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WO2019114793A1 - Anticorps egfr, son procédé de préparation et son application - Google Patents

Anticorps egfr, son procédé de préparation et son application Download PDF

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Publication number
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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Prior art keywords
seq
amino acid
sequence
sequence listing
acid sequence
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English (en)
Chinese (zh)
Inventor
张莹
刘思琪
杨翠青
刘礼乐
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Xdcexplorer (shanghai) Co Ltd
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Xdcexplorer (shanghai) Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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

L'invention concerne un anticorps EGFR, son procédé de préparation et son application. L'anticorps comprend une région déterminant la complémentarité (CDR) : une ou plusieurs parmi une chaîne lourde CDR1, une chaîne lourde CDR2, ou une chaîne lourde CDR3, et/ou une ou plusieurs parmi une chaîne légère CDR1, une chaîne légère CDR2, ou une chaîne légère CDR3 de l'anticorps EGFR. L'anticorps EGFR présente une affinité élevée vis-à-vis des protéines EGFR et un effet de destruction cytotoxique vis-à-vis des cellules positives de l'EGFR, et peut donc être appliqué dans la préparation de médicaments destinés au traitement de tumeurs et analogues.
PCT/CN2018/120906 2017-12-13 2018-12-13 Anticorps egfr, son procédé de préparation et son application Ceased WO2019114793A1 (fr)

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PH12022552826A1 (en) * 2020-04-28 2024-03-11 Sinocelltech Ltd TgfãŸr2 extracellular domain truncated molecule, fusion protein of tgfãŸr2 extracellular domain truncated molecule and anti-egfr antibody, and anti-tumor use of fusion protein
CN111848806B (zh) * 2020-06-18 2022-06-10 广东安普泽生物医药股份有限公司 Egfr-cd3双功能抗体及其应用
CN113980128B (zh) * 2020-07-27 2023-06-16 中元汇吉生物技术股份有限公司 一种抗人甘胆酸单克隆抗体及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101535343A (zh) * 2006-08-09 2009-09-16 格黎卡特生物技术股份公司 结合egfr的抗原结合分子，编码其的载体，及其应用
WO2014151649A1 (fr) * 2013-03-15 2014-09-25 Alper Biotech, Llc Anticorps monoclonaux contre l'egfr et leurs utilisations
CN104411722A (zh) * 2012-06-25 2015-03-11 瑞泽恩制药公司 抗egfr抗体及其使用
CN104955838A (zh) * 2012-11-21 2015-09-30 詹森生物科技公司 双特异性EGFR/c-Met抗体
CN106459200A (zh) * 2014-03-21 2017-02-22 艾伯维公司 抗‑egfr抗体及抗体药物偶联物
CN106714830A (zh) * 2014-05-30 2017-05-24 上海复宏汉霖生物技术股份有限公司 抗表皮生长因子受体(egfr)抗体
CN107253992A (zh) * 2010-05-27 2017-10-17 根马布股份公司 针对her2的单克隆抗体

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8168181B2 (en) * 2006-02-13 2012-05-01 Alethia Biotherapeutics, Inc. Methods of impairing osteoclast differentiation using antibodies that bind siglec-15
EP2132229B1 (fr) * 2007-03-01 2016-05-11 Symphogen A/S Compositions d'anticorps recombinants dirigés contre le récepteur du facteur de croissance épidermique
AU2013224591A1 (en) * 2012-02-22 2014-07-24 Alethia Biotherapeutics Inc. Co-use of a clusterin inhibitor with an EGFR inhibitor to treat cancer
KR20150036274A (ko) * 2012-07-19 2015-04-07 알레시아 바이오쎄라퓨틱스 인코포레이티드 항-siglec-15 항체
PT3239177T (pt) * 2014-11-25 2021-05-14 Pharmabcine Inc Novo anticorpo egfrviii e composição compreendendo o mesmo
CN106800599B (zh) * 2016-12-05 2021-03-23 中国人民解放军第二军医大学 抗人EGFR和Notch多特异性抗体、其制备方法及用途
CN108484772A (zh) * 2018-04-11 2018-09-04 中国人民解放军军事科学院军事医学研究院 抗her2抗原的人源化抗体h5l5及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101535343A (zh) * 2006-08-09 2009-09-16 格黎卡特生物技术股份公司 结合egfr的抗原结合分子，编码其的载体，及其应用
CN107253992A (zh) * 2010-05-27 2017-10-17 根马布股份公司 针对her2的单克隆抗体
CN104411722A (zh) * 2012-06-25 2015-03-11 瑞泽恩制药公司 抗egfr抗体及其使用
CN104955838A (zh) * 2012-11-21 2015-09-30 詹森生物科技公司 双特异性EGFR/c-Met抗体
WO2014151649A1 (fr) * 2013-03-15 2014-09-25 Alper Biotech, Llc Anticorps monoclonaux contre l'egfr et leurs utilisations
CN106459200A (zh) * 2014-03-21 2017-02-22 艾伯维公司 抗‑egfr抗体及抗体药物偶联物
CN106714830A (zh) * 2014-05-30 2017-05-24 上海复宏汉霖生物技术股份有限公司 抗表皮生长因子受体(egfr)抗体

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AKBARI, B. ET AL.: "Design, Expression and Evaluation of a Novel Humanized Single Chain Antibody Against Epidermal Growth Factor Receptor(EGFR", PROTEIN EXPRESSION AND PURIFICATION, vol. 127, 11 June 2016 (2016-06-11), pages 8 - 15, XP029690608, DOI: doi:10.1016/j.pep.2016.06.001 *
KANG, H.N. ET AL.: "ER2, A Novel Human Anti-EGFR Monoclonal Antibody Inhibit Tumoractivity in Non-small Cell Lung Cancer Models", LUNG CANCER, vol. 95, 31 May 2016 (2016-05-31), pages 57 - 64, XP029489413, DOI: doi:10.1016/j.lungcan.2016.02.013 *

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