of resistance to cetuximab and panitumumab in colorectal cancer.J Clin Oncol.2010；28：1254-1261)。
因此，本领域亟需具有更高生物学活性的抗表皮生长因子受体的抗体类药物，尤其是对KRAS突变体有疗效的抗体类药物，例如双特异性抗体药物，抗体偶联药物以及嵌合抗原受体T细胞免疫疗法等以进一步提高疗效。
发明内容
本发明所要解决的技术问题是为了克服目前缺少EGFR抗体的缺陷，提供一种亲和力高、特异性强的EGFR抗体及其制备方法和应用。所述的EGFR抗体，与EGFR蛋白 具有高度亲和力，对EGFR阳性细胞进行细胞毒杀伤作用，因此能够运用于***等药物的制备中。
本发明采用杂交瘤技术，综合运用多种免疫方法，利用有效的细胞水平功能性实验进行筛选，开发出了一组高亲和力、高生物学活性和高多样性的EGFR先导抗体。然后通过分子生物学方法测序获知EGFR抗体的重链可变区和EGFR抗体的轻链可变区的氨基酸序列。
本发明通过杂交瘤技术得到的抗体或抗原结合区均含有一条抗体轻链可变区和一条重链可变区，每一可变区均含有CDR1、CDR2、CDR3三个结构域。用于鉴定抗体分子内重链可变区和轻链可变区，及抗体可变区氨基酸序列内的CDR的方法和技术是本领域熟知的，并且可以用于识别在此公开的特定抗体可变区氨基酸序列内的CDR。可用于识别的CDR的边界示例约定包括，例如，Kabat定义，Chothia定义，和AbM定义。概括地说，所述的Kabat定义基于序列多样性，所述Chothia定义基于结构环区域的位置，AbM定义是Kabat定义和Chothia方法之间的折中。其它公共数据库，也可用于鉴定抗体中的CDR序列。本发明中CDRs的编号依据的是Chothia定义。
本发明提供一种EGFR抗体，其包括互补决定区(CDR)：重链CDR1、重链CDR2和重链CDR3中的一种或多种，和/或，EGFR抗体的轻链CDR1、轻链CDR2和轻链CDR3中的一种或多种，其中，所述重链CDR1的氨基酸序列如序列表中SEQ ID No.2、SEQ ID No.10、SEQ ID No.18、SEQ ID No.26、SEQ ID No.34、SEQ ID No.42、SEQ ID No.50、SEQ ID No.58、SEQ ID No.66、SEQ ID No.74、SEQ ID No.82或SEQ ID No.90所示；所述重链CDR2的氨基酸序列如序列表中SEQ ID No.3、SEQ ID No.11、SEQ ID No.19、SEQ ID No.27、SEQ ID No.35、SEQ ID No.43、SEQ ID No.51、SEQ ID No.59、SEQ ID No.67、SEQ ID No.75、SEQ ID No.83或SEQ ID No.91所示；所述重链CDR3的氨基酸序列如序列表中SEQ ID No.4、SEQ ID No.12、SEQ ID No.20、SEQ ID No.28、SEQ ID No.36、SEQ ID No.44、SEQ ID No.52、SEQ ID No.60、SEQ ID No.68、SEQ ID No.76、SEQ ID No.84或SEQ ID No.92所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.6、SEQ ID No.14、SEQ ID No.22、SEQ ID No.30、SEQ ID No.38、SEQ ID No.46、SEQ ID No.54、SEQ ID No.62、SEQ ID No.70、SEQ ID No.78、SEQ ID No.86或SEQ ID No.94所示；所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.7、SEQ ID No.15、SEQ ID No.23、SEQ ID No.31、SEQ ID No.39、SEQ ID No.47、SEQ ID No.55、SEQ ID No.63、SEQ ID No.71、SEQ ID No.79、SEQ ID No.87或SEQ ID No.95所示；所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.8、SEQ ID No.16、SEQ ID No.24、SEQ ID No.32、SEQ  ID No.40、SEQ ID No.48、SEQ ID No.56、SEQ ID No.64、SEQ ID No.72、SEQ ID No.80、SEQ ID No.88或SEQ ID No.96所示；
或者，所述重链CDR1的氨基酸序列与如序列表中SEQ ID No.2、SEQ ID No.10、SEQ ID No.18、SEQ ID No.26、SEQ ID No.34、SEQ ID No.42、SEQ ID No.50、SEQ ID No.58、SEQ ID No.66、SEQ ID No.74、SEQ ID No.82、SEQ ID No.90所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述重链CDR2的氨基酸序列与如序列表中SEQ ID No.3、SEQ ID No.11、SEQ ID No.19、SEQ ID No.27、SEQ ID No.35、SEQ ID No.43、SEQ ID No.51、SEQ ID No.59、SEQ ID No.67、SEQ ID No.75、SEQ ID No.83、SEQ ID No.91所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述重链CDR3的氨基酸序列与如序列表中SEQ ID No.4、SEQ ID No.12、SEQ ID No.20、SEQ ID No.28、SEQ ID No.36、SEQ ID No.44、SEQ ID No.52、SEQ ID No.60、SEQ ID No.68、SEQ ID No.76、SEQ ID No.84、SEQ ID No.92所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述轻链CDR1的氨基酸序列与如序列表中SEQ ID No.6、SEQ ID No.14、SEQ ID No.22、SEQ ID No.30、SEQ ID No.38、SEQ ID No.46、SEQ ID No.54、SEQ ID No.62、SEQ ID No.70、SEQ ID No.78、SEQ ID No.86、SEQ ID No.94所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述轻链CDR2的氨基酸序列与如序列表中SEQ ID No.7、SEQ ID No.15、SEQ ID No.23、SEQ ID No.31、SEQ ID No.39、SEQ ID No.47、SEQ ID No.55、SEQ ID No.63、SEQ ID No.71、SEQ ID No.79、SEQ ID No.87、SEQ ID No.95所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述轻链CDR3的氨基酸序列与如序列表中SEQ ID No.8、SEQ ID No.16、SEQ ID No.24、SEQ ID No.32、SEQ ID No.40、SEQ ID No.48、SEQ ID No.56、SEQ ID No.64、SEQ ID No.72、SEQ ID No.80、SEQ ID No.88、SEQ ID No.96所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示。
较佳地，所述重链CDR1的氨基酸序列如序列表中SEQ ID No.2所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.3所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.4所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.10所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.11所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.12所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.18所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.19所示，且所述重链CDR3的氨基酸序列如序列中表SEQ ID No.20所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.26所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID  No.27所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.28所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.34所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.35所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.36所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.42所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.43所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.44所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.50所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.51所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.52所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.58所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.59所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.60所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.66所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.67所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.68所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.74所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.75所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.76所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.82所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.83所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.84所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.90所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.91所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.92所示；
所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.6所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.7所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.8所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.14所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.15所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.16所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.22所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.23所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.24所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.30所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.31所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.32所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.38所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.39所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.40所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.46所示，所述轻链CDR2的氨基酸序列如序 列表中SEQ ID No.47所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.48所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.54所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.55所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.56所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.62所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.63所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.64所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.70所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.71所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.72所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.78所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.79所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.80所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.86所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.87所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.88所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.94所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.95所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.96所示。
更佳地，所述EGFR抗体包括含有所述CDR的EGFR抗体重链可变区和/或EGFR抗体的轻链可变区，所述重链可变区的氨基酸序列如序列表中SEQ ID No.1、SEQ ID No.9、SEQ ID No.17、SEQ ID No.25、SEQ ID No.33、SEQ ID No.41、SEQ ID No.49、SEQ ID No.57、SEQ ID No.65、SEQ ID No.73、SEQ ID No.81或SEQ ID No.89所示；所述轻链可变区的氨基酸序列如序列表中SEQ ID No.5、SEQ ID No.13、SEQ ID No.21、SEQ ID No.29、SEQ ID No.37、SEQ ID No.45、SEQ ID No.53、SEQ ID No.61、SEQ ID No.69、SEQ ID No.77、SEQ ID No.85或SEQ ID No.93所示。
进一步更佳地，所述重链可变区的氨基酸序列如序列表中SEQ ID No.1所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.5所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.9所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.13所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.17所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.21所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.25所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.29所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.33所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.37所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.41所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.45所示；所 述重链可变区的氨基酸序列如序列表中SEQ ID No.49所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.53所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.57所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.61所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.65所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.69所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.73所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.77所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.81所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.85所示；或，所述重链可变区的氨基酸序列如序列表中SEQ ID No.89所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.93所示。
综上，本发明提供了一组与EGFR反应的抗体或抗原结合片段：
在一系列的实例中，本发明提供了一组与EGFR反应的抗体或抗原结合片段：
一条重链可变区，其氨基酸序列号为1和基因序列号为97，包含重链CDR1，CDR2，CDR3，其氨基酸序列号分别为2、3、4；
一条轻链可变区，其氨基酸序列号为5和基因序列号为98，包含轻链CDR1，CDR2，CDR3，其氨基酸序列号分别为6、7、8；
一条重链可变区氨基酸序列号为9和基因序列号为99，包含重链CDR1，CDR2，CDR3，其氨基酸序列号分别为10、11、12；
一条轻链可变区，其氨基酸序列号为13和基因序列号为100，包含轻链CDR1，CDR2，CDR3，其氨基酸序列号分别为14，15，16；
一条重链可变区，其氨基酸序列号为17和基因序列号为101，包含重链CDR1，CDR2，CDR3，其氨基酸序列号分别为18、19、20；
一条轻链可变区，其氨基酸序列号为21和基因序列号为102，包含轻链CDR1，CDR2，CDR3，其氨基酸序列号分别为22、23、24；
一条重链可变区，其氨基酸序列号为25和基因序列号为103，包含重链CDR1，CDR2，CDR3，其氨基酸序列号分别为26、27、28；
一条轻链可变区，其氨基酸序列号为29和基因序列号为104，包含轻链CDR1，CDR2，CDR3，其氨基酸序列号分别为30、31、32。
一条重链可变区，其氨基酸序列号为33和基因序列号为105，包含重链CDR1，CDR2，CDR3，其氨基酸序列号分别为34、35、36；
一条轻链可变区，其氨基酸序列号为37和基因序列号为106，包含重链CDR1，CDR2，CDR3，其氨基酸序列号分别为38、39、40；
一条重链可变区，其氨基酸序列号为41和基因序列号为107，包含重链CDR1，CDR2，CDR3，其氨基酸序列号分别为42、43、44；
一条轻链可变区，其氨基酸序列号为45和基因序列号为108，包含重链CDR1，CDR2，CDR3，其氨基酸序列号分别为46、47、48；
一条重链可变区，其氨基酸序列号为49和基因序列号为109，包含重链CDR1，CDR2CDR3，其氨基酸序列号分别为50、51、52；
一条轻链可变区，其氨基酸序列号为53和基因序列号为110，包含重链CDR1，CDR2，CDR3，其氨基酸序列号分别为54、54、56；
一条重链可变区，其氨基酸序列号为57和基因序列号为111，包含重链CDR1，CDR2，CDR3，其氨基酸序列号分别为58、59、60；
一条轻链可变区，其氨基酸序列号为61和基因序列号为112，包含重链CDR1，CDR2，CDR3，其氨基酸序列号分别为62、63、64；
一条重链可变区，其氨基酸序列号为65和基因序列号为113，包含重链CDR1，CDR2，CDR3，其氨基酸序列号分别为66、67、68；
一条轻链可变区，其氨基酸序列号为69和基因序列号为114，包含重链CDR1，CDR2，CDR3，其氨基酸序列号分别为70、71、72；
一条重链可变区，其氨基酸序列号为73和基因序列号为115，包含重链CDR1，CDR2，CDR3，其氨基酸序列号分别为74、75、76；
一条轻链可变区，其氨基酸序列号为77和基因序列号为116，包含重链CDR1，CDR2，CDR3，其氨基酸序列号分别为78、79、80；
一条重链可变区，其氨基酸序列号为81和基因序列号为117，包含重链CDR1，CDR2，CDR3，其氨基酸序列号分别为82、83、84；
一条轻链可变区，其氨基酸序列号为85和基因序列号为118，包含重链CDR1，CDR2，CDR3，其氨基酸序列号分别为86、87、88；
一条重链可变区，其氨基酸序列号为89和基因序列号为119，包含重链CDR1，CDR2，CDR3，其氨基酸序列号分别为90、91、92；
一条轻链可变区，其氨基酸序列号为93和基因序列号为120，包含重链CDR1，CDR2，CDR3，其氨基酸序列号分别为94、95、96。
较佳地，所述的EGFR抗体还包括构架区，所述构架区包括重链构架区和/或轻链构架区；较佳地，所述重链构架区为人或鼠抗体重链构架区，和/或，所述轻链构架区为人或鼠抗体轻链构架区；更佳地，所述重链构架区为人抗体重链构架区，且所述轻链构架 区为人抗体轻链构架区。
较佳地，所述的EGFR抗体还包括抗体重链恒定区和/或抗体轻链恒定区，所述的抗体重链恒定区优选人源或小鼠源抗体重链恒定区，所述的抗体轻链恒定区优选人源或小鼠源抗体轻链恒定区。
所述的EGFR抗体优选EGFR的单克隆抗体、抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体、单域抗体或单区抗体。
所述单克隆抗体可以由多种途径和技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。
所述的抗体全长蛋白为本领域常规的抗体全长蛋白，其包括重链可变区、轻链可变区、重链恒定区和轻链恒定区。所述的重链可变区和轻链可变区与人源重链恒定区和人源轻链恒定区构成全人源抗体全长蛋白。较佳地，所述的抗体全长蛋白为IgG1、IgG2、IgG3或IgG4。
所述的单链抗体为本领域常规的单链抗体，其包括重链可变区、轻链可变区和15～20个氨基酸的短肽。
所述的抗原抗体结合域蛋白质片段为本领域常规的抗原抗体结合域蛋白质片段，其包括轻链可变区、轻链恒定区和重链恒定区的Fd段。较佳地，所述的抗原抗体结合域蛋白质片段为Fab和F(ab’)。
所述的单域抗体为本领域常规的单域抗体，其包括重链可变区和重链恒定区。
所述的单区抗体为本领域常规的单区抗体，其仅包括重链可变区。
其中，所述EGFR抗体的制备方法为本领域常规的制备方法。所述制备方法较佳地为：从重组表达该EGFR抗体的表达转化体中分离获得或者通过人工合成蛋白质序列获得。所述的从重组表达该EGFR抗体的表达转化体中分离获得优选如下方法：将编码所述EGFR抗体并且带有点突变的核酸分子克隆到重组载体中，将所得重组载体转化到转化体中，得到重组表达转化体，通过培养所得重组表达转化体，即可分离纯化获得所述EGFR抗体。
本发明还提供一种核酸，其编码上述的EGFR抗体。
较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.97、SEQ ID No.99、SEQ ID No.101、SEQ ID No.103、SEQ ID No.105、SEQ ID No.107、SEQ ID No.109、SEQ ID No.111、SEQ ID No.113、SEQ ID No.115、SEQ ID No.117或SEQ ID No.119所示；和/或，编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.98、 SEQ ID No.100、SEQ ID No.102、SEQ ID No.104、SEQ ID No.106、SEQ ID No.108、SEQ ID No.110、SEQ ID No.112、SEQ ID No.114、SEQ ID No.116、SEQ ID No.118或SEQ ID No.120所示。
更佳地，编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.97所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.98所示；编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.99所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.100所示；编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.101所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.102所示；编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.103所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.104所示；编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.105所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.106所示；编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.107所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.108所示；编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.109所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.110所示；编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.111所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.112所示；编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.113所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.114所示；编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.115所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.116所示；编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.117所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.118所示；或，编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.119所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.120所示。
本发明还提供一种包含上述的核酸的重组表达载体。其中所述重组表达载体可通过本领域常规方法获得，即：将本发明所述的核酸分子连接于各种表达载体上构建而成。所述的表达载体为本领域常规的各种载体，只要其能够容载前述核酸分子即可。所述载体较佳地包括：各种质粒、粘粒、噬菌体或病毒载体等。
本发明还提供一种包含上述的重组表达载体的重组表达转化体。
其中，所述重组表达转化体的制备方法为本领域常规的制备方法，较佳地为：将上 述重组表达载体转化至宿主细胞中制得。所述的宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核酸可被有效表达即可。较佳地，所述宿主细胞为E.coli TG1或BL21细胞(表达单链抗体或Fab抗体)，或者CHO-K1细胞(表达全长IgG抗体)。将前述重组表达质粒转化至宿主细胞中，即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。
本发明还提供一种EGFR抗体的制备方法，其包括如下步骤：培养上述的重组表达转化体，从培养物中获得EGFR抗体。
本发明还提供一种药物组合物，其包括上述的EGFR抗体以及药学上可接受的载体。
所述的药学可接受的载体为本领域常规的载体，所述的载体可以为任意合适的生理学或药学上可接受的药物辅料。所述的药物辅料为本领域常规的药物辅料，较佳地包括药学上可接受的赋形剂、填充剂或稀释剂等。更佳地，所述的药物组合物包括0.01～99.99％的上述EGFR或者上述免疫偶联物，和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
本发明所述的药物组合物的给药途径较佳地为肠胃外施用、注射给药或口服给药。所述注射给药较佳地包括静脉注射、肌肉注射、腹腔注射、皮内注射或皮下注射等途径。所述的药物组合物为本领域常规的各种剂型，较佳地为固体、半固体或液体的形式，即可以为水溶液、非水溶液或混悬液，更佳的为片剂、胶囊、颗粒剂、注射剂或输注剂等。更佳地为经由血管内、皮下、腹膜内或肌内施用。较佳地，所述药物组合物还可以作为气雾剂或粗喷雾剂施用，即经鼻施用；或者，鞘内、髓内或心室内施用。更佳地，所述的药物组合物还可以透皮、经皮、局部、肠内、***内、舌下或经直肠施用。
本发明所述的药物组合物的给药剂量水平可以根据达到所需诊断或治疗结果的组合物量而调整。施用方案也可以为单次注射或多次注射，或进行调整。所选择的剂量水平和方案依赖于包括所述药物组合物的活性和稳定性(即，半衰期)、制剂、施用途径、与其他药物或治疗的组合、待检测和/或治疗的疾病或病症、以及待治疗的受试者的健康状况和先前医疗史等各种因素而进行合理地调整。
对于本发明的所述药物组合物的治疗有效剂量可以最初在细胞培养实验或动物模型例如啮齿类动物、兔、犬、猪和/或灵长类动物中进行估计。动物模型也可以用于测定合适的施用浓度范围和途径。随后可以用于确定在人中施用的有用剂量和途径。一般地，施用有效量或剂量的确定和调整以及何时和如何进行此类调整的评估为本领域技术人员 已知。
对于组合疗法，上述EGFR抗体、上述免疫偶联物和/或另外的治疗或诊断剂可以各自作为单一药剂，在适合于执行预期治疗或诊断的任何时间范围内进行使用。因此，这些单一药剂可以基本上同时(即作为单一制剂或在数分钟或数小时内)或以按顺序连续施用。例如，这些单一药剂可以在一年内，或10、8、6、4或2个月内，或4、3、2、或1周内，或5、4、3、2或1天内施用。
关于制剂、剂量、施用方案和可测量的治疗结果的另外指导，参见Berkow等人(2000)The Merck Manual of Medical Information(Merck医学信息手册)和Merck&Co.Inc.,Whitehouse Station,New Jersey；Ebadi(1998)CRC Desk Reference of Clinical Pharmacology(临床药理学手册)等著作。
本发明还提供一种上述EGFR抗体或者上述药物组合物在制备抗肿瘤药物中的应用。
本发明还提供一种检测过表达EGFR蛋白的细胞的方法，所述方法包括如下的步骤：将上述EGFR抗体与待检样品在体外接触，检测所述的EGFR抗体与所述待检样品的结合即可。
本发明还提供一种检测过表达EGFR蛋白的细胞的组合物，所述组合物包括上述EGFR抗体作为活性成分。
本发明还提供一种上述EGFR抗体、或者上述药物组合物在制备预防或治疗与EGFR表达或功能异常相关的疾病的药物中的应用；较佳地，所述的与EGFR表达或功能异常相关的疾病为肿瘤，所述肿瘤优选膀胱癌、脑癌、头颈癌、胰腺癌、肺癌、乳腺癌、卵巢癌、结肠癌、***癌或者肾癌。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
所述的EGFR抗体，与EGFR蛋白具有高度亲和力，经ELISA检测，可以与人EGFR蛋白和食蟹猴EGFR蛋白结合，与小鼠EGFR蛋白不结合；经FACS检测，可以识别肿瘤组织中的人EGFR蛋白。另外，对EGFR阳性细胞进行细胞毒杀伤作用，因此能够运用于***等药物的制备中。
附图说明
图1为人EGFR蛋白转染的CHO-K1细胞FACS筛选检测结果。
图2为人EGFR蛋白转染的293F细胞FACS筛选检测结果。
图3A和3B为ELISA检测EGFR蛋白免疫后小鼠血清抗体效价情况。
图4为FACS检测EGFR抗体与A431的结合反应。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
治疗用单克隆抗体可由多种技术和途径进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等。但是通过杂交瘤技术制备单抗，仍然是目前治疗用单抗制备方法的主流。本发明根据目前最新的单抗技术进展，采用优化的杂交瘤技术，来制备所需的抗EGFR抗体。
传统的杂交瘤制备技术由Kohler and Milstein在40年前建立(Kohler and Milstein1975，Nature 256:495)，现在已广泛应用于科研、诊断、治疗等许多相关的单克隆抗体的制备和生产之中。其基本方法虽然延用至今，但在许多方面都有所变化、改进和创新，包括不同品系动物如转基因动物的使用、电融合技术的引入、高效筛选技术设备的应用如ClonePix设备等，使杂交瘤技术的应用更多样化和更加高效。从常规动物如小鼠等制备的单抗，可以通过常规分子生物学方法克隆抗体重链可变区和轻链可变区基因，可变区基因可以嫁接到人源抗体恒定区基因从而形成人鼠嵌合抗体(U.S.Pat.No.4,816,567，Cabilly et al),以大大降低人体使用时的免疫原性。更进一步，鼠源抗体可变区的CDR结构域可以嫁接到人源抗体架构上，从而使鼠源抗体成分降低到5％以下，大大增加了抗体在人体内使用的安全性。这一途径得到抗体称为人源化抗体，并且是目前抗体药物市场的主要产品(U.S.Pat.No.5,225,539to 55,Winter,and U.S.Pat.Nos.5,530,101；5,585,089；5,693,762and 6,180,370to Queen et al。
本发明利用单克隆抗体技术，综合运用多种免疫方法，利用有效的细胞水平功能性实验开发出了一组高亲和力、高生物学活性和高多样性的EGFR先导抗体。这些抗体显示出优异的性质，能够与人EGFR的胞外区相结合。然后通过分子生物学方法测序获知EGFR抗体的重链可变区和EGFR抗体的轻链可变区的氨基酸序列。
实施例1 EGFR抗体的制备
(一)、免疫原A的制备
将含有编码人源EGFR蛋白胞外区氨基酸序列(NCBI：NP_005219.2)的核苷酸序列克隆到带有人IgG Fc片段(hFc)的pCpC载体(购自Invitrogen，V044-50)并按已建立的标准分子生物学方法制备质粒，具体方法参见Sambrook,J.,Fritsch,E.F.,and Maniatis,T.(1989).Molecular Cloning:A Laboratory Manual,Second Edition(Plainview,New York:Cold Spring Harbor Laboratory Press)。对HEK293细胞(购自Invitrogen)进行瞬时转染(PEI，Polysciences)并使用FreeStyle TM 293(Invitrogen)在37℃下进行扩大培养。4天后收集细胞培养液，离心去除细胞成分，得含EGFR蛋白胞外区的培养上清液。将培养上清液上样到蛋白A亲和层析柱(Mabselect Sure，购自GE Healthcare)，同时用紫外(UV)检测仪监测紫外吸收值(A280nm)的变化。上样后用PBS磷酸盐缓冲液(pH7.2)清洗蛋白A亲和层析柱直到紫外吸收值回到基线，然后用0.1M甘氨酸盐酸(pH2.5)洗脱，收集从蛋白A亲和层析柱上洗脱下来的带hFc标签的EGFR蛋白(EGFR-hFc)，用PBS磷酸盐缓冲液(pH7.2)在4℃冰箱透析过夜。透析后的蛋白经0.22微米无菌过滤后分装于-80℃保存，即获得纯化的免疫原A。
(二)、免疫原B的制备
编码人源EGFR全长氨基酸序列(NCBI：NP_005219.2)的核苷酸序列被克隆到pIRES载体(购自Clontech)并制备质粒。对HEK293细胞系和CHO-K1细胞系(均购自Invitrogen)进行质粒转染(PEI，购自Polysciences)后，在含0.5μg/ml的含10％(w/w)胎牛血清的DMEM培养基中选择性培养2周，用有限稀释法在96孔培养板中进行亚克隆，并置于37℃，5％(v/v)CO 2培养，大约2周后选择部分单克隆孔扩增到6孔板中。对扩增后的克隆用已知的EGFR抗体(购自Absoluteantibody，#Ab00184-1.1)经流式细胞分析法进行筛选。选择长势较好、荧光强度较高、单克隆的细胞系继续扩大培养并液氮冻存，即获得免疫原B。具体选择结果如表1和图1所示，IgG亚型对照为小鼠IgG对照。表1,2说明，已经制得EGFR阳性表达的CHO-K1细胞系，以及EGFR阳性表达的HEK293F细胞系。图1中，横坐标为细胞荧光强度，纵坐标为细胞数。图1的结果说明，CHO-K1-hEGFR 3G2为EGFR高水平表达细胞株。
表1 人源EGFR蛋白的CHO-K1稳转细胞系FACS检测结果
Figure PCTCN2018120906-appb-000001
Figure PCTCN2018120906-appb-000002
(三)、杂交瘤细胞的制备和抗体筛选
A、免疫原A免疫采用6～8周龄BALB/cAnNCrl小鼠或SJL/JorllcoCrl小鼠(购自上海斯莱克公司)，小鼠在SPF条件下饲养。初次免疫时，免疫原A蛋白用弗氏完全佐剂乳化后腹腔注射0.25毫升，即每只小鼠注射50微克免疫原A蛋白。加强免疫时，免疫原A蛋白用弗氏不完全佐剂乳化后腹腔注射0.25毫升，即每只小鼠注射50微克免疫原A蛋白。初次免疫与第一次加强免疫之间间隔2周，以后每次加强免疫之间间隔3周。每次加强免疫1周后采血，用ELISA和FACS检测血清中免疫原A的抗体效价和特异性，结果如图3和表2、3所示。表2、3说明，经人源EGFR-hFc免疫的小鼠的免疫后血清对免疫原均有不同程度的结合，呈现抗原抗体反应，其中最高稀释度在一百万左右。其中空白对照为1％(w/w)BSA，其中批次指第二次加强免疫后第七天的小鼠血清，表中的数据为OD 450nm值。
表2 ELISA检测EGFR蛋白免疫后Balb/c小鼠血清抗体效价
Figure PCTCN2018120906-appb-000003
表3 ELISA检测EGFR蛋白免疫后SJL小鼠血清抗体效价
Figure PCTCN2018120906-appb-000004
B、免疫原B免疫采用6～8周龄BALB/cAnNCrl小鼠或SJL/JorllcoCrl小鼠(购自上海斯莱克公司)，小鼠在SPF条件下饲养。含有编码人源EGFR全长氨基酸序列的核苷酸序列的pIRES质粒[参见实施例1步骤(二)]转染HEK293细胞系，得含有人源EGFR的HEK293稳定细胞系(转染使用X-treme GENE HP DNA Transfection Reagent，购自Roche公司，货号Cat#06 366 236 001，并按说明书操作)在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用DMEM基础培养基(购自Invitrogen)洗涤2次，然后用无酶细胞解离液(购自Invitrogen)37℃处理直至细胞从培养皿壁上可脱落，收集细胞。用DMEM基础培养基洗涤2次，进行细胞计数后将细胞用磷酸盐缓冲液(pH7.2)稀释至2╳10 7细胞每毫升。每只小鼠每次免疫时腹腔注射0.5毫升细胞悬液。第一次与第二次免疫之间间隔2周，以后每次免疫间隔3周。除第一次免疫以外，每次免疫1周后采血，用FACS检测血清中抗体效价和特异性。在第二次加强免疫后，FACS检测血清抗体效价达到1:1000以上。
A～B步骤完成前，将所选择的每只小鼠最后一次免疫腹腔注射100微克纯化的免疫原A(原A进行免疫反应的小鼠)或免疫原B针对免疫原B进行免疫反应的小鼠)，5天后处死小鼠，收集脾细胞。加入NH 4OH至终浓度1％(w/w)，裂解脾细胞中掺杂的红细胞，获得脾细胞悬液。用DMEM基础培养基1000转每分钟离心清洗细胞3次，然后按照活细胞数目5:1比率与小鼠骨髓瘤细胞SP2/0(购自ATCC)混合，采用高效电融合方法(参见METHODS IN ENZYMOLOGY,VOL.220)进行细胞融合。融合后的细胞稀释到含20％胎牛血清、1╳HAT的DMEM培养基中，所述百分比为质量百分比。然后按1╳10 5/200微升每孔加入到96孔细胞培养板中，放入5％CO 2、37℃培养箱中，所述百分比为体积百分比。14天后用ELISA和Acumen(微孔板细胞检测法)筛选细胞融合板上清，将ELISA中OD 450nm>1.0和Acumen中MFI值>100的阳性克隆扩增到24孔板，在含10％(w/w)HT胎牛血清，DMEM(invitrogen)在37℃，5％(v/v)CO 2条件下扩大培养。培养3天后取24孔板中扩大培养的培养液进行离心，收集上清液，对上清液进行抗体亚型分析，用ELISA、FACS确定对EGFR蛋白和EGFR阳性细胞的结合活性(结合活性的检测方法请分别参见实施例3A和实施例3B)，以及抗小鼠抗体-MMAF间接细胞毒杀伤实验(间接细胞毒杀伤活性检测方法请分别参见实施例4)。
根据24孔板筛选结果，挑选ELISA实验中OD 450nm>1.0、FACS实验中MFI值>50和间接细胞毒杀伤实验中杂交瘤细胞培养上清对EGFR阳性细胞杀伤率达到50％的杂交瘤细胞为符合条件的阳性克隆，选择符合条件的杂交瘤细胞用有限稀释法在96孔板进行亚克隆，在含10％(w/w)FBS的DMEM培养基中(购自invitrogen)37℃，5％(v/v) CO 2条件下培养。亚克隆后10天用ELISA和Acumen进行初步筛选，挑选单个阳性单克隆扩增到24孔板继续培养。3天后用FACS确定抗原结合阳性并用抗小鼠抗体-MMAF间接细胞毒杀伤实验评估生物活性(评估标准为ELISA实验中OD 450nm>1.0、FACS实验中MFI值>50和间接细胞毒杀伤实验中杂交瘤细胞培养上清对EGFR阳性细胞杀伤率达到50％)。
根据24孔板样品检测结果，挑选出最优的克隆，并于含10％(w/w)FBS的DMEM培养基中(购自invitrogen)在37℃，5％(v/v)CO 2条件下将该最优的克隆进行扩大培养，液氮冻存即得本发明杂交瘤细胞，并可用于后续的抗体生产和纯化。
实施例2 先导抗体的生产和纯化
杂交瘤细胞产生的抗体浓度较低，大约仅1-10μg/毫升，浓度变化较大。且培养基中细胞培养所产生的多种蛋白和培养基所含胎牛血清成分对很多生物活性分析方法都有不同程度的干扰，因此需要进行小规模(1-5毫克)抗体生产纯化。
将实施例1所得的杂交瘤细胞接种到T-75细胞培养瓶并用生产培养基(Hybridoma serum free medium，购自Invitrogen公司)驯化传代3代。待其生长状态良好，接种细胞培养转瓶。每个2升的培养转瓶中加入200毫升生产培养基，接种细胞密度为1.0╳10 5/毫升。盖紧瓶盖，将转瓶置于37℃培养箱中的转瓶机上，转速3转/分钟。连续旋转培养14天后，收集细胞培养液，过滤去除细胞，并用0.45微米的滤膜过滤至培养上清液澄清。澄清的培养上清液可马上进行纯化或-30℃冻存。
澄清的杂交瘤细胞的培养上清液(200mL)中的单克隆抗体用2mL蛋白A柱(购自GE Healthcare)纯化。蛋白G柱先用平衡缓冲液(PBS磷酸缓冲液，pH7.4)平衡，然后将澄清的培养上清液上样到蛋白A柱，控制流速在3mL/分钟。上样完毕后用平衡缓冲液清洗蛋白G柱，平衡缓冲液的体积为4倍蛋白A柱柱床体积。用洗脱液(0.1M柠檬酸钠缓冲液，pH3.5)洗脱结合在蛋白A柱上的EGFR抗体，用紫外检测器监测洗脱情况(A280紫外吸收峰)。收集洗脱的抗体，加入10％1.0M Tris-HCl缓冲液中和pH，所述百分比为体积百分比，然后立即用PBS磷酸缓冲液透析过夜，第二天换液1次并继续透析3小时。收集透析后的EGFR抗体，用0.22微米的滤器进行无菌过滤，无菌保存，即得纯化的EGFR抗体。
将纯化的EGFR抗体进行蛋白浓度(A280/1.4)、纯度、内毒(Lonza试剂盒)等检测分析，结果如表4所示，结果发现，抗体最终产品内毒素浓度在1.0EU/毫克以内。
表4 纯化的EGFR抗体检测分析
克隆号 抗体纯度 蛋白浓度 内毒素
    (毫克/毫升) (EU/毫克)
51B12D10 >95％ 0.96 <1.0
67A6F9 >95％ 0.71 <1.0
80E11E12 >95％ 1.25 <1.0
75F10C9 >95％ 0.74 <1.0
80E12B7 >95％ 0.51 <1.0
74B3D4 >95％ 4.23 <1.0
122E4D8 >95％ 1.14 <1.0
122E4B6 >95％ 1.32 <1.0
126C3B4 >95％ 2.06 <1.0
128C5G12 >95％ 1.27 <1.0
132E5F6 >95％ 1.38 <1.0
132E8H6 >95％ 2.24 <1.0
实施例3 先导抗体的检定
A、流式细胞实验(FACS)检测抗体与EGFR表达细胞的结合
将实施例1步骤(二)中所述含有编码人源EGFR全长氨基酸序列的核苷酸序列导入CHO-K1细胞株得含人EGFR的CHO-K1稳定细胞株(此处称为CHO-K1-hEGFR稳定细胞株)，然后在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用PBS缓冲液(Phosphate buffer saline)(购自Invitrogen)洗涤2次，然后用无酶细胞解离液(Versene solution:购自Life technology公司)处理和收集细胞。用PBS缓冲液洗涤细胞2次，进行细胞计数后将细胞用PBS缓冲液稀释至2×10 6细胞每毫升，加入2％小牛血清封闭液，所述百分比为质量百分比，室温孵育15分钟，然后用PBS缓冲液离心洗涤2次。将收集的细胞用FACS缓冲液(PBS+2％FBS，所述百分比为质量百分比)悬浮至3×106细胞/mL，按每孔100微升加入到96孔FACS反应板中，加入实施例2所得的纯化的EGFR抗体待测样品每孔100微升，4℃孵育1小时。用FACS缓冲液离心洗涤2次，加入每孔100微升荧光(Alexa 488)标记的二抗(购自Invitrogen)，4℃孵育1小时。用FACS缓冲液离心洗涤3次，加入每孔100微升固定液[4％(v/v)多聚甲醛]悬浮细胞，10分钟后用FACS缓冲液离心洗涤2次。用100微升FACS缓冲液悬浮细胞，用FACS(FACS Calibur，购自BD公司)检测和分析结果。结果如图4和表5所示，表5说明，待测抗体可结合 细胞表面的EGFR。其中IgG对照为鼠IgG，表中的数据为MFI所测细胞群的平均荧光强度值。
表5 FACS检测EGFR抗体与A431(人肿瘤细胞系，购自ATCC)的结合反应
Figure PCTCN2018120906-appb-000005
实施例4 轻重链可变区氨基酸序列测定
总RNA分离：将实施例1亚克隆培养所得的上清液检验过抗原结合后(即经过实施例3～5的检定和活性测定后)，通过离心搜集5×10 7个杂交瘤细胞，加入1mL Trizol混匀并转移到1.5mL离心管中，室温静置5分钟；加0.2mL氯仿，振荡15秒，静置2分钟后于4℃，12000g离心5分钟，取上清转移到新的1.5mL离心管中；加入0.5mL异丙醇，将管中液体轻轻混匀，室温静置10分钟后于4℃，12000g离心15分钟，弃上清；加入1mL 75％乙醇(所述百分比为体积百分比)，轻轻洗涤沉淀，4℃，12000g离心5分钟后弃上清，将沉淀物晾干，加入DEPC处理过的H2O溶解(55℃水浴促进溶剂10分钟)，即得总RNA。
逆转录与PCR：取1μg总RNA，配置20μl体系，加入逆转录酶后于42℃反应60分钟，于7℃反应10分钟终止反应。配置50μlPCR体系，包括1μlcDNA、每种引物25pmol、1μl DNA聚合酶以及相配的缓冲体系、250μmol dNTPs；设置PCR程序，预变性95℃3分钟，变性95℃30秒，退火55℃30秒，延伸72℃35秒，35个循环后再额外于72℃延伸5分钟，得PCR产物。其中逆转录所用的试剂盒为PrimeScript RT Master Mix，购自Takara，货号RR036；PCR所用的试剂盒包括Q5超保真酶，购自NEB，货号M0492。
克隆与测序：取5μl PCR产物进行琼脂糖凝胶电泳检测，将检测阳性样品使用柱回收试剂盒纯化，其中回收试剂盒为
Figure PCTCN2018120906-appb-000006
Gel&PCR Clean-up，购自MACHEREY- NAGEL，货号740609。进行连接反应：样品50ng，T载体50ng，连接酶0.5μl，缓冲液1μl，反应体系10μl，于16℃反应半小时得连接产物，其中连接的试剂盒为T4DNA连接酶，购自NEB，货号M0402；取5μl连接产物加入100μl的感受态细胞(Ecos 101competent cells,购自Yeastern，货号FYE607)中，冰浴5分钟，而后于42℃水浴热激1分钟，放回冰上1分钟后加入650μl无抗生素SOC培养基，于37℃摇床上以200RPM的速度复苏30分钟，取出200μl涂布于含抗生素的LB固体培养基上于37℃孵箱过夜培养；次日，使用T载体上引物M13F和M13R配置30μlPCR体系，进行菌落PCR，用移液器枪头蘸取菌落于PCR反应体系中吹吸，并吸出0.5μl点于另一块含100nM氨苄青霉素的LB固体培养皿上以保存菌株；PCR反应结束后，取出5μl进行琼脂糖凝胶电泳检测，将阳性样品进行测序和分析(其中CDR的划分是根据Chothia定义体系)。测序结果如表9～10所示。
表6 EGFR抗体蛋白序列编号
Figure PCTCN2018120906-appb-000007
其中，表6中的数字即为序列表中序列号，如51B12D10的重链蛋白可变区的氨基酸序列为SEQ ID No.1，而51B12D10的重链蛋白可变区中CDR1的氨基酸序列为SEQ ID No.2。
表8 EGFR抗体基因(DNA)序列编号
克隆号 重链蛋白可变区 轻链蛋白可变区
51B12D10 97 98
67A6F9 99 100
80E11E12 101 102
75F10C9 103 104
80E12B7 105 106
74B3D4 107 108
122E4D8 109 110
122E4B6 111 112
126C3B4 113 114
128C5G12 115 116
132E5F6 117 118
132E8H6 119 120
其中，表8中的数字即为序列表中序列号，如编码51B12D10的重链蛋白可变区的核苷酸序列为SEQ ID No.97。
编码51B12D10的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.97中的第76位至第99位；
编码51B12D10的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.97中的第157位至第171位；
编码51B12D10的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.97中的第295位至第318位；
编码51B12D10的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.98中的第70位至第99位；
编码51B12D10的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.98中的第145位至第165位；
编码51B12D10的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.98中的第262位至第288位；
编码67A6F9的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.99中的第76位至第96位；
编码67A6F9的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.99中的第154位至第171位；
编码67A6F9的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.99中的第295位至第330位；
编码67A6F9的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.100中的第70位至第117位；
编码67A6F9的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.100中 的第163位至第183位；
编码67A6F9的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.100中的第280位至第306位；
编码80E11E12的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.101中的第76位至第96位；
编码80E11E12的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.101中的第154位至第171位；
编码80E11E12的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.101中的第295位至第324位；
编码80E11E12的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.102中的第70位至第117位；
编码80E11E12的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.102中的第163位至第183位；
编码80E11E12的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.102中的第280位至第309位。
编码75F10C9的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.103中的第76位至第96位；
编码75F10C9的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.103中的第154位至第171位；
编码75F10C9的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.103中的第295位至第318位；
编码75F10C9的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.104中的第70位至第102位；
编码75F10C9的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.104中的第148位至第168位；
编码75F10C9的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.104中的第265位至第291位。
编码80E12B7的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.105中的第76位至第96位；
编码80E12B7的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.105中的第154位至第171位；
编码80E12B7的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.105中的第295位至第324位；
编码80E12B7的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.106中的第70位至第99位；
编码80E12B7的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.106中的第145位至第165位；
编码80E12B7的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.106中的第262位至第288位。
编码74B3D4的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.107中的第76位至第96位；
编码74B3D4的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.107中的第154位至第171位；
编码74B3D4的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.107中的第295位至第327位；
编码74B3D4的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.108中的第70位至第99位；
编码74B3D4的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.108中的第145位至第165位；
编码74B3D4的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.109中的第262位至第288位。
编码122E4D8的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.109中的第76位至第96位；
编码122E4D8的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.109中的第154位至第171位；
编码122E4D8的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.109中的第295位至第318位；
编码122E4D8的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.110中的第70位至第102位；
编码122E4D8的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.110中的第148位至第168位；
编码122E4D8的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.110 中的第265位至第291位。
编码122E4B6的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.111中的第76位至第96位；
编码122E4B6的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.111中的第154位至第171位；
编码122E4B6的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.111中的第295位至第318位；
编码122E4B6的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.112中的第70位至第102位；
编码122E4B6的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.112中的第148位至第168位；
编码122E4B6的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.112中的第265位至第291位。
编码126C3B4的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.113中的第76位至第96位；
编码126C3B4的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.113中的第154位至第171位；
编码126C3B4的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.113中的第295位至第318位；
编码126C3B4的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.114中的第70位至第120位；
编码126C3B4的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.114中的第166位至第186位；
编码126C3B4的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.114中的第283位至第309位。
编码128C5G12的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.115中的第76位至第96位；
编码128C5G12的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.115中的第154位至第171位；
编码128C5G12的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.115中的第295位至第318位；
编码128C5G12的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.116中的第70位至第117位；
编码128C5G12的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.116中的第163位至第183位；
编码128C5G12的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.116中的第280位至第306位。
编码132E5F6的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.117中的第76位至第96位；
编码132E5F6的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.117中的第154位至第171位；
编码132E5F6的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.117中的第295位至第324位；
编码132E5F6的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.118中的第70位至第102位；
编码132E5F6的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.118中的第148位至第168位；
编码132E5F6的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.118中的第265位至第291位。
编码132E8H6的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.119中的第76位至第96位；
编码132E8H6的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.119中的第154位至第171位；
编码1132E8H6的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.119中的第295位至第330位；
编码132E8H6的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.120中的第70位至第102位；
编码132E8H6的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.120中的第148位至第168位；
编码132E8H6的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.120中的第265位至第288位。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅 是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (19)

  1. 一种EGFR抗体，其特征在于，其包括互补决定区(CDR)：重链CDR1、重链CDR2和重链CDR3中的一种或多种，和/或，轻链CDR1、轻链CDR2和轻链CDR3中的一种或多种，其中，所述重链CDR1的氨基酸序列如序列表中SEQ ID No.2、SEQ ID No.10、SEQ ID No.18、SEQ ID No.26、SEQ ID No.34、SEQ ID No.42、SEQ ID No.50、SEQ ID No.58、SEQ ID No.66、SEQ ID No.74、SEQ ID No.82或SEQ ID No.90所示；所述重链CDR2的氨基酸序列如序列表中SEQ ID No.3、SEQ ID No.11、SEQ ID No.19、SEQ ID No.27、SEQ ID No.35、SEQ ID No.43、SEQ ID No.51、SEQ ID No.59、SEQ ID No.67、SEQ ID No.75、SEQ ID No.83或SEQ ID No.91所示；所述重链CDR3的氨基酸序列如序列表中SEQ ID No.4、SEQ ID No.12、SEQ ID No.20、SEQ ID No.28、SEQ ID No.36、SEQ ID No.44、SEQ ID No.52、SEQ ID No.60、SEQ ID No.68、SEQ ID No.76、SEQ ID No.84或SEQ ID No.92所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.6、SEQ ID No.14、SEQ ID No.22、SEQ ID No.30、SEQ ID No.38、SEQ ID No.46、SEQ ID No.54、SEQ ID No.62、SEQ ID No.70、SEQ ID No.78、SEQ ID No.86或SEQ ID No.94所示；所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.7、SEQ ID No.15、SEQ ID No.23、SEQ ID No.31、SEQ ID No.39、SEQ ID No.47、SEQ ID No.55、SEQ ID No.63、SEQ ID No.71、SEQ ID No.79、SEQ ID No.87或SEQ ID No.95所示；所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.8、SEQ ID No.16、SEQ ID No.24、SEQ ID No.32、SEQ ID No.40、SEQ ID No.48、SEQ ID No.56、SEQ ID No.64、SEQ ID No.72、SEQ ID No.80、SEQ ID No.88或SEQ ID No.96所示；
    或者，所述重链CDR1的氨基酸序列与如序列表中SEQ ID No.2、SEQ ID No.10、SEQ ID No.18、SEQ ID No.26、SEQ ID No.34、SEQ ID No.42、SEQ ID No.50、SEQ ID No.58、SEQ ID No.66、SEQ ID No.74、SEQ ID No.82、SEQ ID No.90所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述重链CDR2的氨基酸序列与如序列表中SEQ ID No.3、SEQ ID No.11、SEQ ID No.19、SEQ ID No.27、SEQ ID No.35、SEQ ID No.43、SEQ ID No.51、SEQ ID No.59、SEQ ID No.67、SEQ ID No.75、SEQ ID No.83、SEQ ID No.91所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述重链CDR3的氨基酸序列与如序列表中SEQ ID No.4、SEQ ID No.12、SEQ ID No.20、SEQ ID No.28、SEQ ID No.36、SEQ ID No.44、SEQ ID No.52、SEQ ID No.60、SEQ ID No.68、SEQ ID No.76、SEQ ID No.84、SEQ ID No.92所示的氨基酸序列至少有80％的序列同源 性的氨基酸序列所示；所述轻链CDR1的氨基酸序列与如序列表中SEQ ID No.6、SEQ ID No.14、SEQ ID No.22、SEQ ID No.30、SEQ ID No.38、SEQ ID No.46、SEQ ID No.54、SEQ ID No.62、SEQ ID No.70、SEQ ID No.78、SEQ ID No.86、SEQ ID No.94所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述轻链CDR2的氨基酸序列与如序列表中SEQ ID No.7、SEQ ID No.15、SEQ ID No.23、SEQ ID No.31、SEQ ID No.39、SEQ ID No.47、SEQ ID No.55、SEQ ID No.63、SEQ ID No.71、SEQ ID No.79、SEQ ID No.87、SEQ ID No.95所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述轻链CDR3的氨基酸序列与如序列表中SEQ ID No.8、SEQ ID No.16、SEQ ID No.24、SEQ ID No.32、SEQ ID No.40、SEQ ID No.48、SEQ ID No.56、SEQ ID No.64、SEQ ID No.72、SEQ ID No.80、SEQ ID No.88、SEQ ID No.96所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示。
  2. 如权利要求1所述的EGFR抗体，其特征在于，所述重链CDR1的氨基酸序列如序列表中SEQ ID No.2所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.3所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.4所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.10所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.11所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.12所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.18所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.19所示，且所述重链CDR3的氨基酸序列如序列中表SEQ ID No.20所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.26所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.27所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.28所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.34所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.35所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.36所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.42所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.43所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.44所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.50所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.51所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.52所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.58所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.59所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.60所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.66所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.67所示，且所述重链CDR3的氨基酸序列如序列表中 SEQ ID No.68所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.74所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.75所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.76所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.82所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.83所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.84所示；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.90所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.91所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.92所示；
    所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.6所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.7所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.8所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.14所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.15所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.16所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.22所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.23所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.24所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.30所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.31所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.32所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.38所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.39所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.40所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.46所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.47所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.48所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.54所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.55所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.56所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.62所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.63所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.64所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.70所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.71所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.72所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.78所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.79所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.80所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.86所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.87所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.88所示；所 述轻链CDR1的氨基酸序列如序列表中SEQ ID No.94所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.95所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.96所示。
  3. 如权利要求2所述的EGFR抗体，其特征在于，所述EGFR抗体包括含有所述CDR的重链可变区和/或轻链可变区，所述重链可变区的氨基酸序列如序列表中SEQ ID No.1、SEQ ID No.9、SEQ ID No.17、SEQ ID No.25、SEQ ID No.33、SEQ ID No.41、SEQ ID No.49、SEQ ID No.57、SEQ ID No.65、SEQ ID No.73、SEQ ID No.81或SEQ ID No.89所示；所述轻链可变区的氨基酸序列如序列表中SEQ ID No.5、SEQ ID No.13、SEQ ID No.21、SEQ ID No.29、SEQ ID No.37、SEQ ID No.45、SEQ ID No.53、SEQ ID No.61、SEQ ID No.69、SEQ ID No.77、SEQ ID No.85或SEQ ID No.93所示。
  4. 如权利要求3所述的EGFR抗体，其特征在于，所述重链可变区的氨基酸序列如序列表中SEQ ID No.1所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.5所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.9所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.13所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.17所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.21所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.25所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.29所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.33所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.37所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.41所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.45所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.49所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.53所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.57所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.61所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.65所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.69所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.73所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.77所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.81所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.85所示；或，所述重链可变区的氨基酸序列如序列表中SEQ ID No.89所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.93所示。
  5. 如权利要求1或者2所述的EGFR抗体，其特征在于，其还包括构架区，所述构架区包括重链构架区和/或轻链构架区；较佳地，所述重链构架区为人或鼠抗体重链构架 区，和/或，所述轻链构架区为人或鼠抗体轻链构架区；更佳地，所述重链构架区为人抗体重链构架区，且所述轻链构架区为人抗体轻链构架区。
  6. 如权利要求1～5任一项所述的EGFR抗体，其特征在于，所述的EGFR抗体还包括抗体重链恒定区和/或抗体轻链恒定区，所述的抗体重链恒定区优选人源或小鼠源抗体重链恒定区，所述的抗体轻链恒定区优选人源或小鼠源抗体轻链恒定区。
  7. 如权利要求1～6任一项所述的EGFR抗体，其特征在于，所述的EGFR抗体是EGFR的单克隆抗体、抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体、单域抗体或单区抗体。
  8. 一种核酸，其特征在于，其编码如权利要求1～7任一项所述的EGFR抗体。
  9. 如权利要求8所述的核酸，其特征在于，编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.97、SEQ ID No.99、SEQ ID No.101、SEQ ID No.103、SEQ ID No.105、SEQ ID No.107、SEQ ID No.109、SEQ ID No.111、SEQ ID No.113、SEQ ID No.115、SEQ ID No.117或SEQ ID No.119所示；和/或，编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.98、SEQ ID No.100、SEQ ID No.102、SEQ ID No.104、SEQ ID No.106、SEQ ID No.108、SEQ ID No.110、SEQ ID No.112、SEQ ID No.114、SEQ ID No.116、SEQ ID No.118或SEQ ID No.120所示。
  10. 如权利要求9所述的核酸，其特征在于，编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.97所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.98所示；编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.99所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.100所示；编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.101所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.102所示；编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.103所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.104所示；编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.105所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.106所示；编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.107所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.108所示；编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.109所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.110所示；编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.111所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.112所示；编码所述重链可变区的核酸的核苷 酸序列如序列表中SEQ ID No.113所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.114所示；编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.115所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.116所示；编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.117所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.118所示；或，编码所述重链可变区的核酸的核苷酸序列如序列表中SEQ ID No.119所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表中SEQ ID No.120所示。
  11. 一种包含如权利要求8～10中任一项所述的核酸的重组表达载体。
  12. 一种包含如权利要求11所述的重组表达载体的重组表达转化体。
  13. 一种EGFR抗体的制备方法，其包括如下步骤：培养如权利要求12所述的重组表达转化体，从培养物中获得EGFR抗体。
  14. 一种药物组合物，其特征在于，其包括如权利要求1～7中任一项所述的EGFR抗体以及药学上可接受的载体。
  15. 如权利要求14所述的药物组合物，其特征在于，所述的药物组合物包括0.01～99.99％的如权利要求1～7中任一项所述的EGFR抗体和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
  16. 一种如权利要求1～7任一项所述的EGFR抗体或者如权利要求14～15任一项所述的药物组合物在制备抗肿瘤药物中的应用。
  17. 一种检测过表达EGFR蛋白的细胞的方法，其特征在于，包括如下的步骤：如权利要求1～7中任一项所述的EGFR抗体与待检样品在体外接触，检测如权利要求1～7中任一项所述的EGFR抗体与所述待检样品的结合即可。
  18. 一种检测过表达EGFR蛋白的细胞的组合物，其特征在于，其包括如权利要求1～7中任一项所述的EGFR抗体作为活性成分。
  19. 一种如权利要求1～7中任一项所述的EGFR抗体、或者如权利要求14～15所述的药物组合物在制备预防或治疗与EGFR表达或功能异常相关的疾病的药物中的应用；较佳地，所述的与EGFR表达或功能异常相关的疾病为肿瘤，所述肿瘤优选膀胱癌、脑癌、头颈癌、胰腺癌、肺癌、乳腺癌、卵巢癌、结肠癌、***癌或者肾癌。
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