WO2019114804A1 - 一种EGFRvIII抗体及其偶联物、制备方法和应用 - Google Patents

一种EGFRvIII抗体及其偶联物、制备方法和应用 Download PDF

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WO2019114804A1
WO2019114804A1 PCT/CN2018/120959 CN2018120959W WO2019114804A1 WO 2019114804 A1 WO2019114804 A1 WO 2019114804A1 CN 2018120959 W CN2018120959 W CN 2018120959W WO 2019114804 A1 WO2019114804 A1 WO 2019114804A1
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seq
sequence
variable region
amino acid
sequence listing
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张莹
杨翠青
刘思琪
张瑜
王荔娜
刘礼乐
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凯惠科技发展(上海)有限公司
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Priority to US16/772,687 priority Critical patent/US20210032351A1/en
Priority to EP18889359.8A priority patent/EP3753952A4/en
Priority to JP2020532972A priority patent/JP7290645B2/ja
Publication of WO2019114804A1 publication Critical patent/WO2019114804A1/zh

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Definitions

  • the invention relates to the field of biomedicine, in particular to an EGFRvIII antibody and a conjugate thereof, 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).
  • EGFRvIII is one of the mutants, also known as de2-7 EGFR, ⁇ EGFR, or ⁇ 2-7 (Olapade-Olaopa et al., Br. J. Cancer. 82, 186-94, 2000).
  • the mature EGFRvIII mRNA lacks 801 nucleotides of exon 2-7, the corresponding EGFRvIII protein is deleted by 267 amino acids (6-273), and a glycine residue is inserted to form a unique linker peptide. (Wong et al., Proc. Natl. Acad. Sci.
  • EGFRvIII does not bind to the ligand, but it is in a sustained low activation state.
  • EGFRvIII can significantly promote the growth of glioma cells (Nishikawa et al., Proc. Natl. Acad. Sci. U.S.A. 91, 7727-31, 1994).
  • expression of EGFRvIII can transform NIH3T3 cells and MCF-7 cells to produce carcinogenicity (Batra et al., Cell Growth Differ. 6, 1251-9, 1995).
  • EGFRvIII can reduce the apoptosis of glioma cells and increase the proliferation of glioma cells to a small extent (Nagane et al ., Cancer Res. 56, 5079-86, 1996).
  • EGFRvIII is specifically expressed in tumor tissues and is not expressed in normal tissues, and thus is a highly specific target in antibody therapy.
  • Monoclonal antibodies are developing into new therapeutic drugs due to their advantages of targeting, specificity, specificity and high affinity.
  • early clinical trials revealed that the use of non-human monoclonal antibodies in humans often results in severe immune responses due to human anti-mouse antibody (HAMA) and human anti-rat antibody (HARA) responses, and antibodies are rapidly cleared.
  • HAMA human anti-mouse antibody
  • HAA human anti-rat antibody
  • antibodies with less immunogenicity were developed, including chimeric antibodies, humanized antibodies, and fully human antibodies.
  • therapeutic monoclonal antibody drugs can be divided into four types: murine antibody (unmanned amino acid sequence), chimeric antibody (60% to 70% humanized amino acid sequence), CDR graft antibody (90% to 95% humanized amino acid sequence) and fully human antibody (100% human amino acid sequence).
  • non-rat monoclonal antibodies can alleviate human anti-mouse antibody responses (HAMA and HARA responses) during human therapy, gradually eliminating the immunogenicity of heterologous antibodies, and maintaining high affinity for antigens.
  • HAMA and HARA responses human anti-mouse antibody responses
  • the pharmacokinetics of the antibodies have been improved, and these antibody drugs have been used in a large amount in the clinic for targeted therapy.
  • Antibody-drug conjugate is a novel biotherapeutic method that links biologically active small molecule drugs to monoclonal antibodies via a chemical link. Monoclonal antibodies serve as carriers to target small molecule drugs. Upon the surface of the target tumor cells, the ADC is endocytosed by the tumor cells to release small molecule drugs, thereby producing specific killing of the tumor cells.
  • antibody-based immunotherapy and chemical-based chemotherapy have been the two major treatment strategies for clinical cancer treatment.
  • Antibodies target antigens overexpressed by tumor cells, and a variety of therapeutic monoclonal antibodies have achieved great clinical success.
  • therapeutic antibodies have good targeting properties, the killing effect is limited.
  • small molecule chemical drugs have an effective killing effect on cancer cells, they also cause the same damage to non-cancer cells. Therefore, the limitations of clinical antibody drugs and small molecule drugs have put forward new requirements for drug development.
  • a new generation of antibody drug conjugates which utilizes the specific binding ability of antibodies to target cells, delivers highly cytotoxic chemical drugs, and achieves targeted and efficient killing of cancer cells.
  • ADC drugs With the advent of new chemical ligation techniques, antibody drug-conjugated drugs began to enter clinical research in the late 1980s, and four ADC drugs have been approved for FDA approval.
  • ADC drugs involves: screening of drug targets, preparation of recombinant antibodies, development of linker technology, and screening optimization of high cytotoxic compounds.
  • EGFRvIII is expressed only in tumor tissues and is not expressed in normal tissues, and thus is a highly specific target in antibody therapy.
  • the technical problem to be solved by the present invention is to overcome the defects of the current lack of EGFRvIII antibody, and to provide an EGFRvIII antibody with high affinity and specificity, a preparation method and application thereof.
  • the EGFRvIII antibody has high affinity with the EGFRvIII protein, and can be conjugated to a small molecule toxin such as MMAF to enter the cell, and cytotoxic killing effect on the EGFRvIII positive cell. Therefore, it can be used in the preparation of drugs for treating tumors and the like.
  • the present inventors used the EGFRvIII protein or a recombinant cell strain overexpressing the EGFRvIII protein as an immunogen, and adopted a conventional hybridoma preparation technique.
  • This technology was established 40 years ago by Kohler and Milstein (Kohler and Milstein 1975, Nature 256:495), and a series of adjustments and improvements were made to obtain a lead antibody for the EGFRvIII antibody.
  • an EGFRvIII antibody with high bio-affinity, such as high affinity to the antibody, conjugated to the small molecule toxin MMAF, and cytotoxic activity against EGFRvIII-positive cells was obtained. .
  • the amino acid sequence of the heavy chain variable region of the EGFRvIII antibody and the light chain variable region of the EGFRvIII antibody was then sequenced by molecular biological methods.
  • the binding regions of an antibody or antigen each comprise a light chain variable region and a heavy chain variable region, each variable region comprising three domains, CDR1, CDR2 and CDR3.
  • the invention provides an EGFRvIII 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 and a light chain One or more of 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, SEQ ID No in the Sequence Listing .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 or SEQ ID No.
  • CDR complementarity determining region
  • 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 No. 51, SEQ ID No. 59, SEQ ID No. 67, SEQ ID No. 75, SEQ ID No. 83, SEQ ID No. 91, SEQ ID No. 99, SEQ ID NO. 184 or SEQ ID
  • the amino acid sequence of the heavy chain CDR3 is SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 20, SEQ ID No. 28, SEQ ID No. 36, SEQ ID in the sequence listing. No. 44, SEQ ID No. 52, SEQ ID No. 60, SEQ ID No.
  • amino acid sequence of the light chain CDR1 is SEQ ID No. 6, SEQ ID No. 14, SEQ in the sequence listing. 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 or SEQ ID No. 102;
  • amino acid sequence of the light chain CDR2 is SEQ ID No. 7, SEQ ID No. 15, SEQ ID No. 23, SEQ ID No in the sequence listing.
  • amino acid sequence of the light chain CDR3 is SEQ ID No. 8, SEQ ID No. 16, SEQ ID No. 24, SEQ ID No. 32, SEQ ID No. 40 in the sequence listing. , SEQ ID No. 48, SEQ ID No. 56, SEQ ID No. 64, SEQ ID No. 72 or SEQ ID No. 80, SEQ ID No. 88, SEQ ID No. 96 or SEQ ID No. 104 ;
  • 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.
  • 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, SEQ ID No. 90, and SEQ ID No. 98 has at least 80 % amino acid sequence of sequence homology; amino acid sequence of the heavy chain CDR2 and SEQ ID No. 3, SEQ ID No. 11, SEQ ID No. 19, SEQ ID No. 27, SEQ in the sequence listing ID No. 35, SEQ ID No. 43, SEQ ID No.
  • amino acid sequence represented by SEQ ID NO. 184 or SEQ ID NO. 186 is represented by an amino acid sequence having at least 80% sequence homology; the amino acid sequence of the heavy chain CDR3 is as SEQ ID No in the sequence listing .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. 9 2.
  • amino acid sequence of SEQ ID No. 100 having at least 80% sequence homology is shown; the amino acid sequence of the light chain CDR1 is SEQ ID No. 6, SEQ ID No. in the sequence listing. 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, The amino acid sequence of SEQ ID No. 86, SEQ ID No. 94, and SEQ ID No. 102 is represented by an amino acid sequence having at least 80% sequence homology; the amino acid sequence of the light chain CDR2 is as in the sequence listing SEQ ID No. 7, SEQ ID No. 15, SEQ ID No. 23, SEQ ID No.
  • SEQ ID No. 39 SEQ ID No. 47, SEQ ID No. 55, SEQ ID No. 63, SEQ ID
  • the amino acid sequence of No. 71, SEQ ID No. 79, SEQ ID No. 87, SEQ ID No. 95, and SEQ ID No. 103 having at least 80% sequence homology is shown;
  • the amino acid sequence of the chain CDR3 is as 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. in the sequence listing. 56, SEQ ID No. 64, SEQ The amino acid sequence of ID No. 72, SEQ ID No. 80, SEQ ID No. 88, SEQ ID No.
  • SEQ ID No. 96, and SEQ ID No. 104 has an amino acid sequence of at least 80% sequence homology; preferably
  • 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, SEQ ID No. 34, SEQ ID No. 42, At least 90% of the amino acid sequences set forth in SEQ ID No. 50, SEQ ID No. 58, SEQ ID No. 66, SEQ ID No. 74, SEQ ID No. 82, SEQ ID No. 90, SEQ ID No. 98
  • the amino acid sequence of the sequence homology is shown;
  • the amino acid sequence of the heavy chain CDR2 is as SEQ ID No. 3, SEQ ID No. 11, SEQ ID No. 19, SEQ ID No.
  • the amino acid sequence of SEQ ID NO. 184 or SEQ ID NO. 186 is represented by an amino acid sequence having at least 90% sequence homology; the amino acid sequence of the heavy chain CDR3 is as SEQ ID No. in the Sequence Listing. 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 represented by SEQ ID No. 92 and SEQ ID No. 100 is represented by an amino acid sequence having at least 90% sequence homology; the amino acid sequence of the light chain CDR1 and SEQ ID No in the sequence listing .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 , the amino acid sequence of SEQ ID No. 78, SEQ ID No. 86, SEQ ID No. 94, SEQ ID No. 102 having at least 90% sequence homology; the light chain CDR2 Amino acid sequence and SEQ ID No. 7, SEQ ID No.
  • 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, SEQ ID No. 50, SEQ ID No. 58, SEQ ID No. 66, SEQ ID No. 74, SEQ ID No. 82, SEQ ID No. 90, SEQ ID No.
  • amino acid sequence of the amino acid sequence having at least 95% sequence homology the amino acid sequence of the heavy chain CDR2 and SEQ ID No. 3, SEQ ID No. 11, SEQ ID No. 19, SEQ ID in the sequence listing 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, as shown in SEQ ID No. 99, SEQ ID NO. 184 or SEQ ID NO.
  • the amino acid sequence of the amino acid sequence having at least 95% sequence homology the amino acid sequence of the heavy chain CDR3 and SEQ ID No. 4, SEQ ID No. 12, SEQ ID No.
  • SEQ ID No. 100 is represented by an amino acid sequence having at least 95% sequence homology; the amino acid sequence of the light chain CDR1 is SEQ ID No. 6, SEQ ID No. in the sequence listing. 14.
  • the amino acid sequence of SEQ ID No. 86, SEQ ID No. 94, and SEQ ID No. 102 is represented by an amino acid sequence having at least 95% sequence homology; the amino acid sequence of the light chain CDR2 is as in the sequence listing 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
  • the amino acid sequence of SEQ ID No. 87, SEQ ID No. 95, and SEQ ID No. 103 having at least 95% sequence homology; the amino acid sequence of the light chain CDR3 and the sequence listing SEQ ID No.
  • amino acid sequence of ID No. 72, SEQ ID No. 80, SEQ ID No. 88, SEQ ID No. 96, and SEQ ID No. 104 has an amino acid sequence of at least 95% sequence homology; further Preferably, 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. in the sequence listing. 42.
  • SEQ ID No. 66, SEQ ID No. 74, SEQ ID No. 82, SEQ ID No. 90, and SEQ ID No. 98 has at least 99% sequence amino acid sequence of sequence homology; amino acid sequence of the heavy chain CDR2 and SEQ ID No. 3, SEQ ID No. 11, SEQ ID No. 19, SEQ ID No. 27 in the sequence listing, 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, SEQ ID No. 99, SEQ ID NO The amino acid sequence of .184 or SEQ ID NO.
  • the amino acid sequence of the heavy chain CDR3 is 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
  • the amino acid sequence of SEQ ID No. 84, SEQ ID No. 92, and SEQ ID No. 100 having at least 99% sequence homology; the amino acid sequence of the light chain CDR1 and the sequence listing 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.
  • SEQ ID No. 62 SEQ An amino acid sequence having at least 99% sequence homology of the amino acid sequence represented by ID No. 70, SEQ ID No. 78, SEQ ID No. 86, SEQ ID No. 94, and SEQ ID No. 102; Amino acid sequence and sequence of light chain CDR2 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, Amino acid sequence of at least 99% sequence homology of the amino acid sequence represented by SEQ ID No. 71, SEQ ID No. 79, SEQ ID No. 87, SEQ ID No. 95, and SEQ ID No.
  • the amino acid sequence of the light chain CDR3 is as 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 in the sequence listing. At least 99% of the amino acid sequence represented by No. 56, SEQ ID No. 64, SEQ ID No. 72, SEQ ID No. 80, SEQ ID No. 88, SEQ ID No. 96, and SEQ ID No. 104 The amino acid sequence of homology is shown.
  • the antibody used in antibody drugs is a monoclonal antibody obtained from a clone of a single antibody-producing cell
  • the antibody is a biological macromolecule and has a very complicated structure, so it is produced, transported, stored, and used in vivo.
  • Various post-translational modifications and degradation reactions occur, such as N-terminal cyclization, glycosylation, deamidation, isomerization, oxidation, fragmentation, disulfide bond mismatch, and the like.
  • These quality attributes may affect antibody stability, biological activity, and bioavailability of the final product, so it is important to control the stability and consistency of product quality.
  • the amino acid sequence of "at least 80% (or 90%, 95%, 99%) of sequence homology" as described in the present invention is obtained by inserting, deleting or replacing the amino acid sequence shown in the aforementioned sequence.
  • PTMs potential post-translational modifications
  • possible analysis of potential post-translational modifications (PTMs) including antibody aggregation, deamidation-based (asparagine deamidation) Analysis of points (NG, NS, NH, etc.), aspartate isoform (DG, DP) sensitive sites, N-glycosylation (N- ⁇ P ⁇ S/T) sensitive sites, and oxidation-sensitive sites replace.
  • deamidation refers to the reaction of the amide of the side chain of asparagine and aspartic acid into a carboxylic acid.
  • glutamine When it is glutamine, the deamidation rate is usually one tenth of that of asparagine, but The mechanism is the same.
  • Isomerization means that the carboxyl group of the side chain of asparagine and aspartic acid is attacked by the electron pair of the nitrogen atom of the residue located on the C-terminal side, resulting in deamidation of asparagine or dehydration of aspartic acid.
  • a stable cyclic imide intermediate which, by cracking, mostly becomes isoaspartic acid and the remainder becomes aspartic acid.
  • an amino acid modification which removes a glutamyl residue and an asparagine residue which is a site for deamidation can be suitably carried out.
  • a site in which the deamidation reaction is promoted that is, a glycine residue in a motif expressed in the form of NG and QG sequences, aspartame An amide residue or a glutamine residue in which (N, Q or G) substitution of any one of the amino acid residues can significantly inhibit the deamidation reaction (WO2003/057881 or WO2005/067620, etc.).
  • a method of culturing the antibody-producing cells or a method of preparing a deamidated reaction to obtain an inhibited antibody can be suitably employed.
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 2 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 3 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR3 The sequence is shown in SEQ ID No. 4 of the Sequence Listing
  • 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 shown in SEQ ID No. 11 of the Sequence Listing.
  • the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 12 of the sequence listing
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No.
  • amino acid sequence of the heavy chain CDR2 is as in the sequence listing.
  • SEQ ID No. 19 and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 20 of the Sequence Listing;
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 26 of the Sequence Listing,
  • the amino acid sequence of the CDR2 of the chain is shown in SEQ ID No. 27 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 28 of the Sequence Listing;
  • the amino acid sequence of the CDR1 of the heavy chain is SEQ ID No.
  • the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No.
  • amino acid sequence of 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, and the amino acid sequence of the heavy chain CDR2 is SEQ ID No. 43 of the Sequence Listing. As shown, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 44 of the Sequence Listing; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 50 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR2 As shown in the Sequence Listing SEQ ID No. 51, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No.
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 58 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 59 of the sequence listing
  • the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 60 of the sequence listing
  • the amino acid sequence of the heavy chain CDR1 is as in the sequence listing.
  • SEQ ID No. 66 the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 67 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 68 of the Sequence Listing
  • the amino acid sequence of the CDR1 of the chain is shown in SEQ ID No.
  • amino acid sequence of the heavy chain CDR2 As shown in SEQ ID No. 75 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 76 of 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 shown in SEQ ID No. 83 of the sequence listing, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 84 of the sequence listing; the amino acid sequence of the heavy chain CDR1 is as in the sequence listing.
  • amino acid sequence of the heavy chain CDR2 As shown in SEQ ID No. 90, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No.
  • the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 92 of the Sequence Listing;
  • the amino acid sequence of the CDR1 of the chain is shown in SEQ ID No. 98 of the Sequence Listing, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 99 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR3 is SEQ ID No.
  • the amino acid sequence of the heavy chain CDR1 is 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. 184 of the Sequence Listing, and the heavy chain is shown.
  • the amino acid sequence of CDR3 is set forth in SEQ ID No.
  • amino acid sequence of the heavy chain CDR2 is set forth in SEQ ID No. 186 of the Sequence Listing
  • amino acid sequence of the heavy chain CDR3 is SEQ ID No. 4 in the Sequence Listing. Shown
  • 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 shown in SEQ ID No. 7 of the Sequence Listing
  • the amino acid sequence of the light chain CDR3 is as in the Sequence Listing.
  • SEQ ID No. 8 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 light The amino acid sequence of the CDR3 of the chain is shown in SEQ ID No. 16 of the Sequence Listing
  • the amino acid sequence of the CDR1 of the light chain is shown in SEQ ID No.
  • amino acid sequence of the CDR2 of the light chain is SEQ ID of the Sequence Listing.
  • amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 24 of the Sequence Listing; the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 30 of the Sequence Listing, the light chain CDR2
  • amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 32 of the Sequence Listing; the amino acid sequence of the light chain CDR1 is SEQ ID No. 38 of the Sequence Listing.
  • amino acid sequence of the light chain CDR2 is shown in SEQ ID No.
  • the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 46 of the Sequence Listing, and the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 47 of the Sequence Listing.
  • the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 48 of the Sequence Listing; the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 54 of the Sequence Listing, and the amino acid sequence of the light chain CDR2 is as The sequence of the SEQ ID No. 55, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 56 of the Sequence Listing; the amino acid sequence of the light chain CDR1 is shown in SEQ ID No.
  • the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 63 of the sequence listing, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 64 of the sequence listing; the amino acid sequence of the light chain CDR1 is as shown in the sequence listing.
  • ID No. 70 the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 71 of the Sequence Listing, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 72 of the Sequence Listing;
  • the amino acid sequence of CDR1 is shown in SEQ ID No. 78 of the Sequence Listing, and the amino acid sequence of the light chain CDR2 is as The SEQ ID No.
  • amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 80 of the Sequence Listing; the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 86 of the Sequence Listing.
  • the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 87 of the Sequence Listing, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 88 of the Sequence Listing; the amino acid sequence of the light chain CDR1 is SEQ ID of the Sequence Listing.
  • amino acid sequence of the light chain CDR2 is shown in SEQ ID No.
  • amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 96 of the Sequence Listing; or The amino acid sequence of the CDR1 of the chain is shown in SEQ ID No. 102 of the Sequence Listing, the amino acid sequence of the CDR2 of the light chain is shown in SEQ ID No. 103 of the Sequence Listing, and the amino acid sequence of the CDR3 of the light chain is SEQ ID No. .104 shows.
  • the EGFRvIII antibody comprises a heavy chain variable region of an EGFRvIII antibody comprising the CDR and/or a light chain variable region of an EGFRvIII antibody, the amino acid sequence of the heavy chain variable region being SEQ ID in the sequence listing 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, SEQ ID No. 89, SEQ ID No. 97, SEQ ID No. 179 or SEQ ID No. 180; amino acid sequence of the light chain variable region SEQ ID No. 5, SEQ ID No.
  • SEQ ID No. 21 SEQ ID No. 29, SEQ ID No. 37, SEQ ID No. 45, SEQ ID No. 53, SEQ ID No. in the sequence listing. 61.
  • 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 variable region of the chain is shown in SEQ ID No. 9 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID No. 13 of the Sequence Listing;
  • the amino acid sequence of the variable region of the heavy chain The sequence of the light chain variable region is shown in SEQ ID No. 21;
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 25, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 33 of the Sequence Listing, 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 of the light chain variable region The sequence is shown in SEQ ID No. 45 of the Sequence Listing; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 49 of the Sequence Listing, and The amino acid sequence of the variable region of the chain is shown in SEQ ID No. 53 of the Sequence Listing; the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID No.
  • the number in Table 1 is the sequence number in the sequence listing, such as the amino acid sequence of the heavy chain protein variable region of 75G7C6 is SEQ ID No. 1, and the amino acid sequence of CDR1 in the heavy chain protein variable region of 75G7C6 is SEQ ID No. 2.
  • the EGFRvIII antibody further comprises a framework region (or a framework region or a framework region), the 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 The framework region is the human antibody light chain framework region.
  • a framework residue is a portion of a light chain variable region or a heavy chain variable region, and is an antibody variable region residue other than a hypervariable residue (a hypervariable residue multi-finger complementarity determining region or CDR) or a CDR residue, It serves as a scaffold for the antigen binding loop (CDR) of this variable domain.
  • the framework residues can be derived from naturally occurring human antibodies, such as the framework regions of human antibodies that are substantially similar to the framework regions of the murine anti-EGFRvIII antibody 75G7C6 or 63A10A7. Artificial framework region sequences representing consensus sequences between individual sequences can also be used. When framework regions for humanization are selected, sequences that are widely present in humans may be superior to less common sequences.
  • Additional mutations in the human framework receptor sequence can be made to restore murine residues believed to be involved in antigen contact and/or residues involved in the structural integrity of the antigen binding site, or to improve antibody expression.
  • Peptide structure prediction can be used to analyze humanized heavy chain variable region and light chain variable region sequences to identify and avoid post-translational protein modification sites introduced by humanized design.
  • the heavy chain framework region is a human antibody heavy chain framework region
  • the human antibody light chain framework region residue may comprise a germline IGKV1- 12.
  • the human antibody heavy chain framework region residues may include germline IGHV7-81, IGHV4-80, IGHV3-79, IGHV(II)-78-1, IGHV5-78, IGHV3-76, IGHV3-73, IGHV3-72, IGHV2.
  • IGHV1-69-2, IGHV2-70D IGHV3-49, IGHV3-43, IGHV1-2, IGHV1-3, IGHV1-8, IGHV1-18, IGHV1-24, IGHV1-45, IGHV1-46, IGHV2 -5, IGHV2-21, IGHV2-26, IGHV2-70, IGHD3-7, IGHV3-9, IGHV3-11, IGHV3-7, IGHV3-7, and IGHD3-9, especially FR1, FR2, FR3 of these lines And the JH fragments JH1, JH2, JH3, JH4, JH4b, JH5 and JH6, in particular the FR4-encoded sequences of these lines, or the consensus sequence of the heavy-chain framework regions.
  • Such framework region sequences can be obtained from public DNA databases including germline antibody gene sequences or published references.
  • the germline DNA sequences of human heavy and light chain variable region genes can be obtained from the "VBase” human germline sequence database (www.mrcco8.com.ac.uk/vbase), and in Kabat, EA et al., 1991. Sequences of Proteins of Immunological Interest, found in the 5th edition.
  • the human acceptor sequence of the humanized EGFRvIII antibody is selected from the human germline exon VH , JH , Vk and Jk sequences, wherein heavy chain variable region of the template is preferably an antibody heavy chain human germline V H exon outer IGHV1-46 * 01, J H exon J H -4; outer or an antibody heavy chain human germline V H exon IGHV1-46 * 01, an outer J H exon J H -6b.
  • the protein further comprises an antibody heavy chain constant region and/or an antibody light chain constant region
  • the antibody heavy chain constant region is conventional in the art, preferably a mouse-derived antibody heavy chain constant region or The human antibody heavy chain constant region, more preferably the human antibody heavy chain constant region.
  • the antibody light chain constant region is conventional in the art, preferably a mouse-derived light chain antibody constant region or a human antibody light chain constant region, more preferably a human antibody light chain constant region.
  • the murine heavy chain constant region constitutes a murine EGFRvIII antibody, and may also constitute an EGFRvIII chimeric antibody with a human heavy chain constant region and a human light chain constant region (wherein the amino acid sequence shown as SEQ ID NO. Mutation of the amino acid sequence shown in NO. 1 was obtained, and NG of the latter CDR2 region was mutated to NA).
  • amino acid sequence of the above chimeric antibody has the sequence determined according to the Kabat definition in the heavy chain variable region represented by SEQ ID NO. 179 or 9, such as SEQ ID NO. 2, 184 and 4 (corresponding to CDR1, 2, respectively) And 3), or the heavy chain CDRs shown in SEQ ID NOS. 10 to 12 and the sequence determined according to the Kabat definition in the light chain variable region represented by SEQ ID NO. 5 or 13 as SEQ ID NO.
  • the human antibody variable region framework is selected, wherein the light chain FR sequence on the antibody light chain variable region, derived from the human germline light chain comprises 1) IGKV1 FR1 -39 * 01 or the IGKV3-11 * 01, FR2, FR3 combined person area, and 2) J K -2 or -4 J K FR4 region of an antibody light chain framework regions; said antibody heavy chain The heavy chain FR sequence on the variable region, derived from the human germline heavy chain sequence comprising 1) FR1, FR2, FR3 region of IGHV1-46*01, and 2) FR4 region of J H -4 or J H -6b A combination of antibody heavy chain framework regions.
  • the selection of the human acceptor framework region should be similar to the framework region of the donor antibody, or the consensus sequence most similar to the variable region subfamily.
  • sequence mutations can be made in the donor and/or acceptor sequences to optimize antigen binding, functionality, codon usage, expression levels, and the like, including introduction of non-human residues into the framework regions.
  • the amino acid sequence of the heavy chain variable region in which the CDR region is grafted into the selected human germline template and the framework region residue is subjected to back mutation is preferably SEQ ID NO. 133, 134, 135, 136, 143. , 144, 145, 146, 147, 141, 142 or 147;
  • the amino acid sequence of the light chain variable region is preferably SEQ ID NO. 137, 138, 139, 140, 148, 149, 150, 151, 152 or 153 Shown.
  • the humanized EGFRvIII antibody of the present invention preferably comprises at least one heavy chain variable region and/or at least one light chain variable region, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO in the Sequence Listing .133, SEQ ID NO. 134, SEQ ID NO. 135, SEQ ID NO. 136, SEQ ID NO. 141, SEQ ID NO. 142, SEQ ID NO. 143, SEQ ID NO. 144, SEQ ID NO. SEQ ID NO. 146 or SEQ ID NO. 147; the light chain variable region sequence is SEQ ID NO. 137, SEQ ID NO. 138, SEQ ID NO. 139, SEQ ID NO. 140, SEQ ID NO. 148, SEQ ID NO. 149, SEQ ID NO. 150, SEQ ID NO. 151, SEQ ID NO. 152 or SEQ ID NO.
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO. 133, SEQ ID NO. 134, SEQ ID NO. 135, SEQ ID NO. 136, SEQ ID NO. 141, SEQ ID NO in the sequence listing. .142, the amino acid sequence of SEQ ID NO. 143, SEQ ID NO. 144, SEQ ID NO. 145, SEQ ID NO. 146 or SEQ ID NO. 147 is represented by an amino acid sequence having at least 80% sequence homology;
  • the light chain variable region sequence is SEQ ID NO. 137, SEQ ID NO. 138, SEQ ID NO. 139, SEQ ID NO. 140, SEQ ID NO. 148, SEQ ID NO. 149, SEQ in the Sequence Listing.
  • the amino acid sequence of the variable region is, for example, SEQ ID No. 154, SEQ ID No. 155, SEQ ID No. 156, SEQ ID No. 157, SEQ ID No. 162, SEQ ID No. 163, SEQ ID in the Sequence Listing.
  • the amino acid sequence of the light chain variable region is, for example, SEQ ID No. 158, SEQ ID No. 159, SEQ ID No. 160, SEQ ID No. 161, SEQ ID No. 169, SEQ ID in the sequence listing.
  • the amino acid sequence encoded by the nucleotide sequence represented by No. 170, SEQ ID No. 171, SEQ ID No. 172, SEQ ID No. 173 or SEQ ID No. 174 has an amino acid sequence of 90% sequence homology; further Preferably, the amino acid sequence of the heavy chain variable region is SEQ ID No. 154, SEQ ID No. 155, SEQ ID No. 156, SEQ ID No. 157, SEQ ID No.
  • amino acid sequence encoded by the nucleotide sequence shown in SEQ ID No. 163, SEQ ID No. 164, SEQ ID No. 165, SEQ ID No. 166, SEQ ID No. 167 or SEQ ID No. 168 A sequence homologous amino acid sequence; the amino acid sequence of the light chain variable region is, as in the sequence listing, SEQ ID No. 158, SEQ ID No. 159, SEQ ID No. 160, SEQ ID No. 161, SEQ The nucleotide sequence encoded by ID No. 169, SEQ ID No. 170, SEQ ID No. 171, SEQ ID No. 172, SEQ ID No. 173 or SEQ ID No.
  • amino acid sequence of 95 has an amino acid sequence of 95.
  • a sequence homologous amino acid sequence; optimally, the amino acid sequence of the heavy chain variable region is, as in the sequence listing, SEQ ID No. 154, SEQ ID No. 155, SEQ ID No. 156, SEQ ID No. .157, SEQ ID No. 162, SEQ ID No. 163, SEQ ID No. 164, SEQ ID No. 165, SEQ ID No. 166, SEQ ID No. 167 or SEQ ID No. 168
  • the amino acid sequence of the sequence-encoded amino acid sequence has 99% sequence homology; the amino acid sequence of the light chain variable region is, as in the sequence listing, SEQ ID No. 158, SEQ ID No. 159, SEQ ID No.
  • the amino acid sequence has 99% sequence homology to the amino acid sequence.
  • the amino acid sequence of the heavy chain variable region is, as in the sequence listing, SEQ ID No. 154, SEQ ID No. 155, SEQ ID No. 156, SEQ ID No. 157, SEQ ID No. 162.
  • the amino acid sequence encoded by the nucleotide sequence represented by SEQ ID No. 163, SEQ ID No. 164, SEQ ID No. 165, SEQ ID No. 166, SEQ ID No. 167 or SEQ ID No. 168 has 80 % sequence homologous amino acid sequence;
  • the amino acid sequence of the light chain variable region is, as in the sequence listing, SEQ ID No. 158, SEQ ID No. 159, SEQ ID No. 160, SEQ ID No.
  • the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID No. 169, SEQ ID No. 170, SEQ ID No. 171, SEQ ID No. 172, SEQ ID No. 173 or SEQ ID No. 174 has an 80% sequence a homologous amino acid sequence; more preferably, as in the sequence listing, SEQ ID No. 154, SEQ ID No. 155, SEQ ID No. 156, SEQ ID No. 157, SEQ ID No. 162, SEQ ID No. 163
  • the amino acid sequence of the light chain variable region is SEQ ID No. 158, SEQ ID No. 159, SEQ ID No. 160, SEQ ID No. 161, SEQ ID No. 169,
  • the amino acid sequence encoded by the nucleotide sequence represented by SEQ ID No. 170, SEQ ID No. 171, SEQ ID No. 172, SEQ ID No. 173 or SEQ ID No. 174 has an amino acid sequence of 90% sequence homology; Further preferably, with SEQ ID No. 154, SEQ ID No. 155, SEQ ID No. 156, SEQ ID No. 157, SEQ ID No. 162, SEQ ID No. 163, SEQ ID No. in the Sequence Listing. 164.
  • the amino acid sequence encoded by the nucleotide sequence represented by SEQ ID No. 165, SEQ ID No. 166, SEQ ID No. 167 or SEQ ID No. 168 has 95% sequence homology; the light
  • the amino acid sequence of the variable region of the chain is, as in the sequence listing, SEQ ID No. 158, SEQ ID No. 159, SEQ ID No. 160, SEQ ID No. 161, SEQ ID No. 169, SEQ ID No. 170,
  • the amino acid sequence encoded by the nucleotide sequence shown by SEQ ID No. 171, SEQ ID No. 172, SEQ ID No. 173 or SEQ ID No. 174 has an amino acid sequence of 95% sequence homology; And SEQ ID No.
  • amino acid sequence encoded by the nucleotide sequence represented by ID No. 165, SEQ ID No. 166, SEQ ID No. 167 or SEQ ID No. 168 has an amino acid sequence of 99% sequence homology; the light chain is variable And the SEQ ID No.
  • the amino acid sequence encoded by the nucleotide sequence shown in .171, SEQ ID No. 172, SEQ ID No. 173 or SEQ ID No. 174 has an amino acid sequence of 99% sequence homology.
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 133 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 137 of the Sequence Listing.
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 133 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 138 of the Sequence Listing;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 133 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No.
  • the amino acid sequence of the variable region is the sequence shown in SEQ ID No. 133 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 140 of the Sequence Listing;
  • the amino acid sequence is the sequence shown in SEQ ID No. 134 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 137 of the Sequence Listing;
  • the amino acid sequence of the heavy chain variable region is as Sequence of SEQ ID No. 134 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as follows
  • the sequence of SEQ ID No. 138; the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No.
  • amino acid sequence of the variable region of the light chain is SEQ ID of the Sequence Listing
  • amino acid sequence of the variable region of the light chain is SEQ ID of the Sequence Listing
  • the sequence shown in No. 139; the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 134 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is SEQ ID No. 140 of the Sequence Listing.
  • the sequence shown; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 135 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 137 of the Sequence Listing.
  • a sequence; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No.
  • amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 138 of the Sequence Listing
  • the amino acid sequence of the variable region of the heavy chain is as shown in SEQ ID No. 135 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 139 of the Sequence Listing
  • the amino acid sequence of the variable region is as shown in SEQ ID No. 135 of the Sequence Listing, and the light chain is variable
  • the amino acid sequence is the sequence shown in SEQ ID No. 140 of the Sequence Listing
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No.
  • amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 140 of the Sequence Listing.
  • the sequence of the heavy chain variable region is as shown in SEQ ID No. 143 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 149 of the Sequence Listing.
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 144 of the Sequence Listing;
  • amino acid sequence of the light chain variable region is as shown in SEQ ID No. 149 of the Sequence Listing;
  • the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 149 of the Sequence Listing; the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 146 of the Sequence Listing, and the light chain
  • the amino acid sequence of the variable region is the sequence shown in SEQ ID No. 149 of the Sequence Listing; the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 147 of the Sequence Listing, and the light chain variable region
  • the amino acid sequence is the sequence shown in SEQ ID No. 149 of the Sequence Listing; the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No.
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 143 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is as in the Sequence Listing SEQ ID No. 150;
  • the amino acid sequence of the heavy chain variable region is as shown in the sequence listing SEQ The sequence shown by ID No. 143, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 151 of the Sequence Listing; the amino acid sequence of the heavy chain variable region is SEQ ID No. in the Sequence Listing.
  • sequence shown by 143, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 152 of the Sequence Listing; the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 143 of the Sequence Listing. a sequence, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 153 of the Sequence Listing; the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 141 of the Sequence Listing, And the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 149 of the Sequence Listing; the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No.
  • the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 150 of the Sequence Listing; the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 141 of the Sequence Listing, and the light chain can be The amino acid sequence of the variable region is as shown in SEQ ID No. 151 of the Sequence Listing; the ammonia of the heavy chain variable region The acid sequence is as shown in SEQ ID No. 141 of the Sequence Listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 152 of the Sequence Listing; the amino acid sequence of the heavy chain variable region a sequence as shown in SEQ ID No.
  • amino acid sequence of the variable region of the light chain is as shown in SEQ ID No. 153 of the Sequence Listing; the amino acid sequence of the variable region of the heavy chain is as in the Sequence Listing
  • the sequence represented by SEQ ID No. 142, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 149 of the Sequence Listing; the amino acid sequence of the heavy chain variable region is SEQ ID No.
  • the sequence shown in .142, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 150 of the Sequence Listing; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 142 of the Sequence Listing.
  • the sequence shown, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 151 of the Sequence Listing; the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 142 of the Sequence Listing. And the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 152 of the Sequence Listing; The amino acid sequence of the variable region of the heavy chain is as shown in SEQ ID No. 142 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 153 of the Sequence Listing; The amino acid sequence of the variable region is the sequence shown in SEQ ID No.
  • amino acid sequence of the variable region of the light chain is the sequence shown in SEQ ID No. 148 of the Sequence Listing; the heavy chain variable region
  • amino acid sequence is the sequence shown in SEQ ID No. 146 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 148 of the Sequence Listing; or, the heavy chain variable region
  • amino acid sequence is the sequence shown in SEQ ID No. 147 of the Sequence Listing, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID No. 148 of the Sequence Listing.
  • the number in Table 1-1 is the sequence number in the sequence listing, such as the amino acid sequence of the heavy chain protein variable region of h75G7C6-1 is SEQ ID No. 133, and the light chain protein variable region of h75G7C6-1 The amino acid sequence is SEQ ID No. 137.
  • CDR1, CDR2 and CDR3 of the heavy chain variable region of c75G7C6-1 are shown in SEQ ID NO. 2, SEQ ID NO. 184 and SEQ ID NO. 4, respectively, in the Sequence Listing.
  • the CDR1, CDR2 and CDR3 of the heavy chain variable region of c75G7C6-2 are shown in SEQ ID NO. 2, SEQ ID NO. 186 and SEQ ID NO. 4, respectively, in the Sequence Listing.
  • the humanized anti-EGFRvIII antibody further comprises a human antibody heavy chain constant region and/or a human antibody anti-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 humanized antibody full length protein.
  • the humanized antibody heavy chain constant region is conventional in the art and may comprise a constant region derived from a human constant region, further comprising a heavy chain constant region of human IgGl, IgG2, IgG3, IgG4 or variants thereof;
  • the humanized antibody light chain constant region is routine in the art and may comprise a constant region derived from a human constant region, further comprising a light chain constant region of a human kappa, lambda chain or variant thereof.
  • the humanized anti-EGFRvIII antibodies of the invention are prepared using any of a variety of methods, including incorporation of complementarity determining regions (CDRs), transplantation, transplantation of shortened CDRs, and transplantation of specificity determining regions (SDRs). , assembled with Frankenstein. Among them, specificity determining regions (SDRs) are residues within CDRs that directly interact with antigen. SDRs correspond to hypervariable residues. See Padlan et al. (1995) FASEB J. 9: 133-139).
  • the human antibody of the present invention is also broadly a class of chimeric antibodies in which the variable region residues responsible for antigen binding, including complementarity determining regions derived from non-human species, shortened complementarity determining regions, or involved in antigen binding. Any other residues; while the remaining variable region residues, eg, residues and constant regions of the framework regions, are at least partially derived from human antibody sequences.
  • a subset of the framework region residues and constant region residues of the humanized antibody can be derived from a non-human source.
  • the variable region of a humanized antibody is also described as a humanized light chain variable region and/or heavy chain variable region.
  • Non-human species are generally species used for immunization with antigens, such as mice, rats, rabbits, non-human primates, or other non-human mammal species.
  • Humanized antibodies are generally less immunogenic than traditional chimeric antibodies and exhibit improved stability upon administration to humans.
  • the humanized antibody also includes a superhumanized antibody, which is a method for preparing a humanized antibody, which does not rely on the human framework sequence as an analysis point, but relies on comparing the normative CDRs of the non-human antibody.
  • the structural type and the CDR structure type of the human antibody particularly the human antibody encoded by the human germline sequence, from which a candidate human antibody sequence from which a suitable human framework sequence can be obtained is identified.
  • a human residue can replace a non-human residue in a CDR, wherein one or more changes have been introduced into the CDRs.
  • the inlay is based on the concept of reducing the amino acid sequence of potential immunogenicity in rodents or other non-human antibodies by reconstituting the solvent-accessible surface of the antibody with a human amino acid sequence.
  • a human amino acid sequence See Padlan (1991) Mol. Immunol. 28: 489-980. Identification of residues of the outer framework regions of solvent accessible residues exposed to the surface in non-human antibodies (the residues are different from the framework regions of human antibodies) Those residues at the same position) and replacing the identified residues with amino acids occupying the same position in the human antibody for inlaying, ie, the embedded antibody, the surface residues of which are predominantly human sequences, and The internal residues are primarily the original murine sequences.
  • Transplantation of CDRs is performed by replacing one or more CDRs of a receptor antibody (eg, a human antibody or other antibody comprising the desired framework residues) with CDRs of a donor antibody (eg, a non-human antibody).
  • the receptor antibody can be selected based on the similarity of the framework residues between the candidate receptor antibody and the donor antibody. For example, according to the Frankenstein method, a human framework region having substantial sequence homology to each framework region of a related non-human antibody is identified, and CDRs of non-human antibodies are grafted onto the complex of these different human framework regions.
  • the above methods can be combined to produce an anti-EGFRvIII antibody of any desired sequence.
  • the EGFRvIII antibody described in the present invention is preferably an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody fragment (scFv), a single domain antibody ( One or more of a single domain antibody, sdAb) and a single-domain antibody, and monoclonal or polyclonal antibodies produced by the above antibodies.
  • 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 of the protein 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 EGFRvIII antibody of the present invention further includes a superhumanized antibody, a diabody, and the like.
  • the preparation method of the EGFRvIII antibody is a conventional preparation method in the art.
  • the preparation method is preferably obtained by isolation from an expression transformant recombinantly expressing the protein or by artificially synthesizing a protein sequence.
  • the method for isolating the expression transformant recombinantly expressing the protein is preferably a method of cloning a nucleic acid molecule encoding the EGFRvIII antibody and having a point mutation into a recombinant vector, and transforming the resulting recombinant vector into a transformant to obtain a recombinant
  • the transformant is expressed, and the EGFRvIII antibody can be obtained by isolation and purification by culturing the resulting recombinant expression transformant.
  • the invention also provides a nucleic acid encoding the EGFRvIII antibody described above.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is SEQ ID No. 105, SEQ ID No. 107, SEQ ID No. 109, SEQ ID No. 111, SEQ ID No in the sequence listing. .113, SEQ ID No. 115, SEQ ID No. 117, SEQ ID No. 119, SEQ ID No. 121, SEQ ID No. 123, SEQ ID No. 125, SEQ ID No. 127, SEQ ID No. 129 SEQ ID No. 185, SEQ ID No. 154, SEQ ID No. 155, SEQ ID No. 156, SEQ ID No. 157, SEQ ID No. 162, SEQ ID No.
  • SEQ ID No. 163, SEQ ID No. 164, SEQ ID No. 165, SEQ ID No. 166, SEQ ID No. 167 or SEQ ID No. 168; and/or the nucleotide sequence of the nucleic acid encoding the light chain variable region is as SEQ ID in the Sequence Listing 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, SEQ ID No. 120, SEQ ID No. 122, SEQ ID No. 124, SEQ ID No. 126, SEQ ID No. 128, SEQ ID No. 130, SEQ ID No. 158, SEQ ID No.
  • nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 105 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in the sequence listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 107, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in ID No. 106.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No.
  • nucleoside of the nucleic acid encoding the light chain variable region is represented by the sequence listing.
  • the acid sequence is shown in SEQ ID No. 110 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 111 of the Sequence Listing, and the nucleic acid encoding the variable region of the light chain
  • the nucleotide sequence is shown in SEQ ID No. 112 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 113 of the Sequence Listing, and the light chain is variable.
  • nucleotide sequence of the nucleic acid of the region is shown in SEQ ID No. 114 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in the Sequence Listing SEQ ID.
  • the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in No. 115, and the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in the sequence of SEQ ID No. 116.
  • SEQ ID No. 117 Listed as SEQ ID No. 117, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No.
  • nucleotide of the nucleic acid encoding the heavy chain variable region The sequence is shown in SEQ ID No. 119 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. 120 of the Sequence Listing; the nucleic acid encoding the heavy chain variable region
  • nucleotide sequence is shown in SEQ ID No. 121 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. 122 of the Sequence Listing; encoding the heavy chain variable region
  • nucleotide sequence of the nucleic acid is shown in SEQ ID No.
  • nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in SEQ ID No. 124 of the Sequence Listing; encoding the heavy chain
  • the nucleotide sequence of the nucleic acid of the variable region is as shown in SEQ ID No. 125 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the variable region of the light chain is, for example, a sequence.
  • SEQ ID No 126 The nucleotide sequence of the nucleic acid encoding the heavy chain variable region is set forth in SEQ ID No.
  • nucleotide sequence of the nucleic acid encoding the light chain variable region is as The nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 129 of the Sequence Listing, and the nucleoside of the nucleic acid encoding the variable region of the light chain is SEQ ID No. 128; The acid sequence is shown in SEQ ID No. 130 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is set forth in SEQ ID No. 185 of the Sequence Listing, and the light chain variable region is encoded. The nucleotide sequence of the nucleic acid is as shown in SEQ ID No.
  • nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 154 of the Sequence Listing, and the light chain is encoded.
  • the nucleotide sequence of the nucleic acid of the variable region is shown in SEQ ID No. 158 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 154 of the Sequence Listing, and the coding institute
  • the nucleotide sequence of the nucleic acid of the variable region of the light chain is shown in SEQ ID No.
  • nucleotide sequence of the nucleic acid encoding the heavy chain variable region As shown in SEQ ID No. 154 of the Sequence Listing, the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 160 of the Sequence Listing; the core of the nucleic acid encoding the heavy chain variable region The nucleotide sequence is shown in SEQ ID No. 154 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in SEQ ID No. 161 of the Sequence Listing; encoding the heavy chain variable region The nucleotide sequence of the nucleic acid is shown in SEQ ID No.
  • nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in SEQ ID No. 158 of the Sequence Listing; encoding the heavy chain can be The nucleotide sequence of the nucleic acid of the variable region is shown in SEQ ID No. 155 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the variable region of the light chain is as shown in SEQ ID No. 159 of the Sequence Listing; The nucleotide sequence of the nucleic acid of the heavy chain variable region is shown in SEQ ID No.
  • nucleotide sequence of the nucleic acid encoding the variable region of the light chain is shown in SEQ ID No. 160 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 155 of the Sequence Listing, and the nucleoside encoding the nucleic acid of the light chain variable region
  • the acid sequence is shown in SEQ ID No. 161 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No.
  • nucleic acid encoding the variable region of the light chain The nucleotide sequence is shown in SEQ ID No. 158 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 156 of the Sequence Listing, and the light chain is variable.
  • the nucleotide sequence of the nucleic acid of the region is shown in SEQ ID No. 159 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 156 of the Sequence Listing, and the light is encoded.
  • nucleotide sequence of the nucleic acid of the chain variable region is shown in SEQ ID No. 160 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 156 of the Sequence Listing, and the coding is performed.
  • the nucleotide sequence of the nucleic acid of the light chain variable region is shown in SEQ ID No. 161 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 157 of the Sequence Listing.
  • nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No.
  • nucleic acid encoding the heavy chain variable region The nucleotide sequence is shown in SEQ ID No. 157 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in SEQ ID No. 159 of the Sequence Listing; encoding the heavy chain variable region
  • nucleotide sequence of the nucleic acid is shown in SEQ ID No. 157 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in SEQ ID No. 160 of the Sequence Listing; encoding the heavy chain can be The nucleotide sequence of the nucleic acid of the variable region is shown in SEQ ID No.
  • nucleotide sequence of the nucleic acid encoding the variable region of the light chain is as shown in SEQ ID No. 161 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid of the heavy chain variable region is shown in SEQ ID No. 164 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in SEQ ID No. 170 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 165 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 encoding the heavy chain variable region is set forth in SEQ ID No. 166 of the Sequence Listing and encodes the light chain variable region
  • the nucleotide sequence of the acid is shown in SEQ ID No. 170 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 167 of the Sequence Listing, and the light chain can be encoded.
  • the nucleotide sequence of the nucleic acid of the variable region is shown in SEQ ID No. 170 of the Sequence Listing; 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 of the light chain variable region is shown in SEQ ID No. 170 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 164 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. 169 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is set forth in SEQ ID No. 164 of the Sequence Listing.
  • nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in SEQ ID No. 171 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is SEQ ID No. And the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 172; encoding the heavy chain variable.
  • nucleotide sequence of the nucleic acid is shown in SEQ ID No. 164 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in SEQ ID No.
  • nucleic acid of the variable region is shown in SEQ ID No. 164 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the variable region of the light chain is shown in SEQ ID No. 174 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid of the variable region of the heavy chain is shown in SEQ ID No. 162 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the variable region of the light chain is shown in SEQ ID No. 170 of the Sequence Listing.
  • nucleotide sequence of the nucleic acid encoding the heavy chain variable region is set forth in SEQ ID No. 162 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. 171; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 162 of the sequence listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in the sequence listing. ID No. 172; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 162 of the Sequence Listing, and the light chain is encoded.
  • nucleotide sequence of the nucleic acid of the variable region is shown in SEQ ID No. 173 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 162 of the Sequence Listing, and the coding institute
  • the nucleotide sequence of the nucleic acid of the variable region of the light chain is shown in SEQ ID No. 174 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 163 of the Sequence Listing.
  • 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 encoding the heavy chain variable region is SEQ ID No. 163 of the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in SEQ ID No. 171 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in the Sequence Listing SEQ ID.
  • the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 172 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in No. 163 Listed in SEQ ID No.
  • nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 173 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid of the heavy chain variable region is shown in SEQ ID No. 163 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the variable region of the light chain is shown in SEQ ID No. 174 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is set forth in SEQ ID No. 166 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. 169 of the Sequence Listing.
  • nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 167 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in the Sequence Listing SEQ ID. No. 169; or the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 168 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region As shown in the Sequence Listing, SEQ ID No. 169.
  • the numbers in Table 1-2 are the sequence numbers in the sequence listing, for example, the nucleotide sequence of the heavy chain protein variable region of h75G7C6-1 is SEQ ID No. 154, and the light chain protein of h75G7C6-1 is variable.
  • the nucleotide sequence of the region is SEQ ID No. 158.
  • the preparation method of the nucleic acid is a preparation method conventional in the art, and preferably includes the steps of obtaining a nucleic acid molecule encoding the above humanized anti-EGFRvIII antibody by gene cloning technology, or obtaining a coding method by artificial full sequence synthesis.
  • a nucleic acid molecule of the above humanized anti-EGFRvIII antibody is obtained by gene cloning technology, or obtaining a coding method by artificial full sequence synthesis.
  • nucleic acids encoding the above-described humanized anti-EGFRvIII antibodies can be suitably introduced as substitutions, deletions, alterations, insertions or additions to provide a homologue of a polynucleotide.
  • a homologue of a polynucleotide of the invention can be made by replacing, deleting or increasing one or more nucleotides of a nucleic acid encoding the humanized anti-EGFRvIII antibody while maintaining antibody activity.
  • the number in Table 2 is the sequence number in the sequence listing, and the nucleotide sequence of the variable region of the heavy chain protein encoding 75G7C6 is SEQ ID No. 105.
  • the complementarity determining regions (CDRs) described in the present invention are residues of an antibody variable region involved in antigen binding.
  • CDRs complementarity determining regions
  • Several numbering systems for identifying CDRs are commonly used, including, for example, Kabat definitions, Chothia definitions, and AbM definitions.
  • the Kabat definition is based on sequence variability
  • the Chothia definition is based on the position of the structural loop region
  • the AbM definition is a compromise between the Kabat and Chothia methods.
  • the light chain variable region has three CDR regions, the CDR1 is located at amino acids 24-34 (CDR1-L), the CDR2 is at amino acids 50-56 (CDR2-L), and the CDR3 is located.
  • Amino acids 89-97 (CDR3-L). Due to the change in the length of the variable region, in the different central or different subgroups, the 27th position may have 1-6 amino acids, and the 95th position may have 1-6 amino acids, which are added to the original number. English letters are numbered, such as: 27A, 27B, 95A, 95B, etc. According to the Kabat definition, the CDRs of the heavy chain variable region are defined by residues at positions 31 and 35B (CDR1-H), positions 50 and 65 (CDR2-H), and positions 95 and 102 (CDR3-H). (according to Kabat numbering).
  • the CDRs of the heavy chain variable region are defined by residues at positions 26 and 32 (CDR1-H), positions 52 and 56 (CDR2-H), and positions 95 and 102 (CDR3-H) (according to the Chothia number).
  • the CDRs of the heavy chain variable region are defined by the residues at positions 26 and 35B (CDR1-H), at positions 50 and 58 (CDR2-H), and at positions 95 and 102 (CDR3-H) (according to Kabat number).
  • a plurality of amino acids may be present at positions 35, 52, 82, and 100, and are numbered A, B, C, and the like. See Martin et al.
  • the CDR numbering system of the following murine antibody is Chothia, and the subsequent humanized antibody adopts the Kabat numbering system.
  • nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 75G7C6 is from position 76 to position 96 in SEQ ID No. 105 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 75G7C6 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 75G7C6 is 295th to 339th in SEQ ID No. 105 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 75G7C6 is the 70th to 105th positions in SEQ ID No. 106 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 75G7C6 is from positions 151 to 171 in SEQ ID No. 106 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region of 75G7C6 is 268th to 294th in SEQ ID No. 106 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 63A10A7 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 63A10A7 is from positions 154 to 171 of SEQ ID No. 107 of the Sequence Listing;
  • nucleotide sequence of the CDR2 encoding the heavy chain protein variable region of c75G7C6-1 is shown in positions 154 to 171 of SEQ ID No. 185 in the Sequence Listing;
  • the nucleotide sequence of CDR2 encoding the heavy chain protein variable region of c75G7C6-2 is shown in positions 154 to 171 of SEQ ID No. 187 in the Sequence Listing.
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 63A10A7 is from positions 295 to 318 of SEQ ID No. 107 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 63A10A7 is from positions 70 to 102 of SEQ ID No. 108 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 63A10A7 is 148th to 168th in SEQ ID No. 108 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 63A10A7 is position 265 to 291 in SEQ ID No. 108 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 64F1F8 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 64F1F8 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 64F1F8 is from positions 295 to 318 in SEQ ID No. 109 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 64F1F8 is from position 70 to position 99 of SEQ ID No. 110 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 64F1F8 is from positions 145 to 165 in SEQ ID No. 110 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 64F1F8 is position 262 to 288 in SEQ ID No. 110 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 7E5-2A9 is from position 76 to position 96 of SEQ ID No. 111 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 7E5-2A9 is from positions 154 to 171 in SEQ ID No. 111 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 7E5-2A9 is from positions 295 to 309 in SEQ ID No. 111 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 7E5-2A9 is from position 70 to position 120 in SEQ ID No. 112 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region of 7E5-2A9 is 166th to 186th in SEQ ID No. 112 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 7E5-2A9 is position 283 to 309 in SEQ ID No. 112 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 43H6A1 is from positions 127 to 150 in SEQ ID No. 113 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 43H6A1 is from position 208 to position 222 in SEQ ID No. 113 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 43H6A1 is 346th to 366th in SEQ ID No. 113 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 43H6A1 is from positions 70 to 102 of SEQ ID No. 114 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 43H6A1 is from positions 148 to 168 of SEQ ID No. 114 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 43H6A1 is from position 265 to position 291 in SEQ ID No. 114 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 46C4A6 is from positions 76 to 96 of SEQ ID No. 115 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region of 46C4A6 is 154th to 171th in SEQ ID No. 115 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 46C4A6 is 295th to 306th in SEQ ID No. 115 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 46C4A6 is from positions 70 to 117 in SEQ ID No. 116 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 46C4A6 is 163th to 183th in SEQ ID No. 116 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 46C4A6 is 280th to 306th in SEQ ID No. 116 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 49G12G5 is from positions 76 to 96 of SEQ ID No. 117 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 49G12G5 is from positions 154 to 171 in SEQ ID No. 117 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 49G12G5 is from positions 295 to 318 of SEQ ID No. 117 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 49G12G5 is the 70th to 105th positions in SEQ ID No. 118 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 49G12G5 is from positions 151 to 171 in SEQ ID No. 118 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 49G12G5 is from positions 268 to 294 in SEQ ID No. 118 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 51G7C2 is from positions 76 to 96 of SEQ ID No. 119 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 51G7C2 is 154th to 171th in SEQ ID No. 119 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 51G7C2 is from positions 295 to 318 of SEQ ID No. 119 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 51G7C2 is the 70th to 105th positions in SEQ ID No. 120 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 51G7C2 is from positions 151 to 171 in SEQ ID No. 120 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 51G7C2 is from positions 268 to 294 in SEQ ID No. 120 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 53D8H4 is from positions 76 to 96 of SEQ ID No. 121 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 53D8H4 is from positions 154 to 171 in SEQ ID No. 121 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 53D8H4 is position 295 to 318 of SEQ ID No. 121 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 53D8H4 is from positions 70 to 102 of SEQ ID No. 122 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 53D8H4 is 148th to 168th in SEQ ID No. 122 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 53D8H4 is from positions 265 to 291 in SEQ ID No. 122 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 54D2G10 is from positions 76 to 96 of SEQ ID No. 123 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 54D2G10 is 154th to 171th in SEQ ID No. 123 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 54D2G10 is 295th to 315th in SEQ ID No. 123 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 54D2G10 is from position 70 to position 99 of SEQ ID No. 124 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 54D2G10 is from positions 145 to 165 in SEQ ID No. 124 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 54D2G10 is position 262 to 288 in SEQ ID No. 124 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 56F10G2 is from position 76 to position 96 of SEQ ID No. 125 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 56F10G2 is 154th to 171th in SEQ ID No. 125 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 56F10G2 is 295th to 315th in SEQ ID No. 125 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 56F10G2 is from positions 70 to 99 of SEQ ID No. 126 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 56F10G2 is from positions 145 to 165 in SEQ ID No. 126 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 56F10G2 is position 262 to 288 in SEQ ID No. 126 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 59G3F12 is from positions 76 to 99 of SEQ ID No. 127 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 59G3F12 is from positions 157 to 171 in SEQ ID No. 127 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 59G3F12 is from position 295 to position 318 in SEQ ID No. 127 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 59G3F12 is from position 70 to position 99 of SEQ ID No. 128 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 59G3F12 is from positions 145 to 165 in SEQ ID No. 128 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 59G3F12 is position 262 to 288 in SEQ ID No. 128 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 64B11F2 is from position 76 to position 96 in SEQ ID No. 129 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 64B11F2 is from positions 154 to 171 in SEQ ID No. 129 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 64B11F2 is 295th to 315th in SEQ ID No. 129 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 64B11F2 is from positions 70 to 102 of SEQ ID No. 130 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 64B11F2 is 148th to 168th in SEQ ID No. 130 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 64B11F2 is position 265 to position 291 in SEQ ID No. 130 of the Sequence Listing.
  • the preparation method of the nucleic acid is a preparation method conventional in the art, and preferably includes the steps of obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial total sequence synthesis. .
  • the base sequence encoding the amino acid sequence of the above protein may be appropriately introduced with a substitution, deletion, alteration, insertion or addition to provide a homologue of a polynucleotide.
  • a homologue of a polynucleotide of the invention can be made by replacing, deleting or increasing one or more bases encoding a gene of the protein sequence within the range of activity of the antibody.
  • the invention also provides a recombinant expression vector comprising the nucleic acid.
  • 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 the EGFRvIII antibody, which comprises the steps of culturing the above recombinant expression transformant, and obtaining the EGFRvIII antibody from the culture.
  • the invention also provides an immunoconjugate comprising the above EGFRvIII antibody covalently attached to a cytotoxic agent.
  • the above 1 equivalent of the protein is linked to the y equivalent of the cytotoxic agent via the x equivalent linker, and has the structure of the formula 1.
  • Ab is the above-mentioned EGFRvIII antibody; L is a linker; D is a cytotoxic agent; the x is a conventional degree of crosslinking in the art, x is a natural number, preferably 1 to 20; y is a natural number greater than, preferably 1 to An integer of 20; x and y are each independently preferably 2w, w is an integer of 1 to 5, further preferably 3 to 4; and the ratio of x and y is preferably 1:1.
  • the L is a linker (or crosslinker or coupling agent) conventional in the art.
  • the L contains two functional groups, a group reactive with an antibody, and a group reactive with a drug (for example, an aldehyde or a ketone).
  • the drug is coupled to the EGFRvIII antibody described above via a linker molecule.
  • the L is released after entering the cell, and includes, but is not limited to, functional groups, active esters, carbonates, carbamates, imine phosphates, anthraquinones, anthraquinones, acetals, orthoesters, amino groups.
  • a small peptide or nucleotide fragment for example, maleimidocaproyl (MC), maleimidocaproyl-L-valine-L-citrulline p-aminobenzyl Alcohol (MC-VC-PAB) or 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC).
  • the L mainly contains the structure of Formula 2, which is the remainder corresponding to the leaving group in L after leaving:
  • Sp 2 is a bond, -S- or -O-, provided that when Alk 2 is a bond, Sp 2 is a bond;
  • Z 1 is H, (C 1 -C 5 )alkyl, or (C 1 -C 5 )alkyl, (C 1 -C 5 )alkoxy, (C 1 -C 4 )thioalkoxy , halogen, nitro, -COOR', -CONHR', -O(CH 2 ) n COOR', -S(CH 2 ) n COOR', -O(CH 2 ) n CONHR' or -S(CH 2 )
  • Sp is a linear or branched divalent or trivalent (C 1 -C 18 ) group, a divalent or trivalent aryl or heteroaryl group, a divalent or trivalent (C 3 -C 18 ) naphthenic group Or a heterocycloalkyl group, a divalent or trivalent aryl or heteroaryl-aryl (C 1 -C 18 ) group, a divalent or trivalent cycloalkyl or heterocycloalkyl-alkyl group ( a C 1 -C 18 ) group, or a divalent or trivalent (C 2 -C 18 ) unsaturated alkyl group, wherein the heteroaryl group is preferably a furyl group, a thienyl group, an N-methylpyrrolyl group, a pyridine group Base, N-methylimidazolyl, oxazolyl, pyrimidinyl.
  • Alk 1 is a branched or unbranched (C 1 -C 5 ) alkylene chain
  • Sp 1 is a bond, -S-, -O-, -CONH-, -NHCO- or -NR', wherein R 'As defined above, the condition is that when Alk 1 is a key, Sp 1 is a key;
  • Ar is composed of (C 1 -C 6 )alkyl, (C 1 -C 5 )alkoxy, (C 1 -C 4 )thioalkoxy, halogen, nitro, -COOR', -CONHR', 1, 2 or 3 groups of -O(CH 2 ) n COOR', -S(CH 2 ) n COOR', -O(CH 2 ) n CONHR' or -S(CH 2 ) n CONHR' Substituted 1,2-, 1,3- or 1,4-phenylene, wherein n and R' are as defined above, or Ar is each from C 1 -C 6 )alkyl, (C 1 -C 5 ) Alkoxy, (C 1 -C 4 )thioalkoxy, halogen, nitro, -COOR', -CONHR', -O(CH 2 ) n COOR', -S(CH 2 ) n COOR', -2 , 3, or
  • Z 1 is (C 1 -C 5 )alkyl, or (C 1 -C 5 )alkyl, (C 1 -C 4 )alkoxy, (C 1 -C 4 )thioalkoxy, halogen , nitro, -COOR', -CONHR', -O(CH 2 ) n COOR', -S(CH 2 ) n COOR', -O(CH 2 ) n CONHR' or -S(CH 2 ) n CONHR '1, 2, or 3 groups are optionally substituted phenyl; Alk 2 and Sp 2 are both bonds; and Sp and Q are as defined above only.
  • the above key means a covalent bond.
  • the L is maleimidocaproyl (MC).
  • the D may be a conventional cytotoxic agent in the art, preferably selected from the group consisting of a cytotoxin, a chemotherapeutic agent, a radioisotope, a therapeutic nucleic acid, an immunomodulator, an anti-angiogenic agent, an antiproliferative pro-apoptotic agent or a lytic enzyme. .
  • cytotoxin is a cytotoxin conventional in the art and generally refers to an active agent which inhibits or prevents cellular function and/or causes cell destruction.
  • active agent which inhibits or prevents cellular function and/or causes cell destruction.
  • cytotoxin selected from the group consisting of antibiotics, inhibitors of tubulin polymerization, alkylating agents, protein synthesis inhibitors, protein kinase inhibitors, phosphatase inhibitors, topoisomerase inhibitors, protein kinases, phosphatases, topoisomerism Enzyme or cyclin.
  • doxorubicin is selected from the group consisting of doxorubicin, daunorubicin, idarubicin, arubicin, zolubicin, mitoxantrone, epirubicin, carbofurin, nogamycin, Menoril, pirarubicin, valrubicin, cytarabine, gemcitabine, flufluuridine, acitretin, enoxacitabine, azacitidine, deoxyfluorouridine, pentazostat Ding, bromouridine, capecitabine, cladribine, decitabine, fluorouridine, fludarabine, glutathione, puromycin, tegafur, thiazolidine nucleoside, trichoderma , cisplatin, carboplatin, cyclophosphamide, dacarbazine, vinblastine, vincristine, bleomycin, nitrogen mustard, prednisone, procarbazine, methotrex
  • the chemotherapeutic agent is a conventional chemotherapeutic agent in the art, preferably selected from the group consisting of an alkylating agent, an alkyl sulfonate chemotherapeutic agent, an aziridine chemotherapeutic agent, a vinyl amide, and a methyl group.
  • Amine chemotherapeutic agents nitrogen mustard, nitrourea chemotherapeutics, antibiotics, antimetabolites, folic acid chemotherapeutics, purine analogs, pyrimidine analogs, androgens, anti-adrenalin, folic acid supplements, and beauty
  • the alkylating agent is a conventional alkylating agent in the art, preferably selected from the group consisting of thiotepa or cyclophosphamide.
  • the alkyl sulfonate chemotherapeutic agent is a conventional alkyl sulfonate chemotherapeutic agent, preferably selected from the group consisting of busulfan, propylene bromide or pipersulfuron.
  • the aziridine chemotherapeutic agent is a conventional aziridine chemotherapeutic agent in the art, preferably selected from the group consisting of aziridine, carbazone, metopril or uridine.
  • the ethylene amide and methyl melamine chemotherapeutic agents are conventional ethylene amide and methyl melamine chemotherapeutic agents, preferably selected from the group consisting of hexamethylene melamine, triethylene melamine, and triethylene ether. Phosphoramide, triethylene thiophosphoramide or trimethylol melamine.
  • the nitrogen mustard is a conventional nitrogen mustard, preferably selected from the group consisting of chlorambucil, naphthyl mustard, estramustine, ifosfamide, nitrogen mustard, and oxygen nitrogen mustard hydrochloride. Phenylalanine mustard, neonitrogen mustard, fentanyl cholesteryl, prednisolone, tromethamine or uracil mustard.
  • the nitrourea chemotherapeutic agent is a nitrourea chemotherapeutic agent conventional in the art, preferably selected from the group consisting of carmustine, chlorourea, formoterol, lomustine, and nimes Ting or ramustine.
  • the antibiotic is a conventional antibiotic in the art, preferably selected from the group consisting of aclarubicin, actinomycin, aflatoxin, azase, bleomycin, actinomycin c, calicheamicin, Carbofurin, erythromycin, cancer, chromomycin, dactinomycin, daunorubicin, ditoxin, 6-diazo-5-oxo-L-norleucine, Roubicin, epirubicin, ebispercin, idarubicin, fascin, mitomycin, mycophenolic acid, nogamycin, oligomycin, pedromycin, kaimycin , puromycin, triiron adriamycin, rhodamine, streptavidin, streptozotocin, tuberculin, umbrel, statstatin or zorubicin.
  • aclarubicin preferably selected from the group consisting of aclarubicin, actinomycin
  • the antimetabolite is a conventional antimetabolite in the art, preferably selected from methotrexate or 5-fluorouracil (5-FU).
  • the folic acid chemotherapeutic agent is a conventional folic acid chemotherapeutic agent in the art, preferably selected from the group consisting of dimethyl folic acid, pterostilbose or trimethoate.
  • the purine analogs are conventional anthraquinone analogs in the art, preferably selected from fludarabine, 6-oxime, thioxime or thioguanine.
  • the pyrimidine analog is a pyrimidine analog conventional in the art, preferably selected from the group consisting of amphetamine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, Deoxyfluorouridine, enoxabine, fluorouridine or 5-EU.
  • the androgen is a conventional androgen in the art, preferably selected from the group consisting of caprotestosterone, alpha-androstanol propionate, anthracycline, megestrol or testosterone.
  • the anti-adrenalin is a conventional anti-adrenalin in the art, preferably selected from the group consisting of anlumet, mitoxantrone or tromethamine.
  • the folic acid supplement is a conventional folic acid supplement in the art, preferably selected from the group consisting of folinic acid, acetolactone, aldophosphamide glucoside, amino keto valeric acid, amsacrine, amoxicilin, specific life.
  • the maytansinol is a conventional maytansinol in the art, preferably selected from the group consisting of maytansin, anspirin, mitoxantrone, mitoxantrone, moppeldol, diamine nitidine, Pentastatin, egg ammonia, pirarubicin, loxoprost, podophyllin, 2-ethyl hydrazide or procarbazine.
  • the polysaccharide complex is a conventional polysaccharide complex in the art, preferably selected from the group consisting of razuxon, rhizomycin, cilostazol, quercetin, fine-chain oxysporic acid, and triammine oxime 2, 2' , 2"-trichlorotriethylamine, trichothecenes, urethane, vindesine, dacarbazine, mannimostatin, dibromomannitol, dibromodusol, piperobbrane, Gacytosine, cytarabine, cyclophosphamide or thiotepa.
  • T-2 toxin More preferably selected from the group consisting of T-2 toxin, fusarium A, porphyrin A or anguidine.
  • the taxane is a conventional purple in the art.
  • Cedarane preferably selected from paclitaxel, non-hydrogenated castor oil, paclitaxel, albumin engineered nanoparticle preparation (American Pharmaceutical Partners, Schaumberg, Illinois), docetaxel, chlorambucil, gemcitabine, 6-sulfur A guanine, guanidine or methotrexate.
  • the platinum analog is a conventional platinum analog, preferably selected from the group consisting of cisplatin, carboplatin, vinblastine, etoposide, ifosfamide, Mitoxantrone, vincristine, nooto, teniposide, edarshasha, daunorubicin, aminopterin, Peitabin ibandronate, CPT-11, topoisomerase inhibitor RFS 2000 or difluoromethylornithine.
  • the retinoid is a retinoid in the field, preferably Retinoic acid.
  • the radioisotope is a conventional radioisotope in the art, preferably, it directly binds to the above EGFRvIII antibody, or binds to the above EGFRvIII antibody by a chelating agent. More preferably, it binds directly to the cysteine residue of the EGFRvIII antibody.
  • the radioisotope is selected from the group consisting of alpha-emitters, beta-emitters and Auger electrons suitable for radiation therapy and positron emitters or gamma-emitters suitable for diagnosis. More preferably, the radioisotope is selected from the group consisting of 18 fluoro, 64 copper, and 65 copper.
  • the therapeutic nucleic acid is a nucleic acid conventional in the art, preferably a gene encoding an immunomodulator, an anti-angiogenic agent, an anti-proliferative agent or a pro-apoptotic agent.
  • the therapeutic agent includes the therapeutic agent, derivatives thereof, and pharmaceutically acceptable salts, acids, and derivatives of the therapeutic agents.
  • the immunomodulatory agent is a conventional immunomodulator in the art, ie, an agent that elicits an immune response, including a humoral immune response (eg, production of an antigen-specific antibody) and a cell-mediated immune response (eg, lymphocyte proliferation).
  • a humoral immune response eg, production of an antigen-specific antibody
  • a cell-mediated immune response eg, lymphocyte proliferation
  • cytokine a cytokine
  • a growth factor eg, a hormone, an anti-hormonal agent, an immunosuppressive agent or a corticosteroid.
  • the cytokine is a cytokine conventional in the art, preferably selected from the group consisting of xanthine, interleukin or interferon.
  • the growth factor is a conventional growth factor in the art, preferably selected from the group consisting of TNF, CSF, GM-CSF or G-CSF.
  • the hormone is a conventional hormone in the art, preferably selected from the group consisting of estrogen, androgen or progestin. More preferably, the estrogen is diethylstilbestrol or estradiol. More preferably, the androgen is testosterone or fluorotestosterone. More preferably, the progestin is megestrol acetate or medroxyprogesterone acetate.
  • the corticosteroids are conventional corticosteroids in the art, preferably selected from prednisone, dexamethasone or cortisone.
  • the antihormonal agent is a conventional antihormonal agent in the art, which can block the action of a hormone on a tumor, inhibit the production of a cytokine, down-regulate the expression of an autoantigen, or mask an immunosuppressant of an MHC antigen. It is preferably selected from the group consisting of an antiestrogenic drug, an antiandrogen or an antinephrine. More preferably, the antiestrogens are selected from the group consisting of tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, travoxifene or toremi Fen.
  • the antiandrogen is selected from the group consisting of flutamide, nilutamide, bicalutamide, leuprolide or goserelin.
  • the immunosuppressant is a conventional immunosuppressant in the art, preferably selected from the group consisting of 2-amino-6 aryl-5 substituted pyrimidines, azathioprine, cyclophosphamide, bromocriptine, danazol, ammonia Phenyl sulfone, glutaraldehyde, anti-idiotypic antibodies against MHC antigens and MHC fragments, cyclosporin A, steroids such as glucocorticosteroids, streptokinase, TGFb, rapamycin, T cell receptor, T cell receptor Fragment, cytokine receptor antagonist or T cell receptor antibody. More preferably, the cytokine receptor antagonist is selected from the group consisting of an anti-interferon antibody, an anti-IL10 antibody, an anti-TNFa
  • the anti-angiogenic agent is a conventional anti-angiogenic agent in the art, preferably selected from the group consisting of a farnesyl transferase inhibitor, a COX-2 inhibitor, a VEGF inhibitor, a bFGF inhibitor, a steroid sulfatase Inhibitor, interleukin-24, thrombospondin, metallospondin protein, class I interferon, interleukin 12, protamine, angiostatin, laminin, endostatin or prolactin fragment. More preferably, it is 2-methoxyestradiol diaminosulphonate (2-MeOE2bisMATE).
  • the anti-proliferative pro-apoptotic agent is a conventional anti-proliferative pro-apoptotic agent in the art, preferably selected from the group consisting of a PPAR- ⁇ activator, a retinoid, a triterpenoid, an EGF receptor inhibitor, and an end.
  • the PPAR-gamma activators are conventional PPAR-gamma activators in the art, preferably cyclopentenone prostaglandins (cyPGs).
  • the triterpenoids are conventional triterpenoids in the art, preferably selected from the group consisting of cyclobungane, lupin, ursane, zitherane, xylan, dammarane, cucurbitacin, lemon A bitter analog or a triterpenoid.
  • the EGF receptor inhibitor is a conventional EGF receptor inhibitor in the art, preferably selected from the group consisting of HER4, rapamycin or 1,25-dihydroxycholecalciferol (vitamin D).
  • the iron chelate is a conventional iron chelate compound, preferably 3-aminopyridine-2-formaldehyde thiosemicarbazone.
  • the apoptotic protein is a conventional apoptotic protein in the art, preferably the viral protein 3-VP3 of chicken anemia virus.
  • the PI3K-Akt survival pathway signaling inhibitor is a conventional PI3K-Akt survival pathway signaling inhibitor in the art, preferably UCN-01 or geldanamycin.
  • the cytolytic enzyme is a cytolytic enzyme conventional in the art, preferably an RNase.
  • D is a tubulin synthetase inhibitor - methyl auristatin F, (MMAF), and the linker L is a maleimide group Hexamyl (Maleimidocaproyl, MC), the structure of the immunoconjugate is as shown in Formula 3-1 or 3-2,
  • the mAb is the above EGFRvIII antibody.
  • n is a natural number, preferably an integer of 1 to 20, more preferably 2w, w is an integer of 1 to 5, more preferably 3 to 4;
  • m is from 1 to 10, preferably m is 5, L is maleimidocaproyl-L-valine-L-citrulline p-aminobenzyl alcohol; D is methyl auristatin E(MMAE); wherein n is a natural number, preferably an integer of 1 to 20, more preferably 2w, w is an integer of 1 to 5, and more preferably 3 to 4.
  • the preparation method of the immunoconjugate is conventional in the art, and the preparation method described in Doronina, 2006, Bioconjugate Chem. 17, 114-124 is preferably employed.
  • the method of preparation produces an immunoconjugate having a minimal low coupling fraction (LCF) of less than 10%.
  • the preparation method comprises the steps of: dialyzing the above EGFRvIII antibody through a sodium borate buffer solution having a pH of 6.5 to 8.5, and adding tris(2-carboxyethyl)phosphine (TCEP).
  • TCEP tris(2-carboxyethyl)phosphine
  • the molar ratio of TCEP to the above EGFRvIII antibody is 2-10, and is reduced at room temperature for 2 to 4 hours.
  • the excess TCEP is removed by the G25 desalting filler, and a certain proportion of MC-MMAF is added (the drug/antibody ratio is 5-20). Reaction for 4 hours. Cysteine is added to neutralize the excess drug and excess small molecules are removed by G25.
  • a purified antibody drug conjugate is obtained (for coupling methods, see Doronina, 2006, Bioconjugate Chem. 17, 114-124).
  • the immunoconjugate can be present in any physical form known in the art, preferably a clear solution.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the EGFRvIII antibody or immunoconjugate 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 0.01 to 99.99% of the above EGFRvIII or the above immunoconjugate, and 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 EGFRvIII 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 antibody, the above conjugate 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 EGFRvIII protein, comprising the steps of: contacting the EGFRvIII antibody and the sample to be tested in vitro, and detecting the binding of the EGFRvIII antibody to the sample to be tested. .
  • the present invention also provides a composition for detecting a cell overexpressing an EGFRvIII protein, which comprises the above-described EGFRvIII antibody as an active ingredient.
  • the present invention also provides the use of the above antibody, immunoconjugate or pharmaceutical composition for the preparation of a medicament for preventing or treating a disease associated with EGFRvIII expression or dysfunction; preferably, the expression or dysfunction of EGFRvIII is abnormal
  • a related disease is a tumor, which 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 protein of the present invention is an EGFRvIII antibody which has a high affinity for the EGFRvIII protein and which binds to the extracellular region of the EGFRvIII protein receptor at the protein level and the cellular level.
  • the antibody cross-linking agent obtained by coupling the EGFRvIII antibody with a small molecule compound such as MC-MMAF can effectively inhibit cytotoxicity of EGFRvIII-positive cells.
  • Small molecule toxins, such as MMAF are introduced into cells by endocytosis by EGFRvIII antibody, and small molecule compounds are released by intracellular degradation, thereby playing a cytotoxic role. Therefore, the EGFRvIII antibody can be used for the preparation of an antibody cross-linking drug, effectively killing tumor cells, or can be used in the preparation of a drug for treating tumors.
  • Figure 1 shows the results of FACS screening of CHO-K1 cells transfected with human EGFRvIII protein.
  • Figure 2 shows the results of FACS screening assay of human EGFR protein transfected CHO-K1 cells.
  • Figure 3 shows the results of FACS screening of U87MG cells transfected with human EGFRvIII protein.
  • Figure 4 shows the results of FACS screening of 293F cells transfected with human EGFRvIII protein.
  • Figures 5A and 5B show the serum antibody titer of mice after ELISA detection of EGFRvIII protein immunization.
  • Figure 6 is a ELISA showing the binding reaction of EGFRvIII antibody to human EGFRvIII-hFc protein.
  • Figure 7A shows the binding reaction of EGFRvIII antibody to U87MG-hEGFRvIII by FACS
  • Figure 7B shows the binding reaction of EGFRvIII antibody to A431 by FACS
  • Figure 7C shows the binding reaction of EGFRvIII antibody to U87MG by FACS
  • Figure 7D shows the detection of EGFRvIII antibody and HEB by FACS The binding reaction.
  • Figure 8A shows the cell killing effect of EGFRvIII antibody-MMAF antibody-conjugated drug on U87MG-EGFRvIII
  • Figure 8B shows the cell killing effect of EGFRvIII antibody-MMAF antibody-conjugated drug on A431
  • Figure 8C shows the EGFRvIII antibody-MMAF antibody-conjugated drug pair Cell killing effect of U87MG
  • Figure 8D shows the cell killing effect of EGFRvIII antibody-MMAF antibody conjugate on HEB.
  • Figure 9A shows the binding reaction of EGFRvIII antibody to human normal tissue HFF-1 cell line by FACS
  • Figure 9B shows the binding reaction of EGFRvIII antibody to human normal tissue HFL-I cell line by FACS
  • Figure 9C shows FACS detection of EGFRvIII antibody and human normal The binding reaction of QSG-7701 cell line was organized
  • Figure 9D shows the binding reaction of EGFRvIII antibody to human normal tissue HEEC cell line by FACS
  • Figure 9E shows the binding reaction of EGFRvIII antibody to human normal tissue HEB cell line by FACS
  • Figure 9F shows FACS The binding reaction of EGFRvIII antibody to human normal tissue WPMY-1 cell line was examined
  • Figure 9G shows the binding reaction of EGFRvIII antibody to human normal tissue MCF-10A cell line by FACS.
  • Figure 10A shows the results of immunohistochemical staining of human glioma tissue microarray with EGFR antibody (murine anti-80E11);
  • Figure 10B shows the results of immunohistochemical staining of EGFR antibody (murine anti-80E11) on human normal tissue microarray;
  • Figure 10C shows EGFRvIII antibody (Mouse anti-63A10) immunohistochemical staining results of human glioma tissue microarray;
  • Figure 10D shows the results of immunohistochemical staining of human normal tissue microarray with EGFRvIII antibody (murine anti-63A10);
  • Figure 10E is EGFRvIII antibody (rat anti-75G7) Immunohistochemical staining results of human glioma tissue microarray;
  • Figure 10F shows the results of immunohistochemical staining of human normal tissue microarray by EGFRvIII antibody (murine anti-75G7).
  • Figure 11 shows the binding of chimeric antibodies to EGFRvIII protein by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • Figure 12A shows the binding reaction of EGFRvIII chimeric antibody to U87MG-hEGFRvIII by FACS
  • Figure 12B shows the binding reaction of EGFRvIII chimeric antibody to A431 by FACS
  • Figure 12C shows the binding reaction of EGFRvIII chimeric antibody to U87MG by FACS
  • FACS detects the binding reaction of EGFRvIII chimeric antibody to HEB.
  • Figure 13A shows the cell killing effect of EGFRvIII antibody-MMAF antibody-conjugated drug on U87MG-EGFRvIII
  • Figure 13B shows the cell killing effect of EGFRvIII antibody-MMAF antibody-conjugated drug on A431
  • Figure 13C shows EGFRvIII antibody-MMAF antibody-conjugated drug pair Cell killing effect of U87MG
  • Figure 13D shows the cell killing effect of EGFRvIII antibody-MMAF antibody conjugate on HEB.
  • Figure 14A shows changes in tumor volume of U87MG-EGFRvIII xenograft tumor-bearing mice
  • Figure 14B shows changes in body weight of U87MG-EGFRvIII xenograft tumor-bearing mice.
  • Figure 15 is a ELISA binding activity of an antibody after NG mutation of the heavy chain variable region CDR2 of the 75G7C6 antibody.
  • Figure 16 is an ELISA binding activity of an antibody after NG mutation of the heavy chain variable region CDR3 of the 75G7C6 antibody.
  • FIG. 17A shows the binding activity of the NG-mutated antibody to the CHOK-EGFRvIII cell of the heavy chain variable region CDR2 of the 75G7C6 antibody by FACS;
  • FIG. 17B is the FACS detection of the NG mutant antibody and CHOK-EGFR of the heavy chain variable region CDR2 of the 75G7C6 antibody. Cell binding activity.
  • Figure 18A is a FACS assay for the binding activity of a mutant antibody of CDR3 of the heavy chain variable region CDR3 of the 75G7C6 antibody to SG, EG or DA to CHOK-EGFRvIII cells;
  • FIG. 18B is a DG for detecting the heavy chain variable region CDR3 of the 75G7C6 antibody by FACS.
  • the mutant antibody mutated to SG, EG or DA binds to CHOK-EGFR cells.
  • Figure 19 is a sequence comparison of the humanized anti-EGFRvIII antibody h75G7C6 heavy chain variable region h75G7C6.VH and its variants with the chimeric antibody c75G7C6.VH and the human germline VH exon IGHV1-46*01/JH6b. For the CDR.
  • Figure 20 is a sequence comparison of the humanized anti-EGFRvIII antibody h75G7C6 light chain variable region h75G7C6.VL and its variants with the chimeric antibody c75G7C6.VL and the human germline VL exon IGKV3-11*01/JK4. For the CDR.
  • Figure 21 is a sequence comparison of the humanized anti-EGFRvIII antibody h63A10A7 heavy chain variable region h63A10A7.VH and its variants with the chimeric antibody c63A10A7.VH and the human germline VH exon IGHV1-46*01/JH4. For the CDR.
  • Figure 22 is a sequence comparison of the humanized anti-EGFRvIII antibody h63A10A7 light chain variable region h63A10A7.VL and its variants with chimeric antibody c63A10A7.VL and human germline VL exon IGKV1-39*01/JK2, at For the CDR.
  • Figure 23A-C is a ELISA assay for the binding of humanized antibody h75G7C6 variant to human EGFRvIII-hFc protein.
  • 24A-E are ELISA assays for the binding of humanized antibody h63A10A7 variant to human EGFRvIII-hFc protein.
  • Figure 25A-C is a FACS assay for the binding of humanized antibody h75G7C6 variant to U87MG-EGFRvIII cells.
  • 26A-E are FACS assays for the binding of humanized antibody h63A10A7 variant to U87MG-EGFRvIII cells.
  • Figure 27 is a FACS assay for binding of humanized h75G7C6 variant to tumor cell A431 and normal human primary hepatocytes expressing EGFR on the surface.
  • 28A-B are FACS assays for binding of humanized h63A10A7 variants to tumor cell A431 and normal human primary hepatocytes expressing EGFR on the surface.
  • the room temperature described in the examples is room temperature conventional in the art, and is generally from 10 to 30 °C.
  • the nucleotide sequence containing the amino acid sequence encoding the extracellular region of the human EGFRvIII protein was cloned into the pCpC vector carrying the human IgG Fc fragment (hFc) (purchased from Invitrogen, V044). -50) and prepare plasmids according to established standard molecular biology methods. For details, 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 for transient transfection (PEI, Polysciences), using FreeStyle TM 293 (Invitrogen) were expanded at 37 °C. After 4 days, the cell culture medium was collected, and the cell components were removed by centrifugation to obtain a culture supernatant containing the extracellular region of the EGFRvIII protein. 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.
  • PKI ultraviolet
  • UV ultraviolet
  • 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 EGFRvIII protein (EGFRvIII-hFc) eluted on the column was dialyzed against a PBS phosphate buffer (pH 7.2) at 4 ° C overnight. The dialyzed protein was sterile-filtered at 0.22 ⁇ m and stored at -80 ° C to obtain purified immunogen A.
  • HEK293 cell line U87MG cell line and CHO-K1 cell line (both purchased from Invitrogen) with plasmid transfection (PEI, purchased from Polysciences), 10% (w/w) fetal bovine containing 0.5 ⁇ g/ml
  • PEI plasmid transfection
  • 10% (w/w) fetal bovine containing 0.5 ⁇ g/ml
  • the serum was selectively cultured in DMEM medium for 2 weeks, subcloned in a 96-well culture plate by limiting dilution, and placed in a 5% (v/v) CO 2 culture at 37 ° C, and a partial single was selected after about 2 weeks.
  • the cloned wells were expanded into 6-well plates.
  • the amplified clones were screened by flow cytometry using a known EGFRvIII 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 3 and Figure 1, and the IgG subtype control is a mouse IgG control.
  • Table 3 illustrates that a series of EGFRvIII positively expressed CHO-K1, U87MG and 293F cell lines, and EGFR positively expressed CHO-K1 cell lines have been prepared. In Figs.
  • the abscissa is the fluorescence intensity of the cells, and the ordinate is the number of cells.
  • the results of Figure 1, 2, 3, and 4 indicate that CHO-K1-hEGFRvIII 1G10, CHO-K1-hEGFR 3G2, U87MG-hEGFRvIII 2G2, and 293F-hEGFRvIII 1C10 are high-level expression lines of EGFRvIII; CHO-K1-hEGFR 3G2 is EGFR high level expressed cell lines.
  • the EGFR antibody was also obtained by the same immunization route, mainly for the purpose of demonstrating that the EGFRvIII antibody has better tissue specificity than the EGFR antibody.
  • 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.
  • Table 7-8 shows that the immunized serum of mice immunized with human EGFRvIII-hFc has different degrees of binding to the immunogen, showing an antigen-antibody reaction, wherein the highest dilution is about one million.
  • the blank control was 1% (w/w) BSA, wherein the batch refers to the serum of the mice on the seventh day after the second boost, and the data in the table is the OD 450 nm value.
  • 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 (mouse against immunogen A) or immunogen B (immunized against immunogen B).
  • the mice that responded the 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 to the EGFRvIII protein and the EGFRvIII-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 450nm >1.0, MFI value >50 in FACS and 50% of EGFRvIII positive cells in indirect cytotoxic killing experiments were selected.
  • 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.
  • the concentration of antibodies produced by hybridoma cells is relatively low, about 1 to 10 ⁇ g/ml, and the concentration varies greatly.
  • the various proteins produced by cell culture in the medium and the fetal bovine serum components contained in the medium have different degrees of interference with many biological activity analysis methods, so that small-scale (1 to 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 EGFRvIII 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 EGFRvIII antibody was collected, sterile-filtered with a 0.22 micron filter, and stored aseptically to obtain a purified EGFRvIII antibody.
  • the purified EGFRvIII antibody was subjected to detection analysis of protein concentration (A280/1.4), purity, endotoxin (Lonza kit), and the results are shown in Table 9, and it was found that the endotoxin concentration of the antibody final product was within 1.0 EU/mg.
  • Antibody name Clone number Antibody purity Protein concentration (mg/ml) Endotoxin (EU/mg) Rat anti-75G7 75G7C6 >95% 0.44 ⁇ 1.0 Mouse anti-63A10 63A10A7 >95% 1.26 ⁇ 1.0 Rat anti-64F1 64F1F8 >95% 1.52 ⁇ 1.0 Mouse anti-7E5 7E5-2A9 >95% 0.81 ⁇ 1.0 Rat anti-43H6 43H6A1 >95% 1.08 ⁇ 1.0 Mouse anti-46C4 46C4A6 >95% 1.43 ⁇ 1.0 Mouse anti-49G12 49G12G5 >95% 1.16 ⁇ 1.0 Mouse anti-51G7 51G7C2 >95% 1.07 ⁇ 1.0
  • A enzyme-linked immunosorbent assay (ELISA) to detect the binding of antibodies to EGFRvIII protein
  • the purified EGFRvIII antibody obtained in Example 2 was reacted with a human EGFRvIII-hFc protein.
  • the purified immunogen A (EGFRvIII-hFc) obtained in Example 1 was prepared (for the preparation method, see Step 1 of Example 1 (1) diluted with PBS to a final concentration of 1.0 ⁇ g/mL, and then added to a 96-well ELISA plate at 100 ⁇ l per well. Incubate overnight at 4 °C with plastic film, wash plate twice with wash solution [PBS+0.01% (v/v) Tween20], add blocking solution [PBS+0.01%(v/v)Tween20+ 1% (w/w) BSA] was blocked at room temperature for 2 hours. The blocking solution was discarded, and 100 ⁇ l of the purified EGFRvIII antibody obtained in Example 2 was added per well.
  • the desired cells were expanded to 90% confluence in T-75 cell culture flasks, the medium was aspirated, washed twice with HBSS Balanced Salt Solution (purchased from Invitrogen), and then treated with enzyme-free cells.
  • the cells were treated and collected by Versene solution (purchased from Life Technology).
  • the cells were washed twice with HBSS buffer, and after cell counting, the cells were diluted with HBSS buffer to 2 ⁇ 10 6 cells per ml, and 1% goat serum blocking solution was added, the percentage being mass percentage, and incubation on ice for 30 minutes. It was then washed twice with HBSS buffer.
  • the collected cells were suspended in a FACS buffer (HBSS + 1% BSA, the percentage is a mass percentage) to 2 ⁇ 10 6 cells/mL, and added to a 96-well FACS reaction plate at 100 ⁇ L per well, and the examples were added.
  • the purified EGFRvIII antibody sample to be tested was 100 ⁇ l per well and incubated on ice for 2 hours.
  • 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 on ice 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).
  • FACS Calibur FACS Calibur, available from BD
  • the average fluorescence density (MFI) to the cells was analyzed by software (CellQuest).
  • the data was fitted and analyzed by software (GraphPad Prism 5) to calculate the EC50.
  • the antibody to be tested can bind to the EGFRvIII protein on the surface of U87MG-hEGFRvIII cells (Fig. 7A).
  • A431 cells human epidermal cell carcinoma cell line
  • mouse anti-75G7, 63A10 and 64F1 can bind weakly to wild-type EGFR protein on the surface of A431 cells
  • mouse anti-7E5 Antibody clone 7E5-2A9 does not recognize the wild type EGFR protein and can only specifically recognize the mutant EGFRvIII protein.
  • U87MG cells human glioma cell line
  • HEB cells human glial cell line
  • the test antibody did not bind to U87MG and HEB cells, indicating that the mouse anti-75G7, 63A10 and 64F1 have good specificity, only recognize the mutant EGFRvIII protein in the tumor tissue and the overexpressed wild-type EGFR protein, and do not recognize normal. Wild-type EGFR protein in tissues (Fig. 7C, D).
  • the antibody was dialyzed against Phosphate buffer of Ph6.5-8.5, and a certain proportion of TCEP was added for reduction (TCEP/antibody ratio of 2-10), and it was reduced at room temperature or 37 degrees for 2-4 hours, and the excess was removed by G25 desalting filler.
  • TCEP was added to a certain proportion of MC-MMAF (drug/antibody ratio 5-20) for 4 hours. Cysteine is added to neutralize the excess drug and excess small molecules are removed by G25.
  • a purified antibody drug conjugate is obtained. (For the coupling method, see Doronina, 2006, Bioconjugate Chem. 17, 114-124).
  • the cytotoxic activity was analyzed by analyzing the cross-linking rate, purity, and the like of the drug by HIC. For ease of comparison, the drug cross-linking rate of all antibody conjugates was 8.
  • the obtained antibody conjugates were separately diluted with complete medium, and 96-well cell culture plates were added with 90 ⁇ l of U87MG-EGFRvIII cell suspension at 1000 cells/well, and 10 ⁇ l of different concentrations of antibody drugs were added to each well. The dilution of the conjugate was continued for 5 days, and the cell viability was measured using a CellTiter-Glo kit (purchased from Promega, using the method of reference).
  • IC50 of Table 13-1 refers to a half effective amount in which the activity of the cells is inhibited after the action of the drug, and the cell killing activity can be reflected by detecting the activity of the cells.
  • Figure 8 shows the cytotoxic activity of the antibody drug conjugate against the EGFRvIII-positive recombinant tumor cell line U87MG-EGFRvIII.
  • the results show that in vitro, the antibody to be tested can express U87MG with mutant EGFRvIII protein expression on the surface.
  • -hEGFRvIII cells produced a good killing effect (Fig. 8A).
  • A431 cells human epidermal cell carcinoma cell line
  • mouse anti-75G7, 63A10 and 64F1 had weaker killing effect on A431.
  • U87MG cells human glioma cell line
  • HEB cells human glial cell line
  • Rat anti-75G7, 63A10 and 64F1 had little or no killing effect on U87MG and HEB cells, see Figure 8C, Figure 8D.
  • mouse anti-75G7, 63A10 and 64F1 have good specificity and can specifically kill tumor tissues expressing mutant EGFRvIII protein and overexpressing wild-type EGFR protein, but have no killing effect on normal tissues expressing wild-type EGFR protein.
  • EGFRvIII antibodies were grouped by competition ELISA.
  • the purified antibody to be tested was diluted to 1 ⁇ g/mL with PBS, coated with 96-well high-adsorbing enzyme plate at 50 ⁇ L/well, and coated with 250 ⁇ l of blocking solution at 4 ° C overnight [containing 0.01% (v/v) Tween 20 and 1% (w/w) BSA in PBS] was blocked at room temperature for one hour, and 0.05 ⁇ g/mL of biotin-labeled recombinant EGFRvIII protein was added per well. At the same time, 5 ⁇ g/mL of the competing antibody, the purified EGFRvIII antibody obtained in Example 2, was added and incubated at 25-37 ° C for 1 to 2 hours.
  • the plate was washed 3 times with a washing solution [PBS containing 0.01% (v/v) Tween 20], and HRP (horseradish peroxidase)-labeled streptavidin (purchased from Sigma) was added. After incubation at 37 ° C for 0.5 hour, the plate was washed 3 times with a washing solution [PBS containing 0.01% (v/v) Tween 20]. After adding 100 ⁇ L of TMB substrate per well and incubating for 30 minutes at room temperature, 100 ⁇ L of each well was added to the stop solution (1.0 N HCl). The A450 nm values were read using an ELISA plate reader (SpectraMax 384plus, available from Molecular Device) and the results are shown in FIG. Based on the A450nm values, the competition rates of the antibodies were calculated. The results are shown in Table 13-1. The higher the value of the competition rate, the closer the antigen surface of the two antibodies is.
  • control antibody 02 and the murine anti- 7E5, 63A10 and 64F1 epitopes were competitive and should be the same or similar epitopes. While mouse anti-75G7 is not competitive with control antibody 02, the antigenic epitope of mouse anti-75G7 should be different from control antibody 02; control antibody 01 is AMG-595 (purchased from Amgen) and control antibody 02 is ABT-414 (purchased) Since Abbvie).
  • control antibody 01 and murine anti-7E5, murine anti-64F1, and murine anti-63A10 have the same or similar epitopes, namely CGADYYEMEDGVRKC.
  • the mouse anti-75G7 does not bind to the 35 polypeptides, so the antigenic epitope of the mouse anti-75G7 should be different from the control antibody 01 and the control antibody 02.
  • control antibody 01 was AMG-595 (available from Amgen) and control antibody 02 was ABT-414 (purchased from Abbvie). Therefore, the antigenic epitope of mouse anti-75G7 needs further experimental verification.
  • the EGFRvIII antibody is capable of specifically recognizing EGFR in an activated state, that is, a mutant EGFRvIII in tumor tissues and an overexpressed wild type EGFR, but does not recognize wild type EGFR in normal tissues. Therefore, the antibody has excellent target specificity, can effectively prevent the damage of the antibody and its conjugate to normal human tissues, and effectively collect the antibody and its conjugate at the tumor site, thereby accurately achieving the tumor tissue. Killing and greatly reducing the side effects of drugs on other normal organs.
  • Table 14 is derived from 7 different human normal tissue cell lines
  • the human glioma tissue chip (article number: GL805d) and the human multi-organ normal tissue chip (article number: FDA 999n) were purchased from Xi'an Maria Biotechnology Co., Ltd.
  • the tissue chip was placed in an EDTA solution of pH 9.0 and repaired at high temperature for 5 minutes.
  • Non-immune serum was blocked, and the tissue chip was placed in 5% FBS and allowed to stand at room temperature for 15 minutes.
  • the antibody can be adsorbed by the charged collagen and connective tissue components in the tissue section. This results in background coloration.
  • a common method is to use 2% to 10% sheep serum or 2% to 5% bovine serum albumin for 10 to 30 minutes at room temperature.
  • Mouse anti-80E11 is an antibody against wild-type EGFR. From the perspective of Figures 10A-F, wild-type EGFR is expressed in human glioma tissues and human multiple organ normal tissues, and the expression level in tumor tissues is higher than that in tumor tissues. Normal organization. As shown in Table 15, the positive rate of mouse anti-7E5, murine anti-64F1 and murine anti-63A10 in human glioma tissue microarray was 34%, and the microarray reaction with human multi-organ normal tissue was basically negative, only a few samples were weak. Positive. Rat anti-75G7 was negative for both human glioma tissue microarray and human multi-organ normal tissue chip.
  • mouse anti-75G7 Since the antigen-binding epitope of mouse anti-75G7 is different from other antibodies, it may be because the antigen-binding epitope of mouse anti-75G7 was disrupted during chip processing.
  • the tissue chips used in this experiment were all embedded in paraffin, and this method may not be suitable for detecting mouse anti-75G7 antibodies. We will try to freeze the IHC in the follow-up.
  • the EGFRvIII antibody was able to specifically recognize EGFR in tumor tissues by FACS and IHC experiments, but did not recognize EGFR in normal tissues. Therefore, the antibody has excellent target specificity, can effectively prevent the damage of the antibody and its conjugate to normal human tissues, and effectively collect the antibody and its conjugate at the tumor site, thereby accurately achieving the tumor tissue. Killing and greatly reducing the side effects of drugs on other normal organs.
  • RNA isolation 5 ⁇ 10 7 hybridoma cells obtained in Example 1 were collected by centrifugation, mixed with 1 mL of Trizol, and transferred to a 1.5 mL centrifuge tube, and allowed to stand at room temperature for 5 minutes. 0.2 mL of chloroform was added, shaken for 15 seconds, allowed to stand for 10 minutes, centrifuged at 12000 g for 5 minutes at 4 ° C, and the supernatant was transferred to a new 1.5 mL centrifuge tube. 0.5 mL of isopropanol was added, and the liquid in the tube was gently mixed. After standing at room temperature for 10 minutes, it was centrifuged at 12,000 g for 15 minutes at 4 ° C, and the supernatant was discarded.
  • 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 by reaction at 7 ° C for 10 minutes.
  • a 50 ⁇ L PCR system was configured, including 1 ⁇ L of cDNA, 25 pmol of each primer, 1 ⁇ L of DNA polymerase, and a matching buffer system, 250 ⁇ mol dNTPs; a PCR program was set, predenatured at 95 ° C for 3 minutes, denatured at 95 ° C for 30 seconds, and annealed at 55 ° C for 30 seconds.
  • the extension was carried out at 72 ° C for 35 seconds, and after 35 cycles, an additional 5 minutes at 72 ° C to obtain a PCR product.
  • 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.
  • PCR product 5 ⁇ L was detected by agarose gel electrophoresis, and the positive samples were purified using a column recovery kit, wherein the recovery kit was Gel & PCR Clean-up, available from MACHEREY-NAGEL, Cat. No. 740609.
  • the ligation reaction was carried out: 50 ng of the sample, 50 ng of the T vector, 0.5 ⁇ L of the ligase, 1 ⁇ L of the buffer solution, and 10 ⁇ L of the reaction system, and the reaction product was allowed to react at 16 ° C for half an hour.
  • the ligated kit was T4 DNA ligase, purchased from NEB, catalog 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 bath for 5 minutes, and then at 42 ° C. The bath was heat shocked for 1 minute, placed on ice for 1 minute, and then 650 ⁇ L of antibiotic-free SOC medium was added and resuscitated at 200 RPM for 30 minutes on a 37 ° C shaker. 200 ⁇ L of the solution was applied to an antibiotic-containing LB solid medium and incubated overnight at 37 ° C in an incubator.
  • a 30 ⁇ L PCR system was configured using the primers M13F and M13R on the T vector, colony PCR was performed, and the colony was pipetted in a PCR reaction system, and 0.5 ⁇ L of the LB containing 100 nM ampicillin was aspirated. The strain was preserved on a solid petri dish. After the end of the PCR reaction, 5 ⁇ L was taken for agarose gel electrophoresis detection, and the positive samples were sequenced and analyzed (wherein the CDRs were divided according to the Chothia definition system). The sequencing results are shown in Tables 16-17.
  • the number in Table 17 is the sequence number in the sequence listing, such as the amino acid sequence of the heavy chain protein variable region of 43H6A1 is SEQ ID No. 1, and the amino acid sequence of CDR1 in the heavy chain protein variable region of 43H6A1 is SEQ ID No. 2.
  • the number in Table 18 is the sequence number in the sequence listing, and the nucleotide sequence of the variable region of the heavy chain protein encoding 75G7C6 is SEQ ID No. 105.
  • the recombinant plasmid having a purity increased by mass spectrometry using an alkaline lysis kit purchased from MACHEREY-NAGEL was used at a mass of 500 ⁇ g or more, and filtered through a 0.22 ⁇ m filter (purchased from Millopore) for transfection.
  • Peptone was added the next day to a final concentration of 0.5% (w/v).
  • the antibody titer of the culture solution was measured.
  • the supernatant was collected by centrifugation (3500 RPM, 30 minutes), and filtered through a 0.22 ⁇ m filter to obtain a filtered cell supernatant for purification.
  • the purified EGFRvIII chimeric antibody was assayed for protein concentration (A280/1.4), purity, endotoxicity (Lonza kit), and the results are shown in Table 18. As a result, the endotoxin concentration of the antibody final product was found to be 1.0 EU/mg. Within.
  • A enzyme-linked immunosorbent assay (ELISA) to detect the binding of antibodies to EGFRvIII protein
  • the purified EGFRvIII chimeric antibody obtained in Example 2 was reacted with a human EGFRvIII-hFc protein.
  • the purified immunogen A (EGFRvIII-hFc) obtained in Example 1 was prepared (for the preparation method, see Step 1 of Example 1 (1) diluted with PBS to a final concentration of 1.0 ⁇ g/mL, and then added to a 96-well ELISA plate at 100 ⁇ l per well. Incubate overnight at 4 °C with plastic film, wash plate twice with wash solution [PBS+0.01% (v/v) Tween20], add blocking solution [PBS+0.01%(v/v)Tween20+ 1% (w/w) BSA] was blocked at room temperature for 2 hours. The blocking solution was discarded, and 100 ⁇ l of the purified EGFRvIII chimeric antibody obtained in Example 2 was added per well.
  • the desired cells were expanded to 90% confluence in T-75 cell culture flasks, the medium was aspirated, washed twice with HBSS Balanced Salt Solution (purchased from Invitrogen), and then treated with enzyme-free cells.
  • the cells were treated and collected by Versene solution (purchased from Life Technology).
  • the cells were washed twice with HBSS buffer, and after cell counting, the cells were diluted with HBSS buffer to 2 ⁇ 10 6 cells per ml, and 1% goat serum blocking solution was added, the percentage being mass percentage, and incubation on ice for 30 minutes. It was then washed twice with HBSS buffer.
  • the collected cells were suspended in a FACS buffer (HBSS + 1% BSA, the percentage is a mass percentage) to 2 ⁇ 10 6 cells/mL, and added to a 96-well FACS reaction plate at 100 ⁇ L per well, and the examples were added.
  • the purified EGFRvIII antibody sample to be tested was 100 ⁇ l per well and incubated on ice for 2 hours.
  • 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 on ice 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).
  • FACS Calibur FACS Calibur, available from BD
  • the average fluorescence density (MFI) to the cells was analyzed by software (CellQuest).
  • the data was fitted and analyzed by software (GraphPad Prism 5) to calculate the EC50.
  • the chimeric antibodies to be tested all bind to the EGFRvIII protein on the surface of U87MG-hEGFRvIII cells (Fig. 12A).
  • A431 cells human epidermal cell carcinoma cell line
  • chimeric antibody 75G7, chimeric antibody 63A10 and chimeric antibody 64F1 can be combined with wild-type EGFR protein on the surface of A431 cells.
  • U87MG cells human glioma cell line
  • HEB cells human glial cell line
  • the test antibody did not bind to U87MG and HEB cells, indicating that chimeric antibody 75G7, chimeric antibody 63A10 and chimeric antibody 64F1 have good specificity, only recognize mutant EGFRvIII protein in tumor tissue and wild overexpression.
  • the type EGFR protein does not recognize wild-type EGFR protein in normal tissues (Fig. 12C, D).
  • the obtained antibody conjugates were separately diluted with complete medium, and 96-well cell culture plates were added with 90 ⁇ l of U87MG-EGFRvIII cell suspension at 1000 cells/well, and 10 ⁇ l of different concentrations of antibody drugs were added to each well. The dilution of the conjugate was continued for 5 days, and the cell viability was measured using a CellTiter-Glo kit (purchased from Promega, using the method of reference).
  • IC50 of Table 23 refers to a half effective amount in which the activity of the cells is inhibited after the action of the drug, and the cell killing activity can be reflected by detecting the activity of the cells.
  • Figure 13 shows the cytotoxic activity of the antibody drug conjugate against the EGFRvIII-positive recombinant tumor cell line U87MG-EGFRvIII. The results show that in vitro, the test antibody can express U87MG with mutant EGFRvIII protein expression on the surface. -hEGFRvIII cells produced a good killing effect (Fig. 13A).
  • A431 cells have a large amount of wild-type EGFR protein on the surface.
  • chimeric antibodies 75G7, 63A10 and 64F1 have a weak killing effect on A431.
  • U87MG cells human glioma cell line
  • HEB cells human glial cell line
  • the chimeric antibodies 75G7, 63A10 and 64F1 have little or no killing effect on U87MG and HEB cells, see Figure 13C, Figure 13D.
  • the chimeric antibodies 75G7, 63A10 and 64F1 have good specificity and can specifically kill tumor tissues expressing mutant EGFRvIII protein and overexpressing wild type EGFR protein, but have no killing effect on normal tissues expressing wild type EGFR protein.
  • the construction method of the U87MG-EGFRvIII recombinant cell line is shown in Example 1.
  • 200 ⁇ l (1 ⁇ 10 6 ) of U87MG-EGFRvIII cell suspension was inoculated into the right back of Balb/c nude mice. After 7 days, the tumor grew to 150-250 mm 3 , and the tumor-bearing mice with appropriate tumor volume were selected according to the tumor. Volume The mice were randomized and dosed according to the established schedule. The specific research plan and dosage are shown in Table 22.
  • TGI antitumor effect
  • TGI% 1 - (TRTVn - TRTV1) / (CRTVn - CRTV1) ⁇ 100% (TRTVn: RTV on the last day of the treatment group; CRTV: negative control group RTV).
  • the percentage value of T/C (%) reflects the tumor growth inhibition rate. According to the guidelines of the drug trial center for anti-tumor drugs, TGI% ⁇ 58%, the drug is considered effective.
  • the low dose group (1 mg/kg) also showed better antitumor activity (TGI%>58%).
  • the NG is mutated to QG, and the amino acid sequence of the heavy chain variable region of the 75G7C6 antibody after mutation is shown in SEQ ID No. 180.
  • the DG located in CDR3 is mutated into SG, and the amino acid sequence of the heavy chain variable region of the 75G7C6 antibody after mutation is as shown in SEQ ID No. 181 (the corresponding nucleic acid sequence is shown in SEQ ID No. 188); EG, the amino acid sequence of the heavy chain variable region of the 75G7C6 antibody after mutation is as shown in SEQ ID No. 182 (the corresponding nucleic acid sequence is shown as SEQ ID No.
  • DG is mutated to DA, after mutation 75G7C6
  • the amino acid sequence of the antibody heavy chain variable region is set forth in SEQ ID No. 183 (the corresponding nucleic acid sequence is set forth in SEQ ID No. 132). It is intended to remove the amino acid modification of the asparagine residue and the asparagine residue as a site for deamidation by the above site-directed mutagenesis.
  • chimeric 75G7C6 antibody heavy chain variable region site-directed mutagenesis sequence was subjected to gene synthesis, plasmid construction, cell transfection and antibody purification were carried out as described in Example 8, and the purified antibody was subjected to ELISA and FACS as described in Example 9 for mutation.
  • the chimeric antibody binding activity was identified, and the ELISA identification results are shown in Fig. 15 and Fig. 16 and Table 23, wherein Fig. 15 is the ELISA binding activity of the NG mutant antibody of the 75G7C6 antibody heavy chain variable region CDR2, and Fig.
  • DG which is located in CDR3 affects the binding activity to EGFRvIII-hFc regardless of whether it is mutated into EG, SG or DA.
  • the binding ability of mutant antibody c75G7C6-3 and EGFRvIII-hFc after DG mutation to EG is better than that of wild type.
  • the antibody c75G7C6 decreased 22-fold, while the mutant antibodies c75G7C6-4 and c75G7C6-5 after DG mutation to SG or DA did not bind to EGFRvIII-hFc, indicating that DG mutation into EG, SG or DA seriously affected the antibody and EGFRvIII. -hFc binding.
  • Figures 17A-B and Figures 28A-B and Tables 24 and 25, wherein Figure 17A is the mutant antibody c75G7C6-1 after mutation of NG of the heavy chain variable region CDR2 of the 75G7C6 antibody into NA or QG. c75G7C6-2 and wild-type antibody c75G7C6 and CHOK-EGFRvIII cell binding activity assay, Figure 17B is the 75G7C6 antibody heavy chain variable region CDR2 mutated into NA or QG mutant antibodies c75G7C6-1 and c75G7C6-2 and wild type The antibody c75G7C6 was detected with CHOK-EGFR cell binding activity; FIG.
  • FIG. 18A is the mutant antibodies c75G7C6-3, c75G7C6-4 and c75G7C6-5 and wild type after DG mutation of CDR3 of the heavy chain variable region CDR3 of the 75G7C6 antibody was SG, EG or DA.
  • the antibody c75G7C6 was detected by binding activity to CHOK-EGFRvIII cells
  • FIG. 18B is the mutant antibodies c75G7C6-3, c75G7C6-4 and c75G7C6-5 and wild type after DG mutation of CDR3 of the heavy chain variable region CDR3 of the 75G7C6 antibody was mutated into SG, EG or DA.
  • the antibody c75G7C6 was tested for binding activity to CHOK-EGFR cells.
  • Table 24 summarizes the binding activity of the c75G7C6 wild type antibody and the mutant antibody in CHOK-EGFRvIII cells
  • Table 25 summarizes the binding activity of the c75G7C6 wild type antibody and the mutant antibody in CHOK-EGFR cells.
  • Antibody name Point mutation EC50 (unit: nM) Maximum average fluorescence intensity c75G7C6 Wild type (NG&DG) 10.5 111769 c75G7C6-1 N G /N A 7.452 109947 c75G7C6-2 N G/ Q G 11.64 111041 c75G7C6-3 D G/ E G 24.82 94071 c75G7C6-4 D G/ S G twenty four 52584 c75G7C6-5 D G /D A no 296 Negative control hIgG no no 132.6
  • the binding capacity of EGFRvIII was about 2.5-fold lower than that of the wild-type chimeric antibody c75G7C6, and the maximum mean fluorescence intensity decreased by 16% and 53%, respectively.
  • the mutant antibody c75G7C6-5 after DG mutation into DA did not bind to CHOK-EGFRvIII. Mutation of DG to EG, SG or DA severely affected the binding of antibodies to CHOK-EGFRvIII cells.
  • the chimeric antibody c75G7C6 decreased about 3 fold, and the maximum mean fluorescence intensity decreased by 38%, while the mutant antibodies c75G7C6-4 and c75G7C6-5 after DG mutation to SG or DA weakly or did not bind to CHOK-EGFR, indicating DG Mutation to EG, SG or DA severely affected the binding of antibodies to CHOK-EGFR cells.
  • NG located in the CDR2 of the heavy chain variable region of c75G7C6 can be mutated to NA or QG, and the binding activity of the antibody to the antigen after the mutation is not affected, but the DG located in the CDR3 is mutated into EG. SG or DA affect the binding of the antibody to the antigen, and it is not suitable for mutation. Therefore, the subsequent study of the antibody c75G7C6-1 after mutating NG of CDR2 into NA will be carried out.
  • the human germline antibody heavy and light chain variable region templates that best match the non-CDR regions of chimeric antibodies 63A10A7 and 75G7C6 described above were selected in the Germline database.
  • the human acceptor sequence of the humanized EGFRvIII antibody is selected from the human germline exon VH , JH , Vk and Jk sequences.
  • 75G7C6 template antibody heavy chain variable region of an antibody heavy chain human germline outer V H exon IGHV1-46 * 01, an outer J H exon J H -6b, the light chain variable region templates for human outer V K germline antibody light chain exon IGKV3-11 * 01, J K exon J K -4.
  • Humanized antibody Briefly, humanized generated across the V H or V L domains of synthetic overlapping oligonucleotides and assembled by PCR overlap extension of each domain.
  • Figure 19 is the sequence of the humanized anti-EGFRvIII antibody h75G7C6 heavy chain variable region h75G7C6.VH and its variant and chimeric antibody c75G7C6.VH and human germline VH exon IGHV1-46*01/JH6b
  • Figure 20 is a comparison of the sequence of humanized anti-EGFRvIII antibody h75G7C6 light chain variable region h75G7C6.VL and its variants with chimeric antibody c75G7C6.VL and human germline VL exon IGHV3-11*01/JK4 21 is the sequence comparison of humanized anti-EGFRvIII antibody h63A10A7 heavy chain variable region h63A10A7.VH and its variant with chimeric antibody
  • JH4 YFDYWGQGTLVTVSS (SEQ ID NO. 189).
  • JH6b YYYYYGMDVWGQGTTVTVSS (SEQ ID NO. 190)
  • JK4 LTFGGGTKVEIK (SEQ ID NO. 191).
  • JK2 YTFGQGTKLEIK (SEQ ID NO. 192).
  • the cDNA is synthesized according to the amino acid sequences of the light chain variable region and the heavy chain variable region of each humanized antibody (ie, SEQ ID NO. 154, 155, 156, 157, 158, 159, 160, 161 in the sequence listing, respectively. 162,163,,164,165,166,167,168,169,170,171,172,173,174), heavy chain cDNA was digested with FspAI and AfeI, and light chain cDNA was treated with FspAI and BsiwI.
  • the cDNA fragment is inserted into an expression vector comprising a signal peptide and a human heavy chain antibody IgG1 constant region and an expression vector comprising a signal peptide and a human antibody light chain kappa constant region by FspAI/AfeI or FspAI/BsiwI cleavage sites, respectively.
  • the expression vector was purchased from Invitrogen, and the recombination step was also completed by Shanghai Ruizhi Chemical.
  • the recombinant plasmid was verified by sequencing, and the recombinant plasmid with a purity of 500 ⁇ g was purified by alkaline lysis kit (purchased from MACHEREY-NAGEL). The above was filtered through a 0.22 ⁇ m filter (purchased from Millopore) for transfection.
  • 293E cells Prior to transfection, 293E cells (purchased from Invitrogen) were cultured in medium Freestyle 293 expression medium (purchased from Invitrogen). When transfected, 10% (v/v) F68 (purchased from Invitrogen) was added to Freestyle 293 expression medium to a final concentration of 0.1% (v/v) of F68 to obtain Freestyle 293 expression containing 0.1% (v/v) F68.
  • the shaker was set to 37 ° C, 130 RPM and 8% CO 2 (v/v).
  • Peptone was added the next day to a final concentration of 0.5% (w/v).
  • the antibody titer of the culture solution was measured.
  • the supernatant was collected by centrifugation (3500 RPM, 30 minutes), and filtered through a 0.22 ⁇ m filter to obtain a filtered cell supernatant for purification.
  • A enzyme-linked immunosorbent assay (ELISA) to detect the binding of antibodies to EGFRvIII protein
  • the purified humanized anti-EGFRvIII antibody obtained in Example 12 was reacted with a human EGFRvIII-hFc protein.
  • the purified immunogen A (EGFRvIII-hFc) obtained in Example 1 was prepared (for the preparation method, see Step 1 of Example 1 (1) diluted with PBS to a final concentration of 1.0 ⁇ g/mL, and then added to a 96-well ELISA plate at 100 ⁇ l per well. Incubate overnight at 4 °C with plastic film, wash plate twice with wash solution [PBS+0.01% (v/v) Tween20], add blocking solution [PBS+0.01%(v/v)Tween20+ 1% (w/w) BSA] was blocked at room temperature for 2 hours. The blocking solution was discarded, and 100 ⁇ l of the purified EGFRvIII chimeric antibody obtained in Example 2 was added per well.
  • Figure 24A-E is a purified human source The binding reaction of the h63A10A7 variant to the human EGFRVIII-hFc protein.
  • Table 28 and Table 29 are the EC50 values calculated from the OD 450nm values for the h75G7C6 variant and the h63A10A7 variant, respectively, indicating the purified humanized EGFRvIII antibody variant. It has a good binding to EGFRvIII recombinant protein at the ELISA level.
  • the cells were washed twice with HBSS buffer, and after cell counting, the cells were diluted with HBSS buffer to 2 ⁇ 10 6 cells per ml, and 1% goat serum blocking solution was added, the percentage being mass percentage, and incubation on ice for 30 minutes. It was then washed twice with HBSS buffer. The collected cells were resuspended to 2 ⁇ 10 6 cells/mL with FACS buffer (HBSS + 2% FBS, the percentage is a percentage by volume), and added to a 96-well FACS reaction plate at 100 ⁇ L per well. The purified EGFRvIII antibody sample obtained in Example 12 was subjected to 100 ⁇ l per well and incubated on ice for 2 hours.
  • the cells were washed twice with FACS buffer, and 100 ⁇ l of a 1:1000 diluted fluorescent (Alexa 488)-labeled secondary antibody (purchased from Invitrogen) was added per well, and incubated on ice for 1 hour.
  • the cells were washed three times with FACS buffer, and the cells were resuspended by adding 100 ⁇ l of the fixing solution [4% (v/v) paraformaldehyde] per well, and after 10 minutes, the cells were washed twice with FACS buffer.
  • the cells were resuspended in 100 ⁇ l of FACS buffer, and the results were detected and analyzed by FACS (FACS Calibur, available from BD).
  • the average fluorescence density (MFI) to the cells was analyzed by software (CellQuest).
  • the data was fitted and analyzed by software (GraphPad Prism 5) to calculate the EC50.
  • humanized 75G7C6 and 63A10A7 antibody variants were also able to bind weakly to wild-type EGFR.
  • A431 cells human epidermal cell carcinoma cell line
  • A431 cells overexpressed a large amount of wild-type EGFR protein on the surface, and there was also a certain amount of wild-type EGFR protein expression on the surface of normal human primary hepatocytes, as shown in Figure 27 and Figure 28A-B.
  • the EGFR control antibody Cetuximab, the humanized 75G7C6 antibody variant and the humanized 63A10A7 antibody variant can both bind to the tumor cell A431 overexpressing EGFR, but weakly bind to the wild type EGFR protein on the surface of normal human primary hepatocytes, which may It is related to the spatial location of the antigenic determinants of the humanized 75G7C6 and 63A10A7 antibodies.
  • the variants of the humanized 75G7C6 antibody have different binding ability to the surface EGFR protein of A431 and normal human primary hepatocytes.
  • the test antibody has weak or no binding to normal human primary hepatocytes, and the average fluorescence
  • the intensity MFI value is about 250, which is similar to the negative control hIgG (MFI value 219), but has a higher level of binding to EGFR on the surface of tumor cell A431.
  • the average fluorescence intensity MFI is between 10,000 and 13,000, indicating humanized 75G7C6.
  • the antibody has an EGFR protein that selectively binds to the surface of the tumor cell, and does not bind or weakly bind to the EGFR protein expressed by the normal cell, and the selectivity window is 35-47 times.
  • each variant of the humanized 63A10A7 antibody differs in binding ability to A431 and normal human primary hepatocyte surface EGFR proteins, as shown in Figures 28A-B and Table 33, and some antibodies to be tested, such as the humanized antibody h63A10A7
  • the binding ability of -17 ⁇ 26 to normal human primary hepatocytes is close to that of anti-EGFR antibody (MFI 433), and the average fluorescence intensity MFI ranges from 433 to 677. Therefore, it is considered that this part of the antibody to be tested has no choice for normal cells expressing EGFR.
  • the fluorescence intensity MFI value is about 260, which is similar to the negative control hIgG (MFI value 219), but has a certain level of binding to EGFR on the surface of tumor cell A431.
  • the average fluorescence intensity MFI is 1000-2000, showing partial human origin.
  • the 63A10A7 antibody variant has an EGFR protein that selectively binds to the surface of the tumor cell overexpressing, but does not bind or weakly bind to the EGFR protein expressed by normal cells, and the selectivity window is 5 to 7.5 times.
  • Table 33 FACS detection of binding of humanized h63A10A7 antibody variant to EGFR on tumor cell A431 and normal human primary hepatocytes
  • the anti-human Fc biosensor AHC (purchased from ForteBio) was used in conjunction with the humanized h75G7C6 to be detected on an OctetRED (purchased from Pall) instrument.
  • Antibody variants were then combined with EGFRvIII-his (purchased from Sino Biological) or EGFR-his (purchased from Sino Biological) proteins and detected by biofilm layer optical interference (BLI) technology.
  • BLI technology detects when the molecules immobilized on the sensor interact with the molecules in the solution, the thickness of the biofilm increases, and the interference spectrum curve moves toward the wavelength.
  • the phase shift of the light wave is detected by the workstation in real time, and the phase shift of the light wave is analyzed.
  • the number of molecules on the sensor surface and related concentrations and kinetic data can be quantified.
  • the binding rate (K a ), dissociation rate (K dis ) and binding affinity (K D ) of the humanized h75G7C6 antibody variant to EGFRvIII and EGFR proteins are shown in Table 34 and Table 35, in which the chimeric antibody c75G7C6 was used as a control. .

Abstract

本发明公开了一种EGFRvIII抗体及其偶联物、制备方法和应用。所述抗体包括EGFRvIII抗体的互补决定区(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，其氨基酸序列如本发明中所述。所述EGFRvIII抗体，与EGFRvIII蛋白具有高度亲和力，与小分子毒素如MMAE偶联后能够进入细胞，对EGFRvIII阳性细胞进行细胞毒杀伤作用。因此能够运用于治疗肿瘤等药物的制备中。

Description

一种EGFRvIII抗体及其偶联物、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CN201711329680.1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EGFRvIII抗体及其偶联物、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，Cancer 94:1593-1611，2002)。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、以及细胞的有丝分裂和细胞转移(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)。
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、前列腺癌、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)。
在很大一部分肿瘤样本中可以同时检测到EGFR基因的扩增以及突变。EGFRvIII是 其中一种突变型，也称作de2-7EGFR、ΔEGFR、或Δ2-7(Olapade-Olaopa et al.，Br.J.Cancer.82,186-94，2000)。成熟的EGFRvIII mRNA中缺失了外显子2-7的801个核苷酸，相应的EGFRvIII蛋白缺失了267个氨基酸(6-273)，同时插入了一个甘氨酸残基，形成了一个独特的连接肽(Wong et al.，Proc.Natl.Acad.Sci.U.S.A.89,2965-9，1992；Yamazaki et al.，Jpn.J.Cancer Res.81,773-9，1990；Yamazaki et al.，Mol.Cell.Biol.8,1816-20，1988；and Sugawa et al.，Proc.Natl.Acad.Sci.U.S.A.87,8602-6，1990)。
多种肿瘤类型中报道了EGFRvIII的表达，包括神经胶质瘤、乳腺癌、肺癌、卵巢癌和前列腺癌(Wikstrand et al.，Cancer Res.57,4130-40，1997；Olapade-Olaopa et al.，Br.J.Cancer.82,186-94，2000；Wikstrand,et al.，Cancer Res.55,3140-8，1995；Garcia de Palazzo et al.，Cancer Res.53,3217-20，1993)。EGFRvIII不能与配体结合，但是它处于一种持续的低激活状态。在裸鼠的肿瘤异种移植模型中，EGFRvIII的表达可以显著促进神经胶质瘤细胞的生长(Nishikawa et al.，Proc.Natl.Acad.Sci.U.S.A.91,7727-31，1994)。另外EGFRvIII的表达可以使NIH3T3细胞和MCF-7细胞发生转化，产生致癌性(Batra et al.，Cell Growth Differ.6,1251-9，1995)。EGFRvIII在神经胶质瘤中的作用机制并没有完全清楚，但是根据已有的报道，EGFRvIII可以降低神经胶质瘤细胞的凋亡，并小幅度的提高神经胶质瘤细胞的增殖(Nagane et al.，Cancer Res.56,5079-86，1996)。EGFRvIII特异性的表达于肿瘤组织中，在正常组织中不表达，因此在抗体治疗中是一个高度特异性的靶点。
单克隆抗体由于具有靶向性、特异性、专一性、高亲和力等优势，正发展成为新型治疗药物。然而，早期的临床试验揭示，在人体中使用非人源单克隆抗体，常常因为人抗小鼠抗体(HAMA)和人抗大鼠抗体(HARA)应答，导致严重的免疫反应，抗体被快速清除。随后开发出免疫原性较小的抗体，包括嵌合抗体、人源化抗体和全人源抗体。根据人源化程度不同,治疗性单克隆抗体药物可分为4种：鼠源性抗体(无人源氨基酸序列)、嵌合抗体(60％～70％人源化氨基酸序列)、CDR移植抗体(90％～95％人源化氨基酸序列)以及全人源抗体(100％人源氨基酸序列)。随着人源化程度增加，非鼠源单克隆抗体可以减轻人体治疗过程中人抗鼠抗体反应(HAMA和HARA反应)，逐步消除异源性抗体的免疫原性问题，在保持对抗原高亲和力的同时，改善了抗体的药代动力学，临床上已大量使用这些抗体药物进行靶向治疗。
抗体药物偶联物(antibody-drug conjugate，ADC)是一种新型生物治疗方法，通过一个化学链接将具有生物活性的小分子药物连接到单抗上，单抗作为载体将小分子药物靶向运输到目标肿瘤细胞表面，ADC被肿瘤细胞内吞后释放出小分子药物，从而产生对肿瘤 细胞的特异性杀伤。
近一百年间，基于抗体的免疫疗法与基于化学药物的化学疗法，一直是临床上癌症治疗的两大治疗策略。抗体以肿瘤细胞过度表达的抗原为靶点，多种治疗性单抗已经在临床上取得了巨大成功。在临床实践中，治疗性抗体虽然具有很好的靶向性，但是杀伤作用存在局限性。小分子化学药物虽然具备对癌细胞的高效杀伤作用，但是对非癌细胞也造成同样的伤害。因此临床上抗体药物以及小分子药物各自的局限性，对药物研发提出了新的要求。新一代抗体药物偶联物，利用抗体对靶细胞的特异结合能力，输送高细胞毒的化学药物，实现对癌细胞的靶向高效杀伤。随着新型化学连接技术的出现，抗体药物偶联药在八十年代末开始进入临床研究，目前已经有4个ADC药物经FDA批准上市。
ADC药物的开发涉及：药物靶点的筛选、重组抗体的制备、连接物技术开发以及高细胞毒性化合物的筛选优化等几个方面。EGFRvIII只在肿瘤组织中表达，在正常组织中不表达，因此在抗体治疗中是一个高度特异性的靶点。
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EGFRvIII抗体的缺陷，提供一种亲和力高、特异性强的EGFRvIII抗体及其制备方法和应用。所述的EGFRvIII抗体，与EGFRvIII蛋白具有高度亲和力，与小分子毒素如MMAF偶联后能够进入细胞，对EGFRvIII阳性细胞进行细胞毒杀伤作用。因此能够运用于治疗肿瘤等药物的制备中。
本发明人以EGFRvIII蛋白或者过表达EGFRvIII蛋白的重组细胞株作为免疫原，采用传统的杂交瘤制备技术。该技术由Kohler and Milstein在40年前建立(Kohler and Milstein1975，Nature 256:495)，通过一系列的调整和改进，获得EGFRvIII抗体的先导抗体。再通过对先导抗体的初步生产、纯化和检定，获得具备抗体亲和力高、有与小分子毒素MMAF偶联后能够进入细胞，对EGFRvIII阳性细胞进行细胞毒杀伤作用等优异的生物性特性的EGFRvIII抗体。然后通过分子生物学方法测序获知EGFRvIII抗体的重链可变区和EGFRvIII抗体的轻链可变区的氨基酸序列。
本领域中，抗体或抗原的结合区均含有一条轻链可变区和一条重链可变区，每一个可变区均含有CDR1、CDR2和CDR3三个结构域。
本发明提供一种EGFRvIII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，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、SEQ ID No.90或SEQ ID No.98所示；所述重链CDR2的氨基酸序列如序列表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、SEQ ID No.99、SEQ ID NO.184或SEQ ID NO.186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、SEQ ID No.92或SEQ ID No.100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、SEQ ID No.94或SEQ ID No.102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、SEQ ID No.95或SEQ ID No.103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或SEQ ID No.80、SEQ ID No.88、SEQ ID No.96或SEQ ID No.104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、SEQ ID No.98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、SEQ ID No.99、SEQ ID NO.184或SEQ ID NO.186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、SEQ ID No.100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、SEQ ID  No.102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、SEQ ID No.103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、SEQ ID No.104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、SEQ ID No.98所示的氨基酸序列至少有90％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、SEQ ID No.99、SEQ ID NO.184或SEQ ID NO.186所示的氨基酸序列至少有90％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、SEQ ID No.100所示的氨基酸序列至少有90％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、SEQ ID No.102所示的氨基酸序列至少有90％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、SEQ ID No.103所示的氨基酸序列至少有90％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、SEQ ID No.104所示的氨基酸序列至少有90％的序列同源性的氨基酸序列所示；更佳地，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、SEQ ID No.98所示的氨基酸序列至少有95％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、SEQ ID No.99、SEQ ID NO.184或SEQ ID NO.186所示的氨基酸序列至少有95％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、SEQ ID No.100所示的氨基酸序列至少有95％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、SEQ ID No.102所示的氨基酸序列至少有95％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、SEQ ID No.103所示的氨基酸序列至少有95％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、SEQ ID No.104所示的氨基酸序列至少有95％的序列同源性的氨基酸序列所示；进一步更佳地，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、SEQ ID No.98所示的氨基酸序列至少有99％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、SEQ ID No.99、SEQ ID NO.184或SEQ ID NO.186所示的氨基酸序列至少有99％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、SEQ ID No.100所 示的氨基酸序列至少有99％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、SEQ ID No.102所示的氨基酸序列至少有99％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、SEQ ID No.103所示的氨基酸序列至少有99％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、SEQ ID No.104所示的氨基酸序列至少有99％的序列同源性的氨基酸序列所示。
需知：虽然抗体药物中使用的抗体是由来自单一的抗体产生细胞的克隆得到的单克隆抗体，但抗体是生物大分子，结构非常复杂，因此在生产、运输、储存以及体内使用过程中会发生各种翻译后修饰和降解反应，如N末端环化、糖基化、脱酰胺、异构化、氧化、片段化、二硫键错配等。这些质量属性可能会对最终产品影响抗体稳定性、生物学活性及生物利用度，因此，控制产品质量的稳定性和一致性非常重要。本发明中所述的“至少有80％(或者90％、95％、99％)的序列同源性”的氨基酸序列是通过对前述序列中所示的氨基酸序列进行插入、缺失或者替换获得，以克服上述可能存在的不稳定性问题。例如，对序列进行计算机结构模拟分析，对可能存在的、特别是CDR区的转录后修饰(Potential post-translational modifications，PTMs)位点分析，包括抗体的聚集、脱酰胺基敏感(asparagine deamidation，位点(NG，NS，NH等)、天冬氨酸异构(DG，DP)敏感位点、N糖基化(N-{P}S/T)敏感位点及氧化敏感位点等分析和替换。
其中，脱酰胺是指天冬酰胺和天冬氨酸的侧链的酰胺转变成羧酸的反应，当为谷氨酰胺时，脱酰胺化速度通常是天冬酰胺时的十分之一，但机理是相同的。异构化是指位于天冬酰胺和天冬氨酸的侧链的羧基被位于C末端侧的残基的氮原子电子对攻击，导致天冬酰胺的脱酰胺化或天冬氨酸脱水形成不稳定的环状酰亚胺中间体，该中间体通过开裂，大部分变成异天冬氨酸，剩余部分变成天冬氨酸。另外，据报道，脱酰胺化反应特别容易在天冬酰胺和甘氨酸相邻位点(Asn-Gly)发生(Geiger等人(J.Biol.Chem.1987，262：785-794))。还有报道称，天冬氨酸通过水解反应发生肽链断裂，特别是C端侧存在脯氨酸的序列(Asp-Pro)容易发生酸性条件下的分解(Segalas等，FEBS Letters，1995,371:171- 175)。在这些抗体或蛋白等的保存中发生脱酰胺化、异构化反应成为产品异质性的原因，所以希望尽可能地得到控制。为了抑制脱酰胺化、异构化和水解，可以适当实施去除作为接受脱酰胺化的位点的谷氨酰残基和天冬酰残基的氨基酸修饰。作为除去是特别有效的脱酰胺化位点的一个非限定性方案，优选列举脱酰胺化反应得到促进的位点，即以NG和QG序列的形式表示的模体中的甘氨酸残基，天冬酰胺残基或谷氨酰胺残基，其中任意一个氨基酸残基的(N、Q或G)的取代均可显著抑制脱酰胺化反应(WO2003/057881或WO2005/067620等)。另外，通过调整产生抗体的细胞的培养方法，或可以适当采用制备脱酰胺化反应得到了抑制的抗体的方法。
较佳地，所述重链CDR1的氨基酸序列如序列表SEQ ID No.2所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.3所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.4所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.10所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.11所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.12所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.18所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.19所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.20所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.26所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.27所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.28所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.34所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.35所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.36所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.42所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.43所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.44所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.50所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.51所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.52所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.58所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.59所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.60所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.66所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.67所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.68所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.74所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.75所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.76所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.82所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.83所示，且所述重链CDR3的氨基酸序列如序列表SEQ  ID No.84所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.90所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.91所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.92所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.98所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.99所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.100所示；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.184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；或者，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.186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；
所述轻链CDR1的氨基酸序列如序列表SEQ ID No.6所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.7所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.8所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.14所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.15所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.16所示；或，所述轻链CDR1的氨基酸序列如序列表SEQ ID No.22所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.23所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.24所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.30所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.31所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.32所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.38所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.39所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.40所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.46所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.47所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.48所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.54所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.55所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.56所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.62所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.63所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.64所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.70所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.71所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.72所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.78所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.79所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.80所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.86所示，所述轻链CDR2的氨 基酸序列如序列表SEQ ID No.87所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.88所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.94所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.95所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.96所示；或，所述轻链CDR1的氨基酸序列如序列表SEQ ID No.102所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.103所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.104所示。
更佳地，所述EGFRvIII抗体包括含有所述CDR的EGFRvIII抗体的重链可变区和/或EGFRvIII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、SEQ ID No.89、SEQ ID No.97、SEQ ID No.179或SEQ ID No.180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、SEQ ID No.93或SEQ ID No.101所示。
较佳地，所述重链可变区的氨基酸序列如序列表SEQ ID No.1所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.5所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.9所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.13所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.17所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.21所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.25所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.29所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.33所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.37所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.41所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.45所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.49所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.53所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.57所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.61所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.65所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.69所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.73所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.77所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.81所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.85所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.89所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.93所示； 所述重链可变区的氨基酸序列如序列表SEQ ID No.97所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.101所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.179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；或，所述重链可变区的氨基酸序列如序列表中SEQ ID No.180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。
综上所述，上述氨基酸序列的编号如表1所示：
表1 EGFRvIII抗体蛋白序列编号
Figure PCTCN2018120959-appb-000001
其中，表1中的数字即为序列表中序列号，如75G7C6的重链蛋白可变区的氨基酸序列为SEQ ID No.1，而75G7C6的重链蛋白可变区中CDR1的氨基酸序列为SEQ ID No.2。
较佳地，所述的EGFRvIII抗体还包括构架区(或称框架区或骨架区)，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。构架残基是轻链可变区或重链可变区的一部分，是除高变残基(高变残基多指互补决定区或CDR)或CDR残基外的抗体可变区残基，其用作该可变结构域的抗原结合环(CDR)的支架。构架残基可以衍生自天然存在的人的抗体，例如基本上类似于鼠源抗EGFRvIII抗体75G7C6或63A10A7的构架 区的人抗体的构架区。还可以使用代表个体序列之间的共有序列的人工构架区序列。当选择用于人源化的构架区时，在人类中广泛呈现的序列可能优于较不常见的序列。可以制备人构架受体序列的另外突变，以恢复被认为涉及抗原接触的鼠类残基和/或涉及抗原结合位点的结构完整性的残基，或改善抗体表达。肽结构预测可以用于分析人源化重链可变区和轻链可变区序列，以鉴定和避免由人源化设计引入的翻译后蛋白质修饰位点。
更佳地，当本发明所述的EGFRvIII抗体为人源化抗体或全人源抗体时，所述重链构架区为人抗体重链构架区，人抗体轻链构架区残基可以包含种系IGKV1-12、IGKV1-13、IGKV1-16、IGKV1-17、IGKV1-22、IGKV1-27、IGKV1-32、IGKV1-37、IGKV1-39、IGKV1-5、IGKV1-8、IGKV1-9、IGKV3-34、IGKV2-36、IGKV3-11、IGKV2-40、IGKV3-25、IGKV6-21、IGKV7-3、IGKV5-2、IGKV4-1、IGKV2-4、IGKV2-19、IGKV2-18、IGKV3-20、IGKV2-26和IGKV2-24，特别是这些种系的FR1、FR2、FR3；以及由Jk片段Jk1、Jk2、Jk3、Jk4和Jk5，特别是这些种系的FR4编码的序列。人抗体重链构架区残基可以包含种系IGHV7-81、IGHV4-80、IGHV3-79、IGHV(II)-78-1、IGHV5-78、IGHV3-76、IGHV3-73、IGHV3-72、IGHV2-70、IGHV1-69-2、IGHV2-70D、IGHV3-49、IGHV3-43、IGHV1-2、IGHV1-3、IGHV1-8、IGHV1-18、IGHV1-24、IGHV1-45、IGHV1-46、IGHV2-5、IGHV2-21、IGHV2-26、IGHV2-70、IGHD3-7、IGHV3-9、IGHV3-11、IGHV3-7、IGHV3-7和IGHD3-9，特别是这些种系的FR1、FR2、FR3；以及JH片段JH1、JH2、JH3、JH4、JH4b、JH5和JH6，特别是这些种系的FR4编码的序列，或重链构架区的共有序列。此类构架区序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在"VBase"人种系序列数据库(www.mrcco8.com.ac.uk/vbase)获得，以及在Kabat，E.A等人，1991 Sequences of Proteins of Immunological Interest，第5版中找到。在本发明人源化抗体的一较佳实施例中，所述人源化EGFRvIII抗体的人接受序列选自人种系外显子V H、J H、V k和J k序列，其中，抗体重链可变区的模板优选人种系抗体重链V H外显子的IGHV1-46*01、J H外显子的J H-4；或者人种系抗体重链V H外显子的IGHV1-46*01，J H外显子的J H-6b。抗体轻链可变区的模板优选人种系抗体轻链V K外显子的IGKV1-39*01、J K外显子的/J K-2；或者人种系抗体轻链V K外显子的IGKV3-11*01，J K外显子的J K-4。
较佳地，所述的蛋白质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为本领域常规，较佳地为小鼠源抗体重链恒定区或人源抗体重链恒定区，更佳地为人源抗体重链恒定区。所述的抗体轻链恒定区为本领域常规，较佳地为小鼠源轻链抗体恒定区或人源抗体轻链恒定区，更佳地为人源抗体轻链恒定区。
其中，氨基酸序列如SEQ ID NO.1、9、17、25或者179所示的重链可变区以及序列如SEQ ID NO.5、13、21或者29所示的轻链可变区可与鼠源重链恒定区构成鼠源化EGFRvIII抗体、亦可与人源重链恒定区以及人源轻链恒定区构成EGFRvIII嵌合抗体(其中SEQ ID NO.179所示的氨基酸序列为经SEQ ID NO.1所示的氨基酸序列突变获得，将后者CDR2区的NG突变为NA)。
进一步地，将上述嵌合抗体中氨基酸序列如SEQ ID NO.179或9所示的重链可变区中根据Kabat定义确定的序列如SEQ ID NO.2、184以及4(分别对应CDR1、2和3)，或者SEQ ID NO.10～12所示的重链CDR以及上述序列如SEQ ID NO.5或13所示的轻链可变区中根据Kabat定义确定的序列如SEQ ID NO.6～8或者SEQ ID NO.14～16所示的轻链CDR分别移植到所选人种系模板中，替换人种系模板的CDR区，即得人源化抗体；所述种系模板中的轻链构架区和重链构架区如上所述：人的抗体可变区框架经过选择，其中所述抗体轻链可变区上的轻链FR序列，来源于人种系轻链包含1)IGKV1-39*01或IGKV3-11*01的FR1、FR2、FR3区，和2)J K-2或J K-4的FR4区的人抗体轻链构架区的组合；所述抗体的重链可变区上的重链FR序列，来源于人种系重链序列包含1)IGHV1-46*01的FR1、FR2、FR3区，和2)J H-4或J H-6b的FR4区的人抗体重链构架区的组合。一般而言，人受体构架区的选择应类似于供体抗体的构架区，或最类似于可变区亚家族的共有序列进行选择。移植后，可以在供体和/或受体序列中进行序列突变，以优化抗原结合、功能性、密码子使用、表达水平等，包括将非人残基引入构架区内。
较佳地，上述CDR区被移植到所选人种系模板中且构架区残基经过回复突变后的重链可变区的氨基酸序列优选如SEQ ID NO.133、134、135、136、143、144、145、146、147、141、142或者147所示；轻链可变区的氨基酸序列优选如SEQ ID NO.137、138、139、140、148、149、150、151、152或者153所示。
本发明中所述的人源化EGFRvIII抗体优选包含至少一个重链可变区和/或至少一个轻链可变区，其中，所述重链可变区的氨基酸序列如序列表中SEQ ID NO.133、SEQ ID NO.134、SEQ ID NO.135、SEQ ID NO.136、SEQ ID NO.141、SEQ ID NO.142、SEQ ID NO.143、SEQ ID NO.144、SEQ ID NO.145、SEQ ID NO.146或SEQ ID NO.147所示；所述的轻链可变区序列如序列表中SEQ ID NO.137、SEQ ID NO.138、SEQ ID NO.139、SEQ ID NO.140、SEQ ID NO.148、SEQ ID NO.149、SEQ ID NO.150、SEQ ID NO.151、SEQ ID NO.152或SEQ ID NO.153所示。
或者，所述的重链可变区的氨基酸序列如序列表中SEQ ID NO.133、SEQ ID NO.134、SEQ ID NO.135、SEQ ID NO.136、SEQ ID NO.141、SEQ ID NO.142、SEQ ID NO.143、 SEQ ID NO.144、SEQ ID NO.145、SEQ ID NO.146或SEQ ID NO.147所示的氨基酸序列至少有80％序列同源的氨基酸序列所示；所述的轻链可变区序列如序列表中SEQ ID NO.137、SEQ ID NO.138、SEQ ID NO.139、SEQ ID NO.140、SEQ ID NO.148、SEQ ID NO.149、SEQ ID NO.150、SEQ ID NO.151、SEQ ID NO.152或SEQ ID NO.153所示的氨基酸序列至少有80％序列同源的氨基酸序列所示；更佳地，所述的重链可变区的氨基酸序列为，与如序列表中SEQ ID No.154、SEQ ID No.155、SEQ ID No.156、SEQ ID No.157、SEQ ID No.162、SEQ ID No.163、SEQ ID No.164、SEQ ID No.165、SEQ ID No.166、SEQ ID No.167或SEQ ID No.168所示的核苷酸序列编码的氨基酸序列有90％序列同源的氨基酸序列；所述的轻链可变区的氨基酸序列为，与如序列表中SEQ ID No.158、SEQ ID No.159、SEQ ID No.160、SEQ ID No.161、SEQ ID No.169、SEQ ID No.170、SEQ ID No.171、SEQ ID No.172、SEQ ID No.173或SEQ ID No.174所示的核苷酸序列编码的氨基酸序列有90％序列同源的氨基酸序列；进一步更佳地，所述的重链可变区的氨基酸序列为，与如序列表中SEQ ID No.154、SEQ ID No.155、SEQ ID No.156、SEQ ID No.157、SEQ ID No.162、SEQ ID No.163、SEQ ID No.164、SEQ ID No.165、SEQ ID No.166、SEQ ID No.167或SEQ ID No.168所示的核苷酸序列编码的氨基酸序列有95％序列同源的氨基酸序列；所述的轻链可变区的氨基酸序列为，与如序列表中SEQ ID No.158、SEQ ID No.159、SEQ ID No.160、SEQ ID No.161、SEQ ID No.169、SEQ ID No.170、SEQ ID No.171、SEQ ID No.172、SEQ ID No.173或SEQ ID No.174所示的核苷酸序列编码的氨基酸序列有95％序列同源的氨基酸序列；最佳地，所述的重链可变区的氨基酸序列为，与如序列表中SEQ ID No.154、SEQ ID No.155、SEQ ID No.156、SEQ ID No.157、SEQ ID No.162、SEQ ID No.163、SEQ ID No.164、SEQ ID No.165、SEQ ID No.166、SEQ ID No.167或SEQ ID No.168所示的核苷酸序列编码的氨基酸序列有99％序列同源的氨基酸序列；所述的轻链可变区的氨基酸序列为，与如序列表中SEQ ID No.158、SEQ ID No.159、SEQ ID No.160、SEQ ID No.161、SEQ ID No.169、SEQ ID No.170、SEQ ID No.171、SEQ ID No.172、SEQ ID No.173或SEQ ID No.174所示的核苷酸序列编码的氨基酸序列有99％序列同源的氨基酸序列.
较佳地，所述的重链可变区的氨基酸序列为，与如序列表中SEQ ID No.154、SEQ ID No.155、SEQ ID No.156、SEQ ID No.157、SEQ ID No.162、SEQ ID No.163、SEQ ID No.164、SEQ ID No.165、SEQ ID No.166、SEQ ID No.167或SEQ ID No.168所示的核苷酸序列编码的氨基酸序列有80％序列同源的氨基酸序列；所述的轻链可变区的氨基酸序列为，与如序列表中SEQ ID No.158、SEQ ID No.159、SEQ ID No.160、SEQ ID No.161、SEQ ID No.169、SEQ ID No.170、SEQ ID No.171、SEQ ID No.172、SEQ ID No.173或SEQ ID  No.174所示的核苷酸序列编码的氨基酸序列有80％序列同源的氨基酸序列；更佳地，与如序列表中SEQ ID No.154、SEQ ID No.155、SEQ ID No.156、SEQ ID No.157、SEQ ID No.162、SEQ ID No.163、SEQ ID No.164、SEQ ID No.165、SEQ ID No.166、SEQ ID No.167或SEQ ID No.168所示的核苷酸序列编码的氨基酸序列有90％序列同源的氨基酸序列；所述的轻链可变区的氨基酸序列为，与如序列表中SEQ ID No.158、SEQ ID No.159、SEQ ID No.160、SEQ ID No.161、SEQ ID No.169、SEQ ID No.170、SEQ ID No.171、SEQ ID No.172、SEQ ID No.173或SEQ ID No.174所示的核苷酸序列编码的氨基酸序列有90％序列同源的氨基酸序列；进一步更佳地，与如序列表中SEQ ID No.154、SEQ ID No.155、SEQ ID No.156、SEQ ID No.157、SEQ ID No.162、SEQ ID No.163、SEQ ID No.164、SEQ ID No.165、SEQ ID No.166、SEQ ID No.167或SEQ ID No.168所示的核苷酸序列编码的氨基酸序列有95％序列同源的氨基酸序列；所述的轻链可变区的氨基酸序列为，与如序列表中SEQ ID No.158、SEQ ID No.159、SEQ ID No.160、SEQ ID No.161、SEQ ID No.169、SEQ ID No.170、SEQ ID No.171、SEQ ID No.172、SEQ ID No.173或SEQ ID No.174所示的核苷酸序列编码的氨基酸序列有95％序列同源的氨基酸序列；最佳地，与如序列表中SEQ ID No.154、SEQ ID No.155、SEQ ID No.156、SEQ ID No.157、SEQ ID No.162、SEQ ID No.163、SEQ ID No.164、SEQ ID No.165、SEQ ID No.166、SEQ ID No.167或SEQ ID No.168所示的核苷酸序列编码的氨基酸序列有99％序列同源的氨基酸序列；所述的轻链可变区的氨基酸序列为，与如序列表中SEQ ID No.158、SEQ ID No.159、SEQ ID No.160、SEQ ID No.161、SEQ ID No.169、SEQ ID No.170、SEQ ID No.171、SEQ ID No.172、SEQ ID No.173或SEQ ID No.174所示的核苷酸序列编码的氨基酸序列有99％序列同源的氨基酸序列。
较佳地，所述重链可变区的氨基酸序列如序列表SEQ ID No.133所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.137所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.133所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.138所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.133所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.139所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.133所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.140所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.134所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.137所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.134所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.138所示的序列；所述重链可变区的氨 基酸序列如序列表SEQ ID No.134所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.139所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.134所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.140所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.135所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.137所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.135所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.138所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.135所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.139所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.135所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.140所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.136所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.137所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.136所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.138所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.136所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.139所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.136所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.140所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.143所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.149所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.144所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.149所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.145所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.149所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.146所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.149所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.147所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.149所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.143所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.148所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.143所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.150所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.143所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.151所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.143所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.152所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.143所示的序列，且所 述轻链可变区的氨基酸序列如序列表SEQ ID No.153所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.141所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.149所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.141所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.150所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.141所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.151所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.141所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.152所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.141所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.153所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.142所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.149所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.142所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.150所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.142所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.151所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.142所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.152所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.142所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.153所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.145所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.148所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.146所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.148所示的序列；或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.147所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.148所示的序列。
综上所述，上述氨基酸序列的编号如表1-1所示。
表1-1.人源化EGFRvIII抗体蛋白序列编号
Figure PCTCN2018120959-appb-000002
Figure PCTCN2018120959-appb-000003
其中，表1-1中的数字即为序列表中序列号，如h75G7C6-1的重链蛋白可变区的氨基酸序列为SEQ ID No.133，而h75G7C6-1的轻链蛋白可变区的氨基酸序列为SEQ ID No.137。
其中，c75G7C6-1的重链可变区的CDR1、CDR2和CDR3分别如序列表中SEQ ID NO.2、SEQ ID NO.184和SEQ ID NO.4所示。
c75G7C6-2的重链可变区的CDR1、CDR2和CDR3分别如序列表中SEQ ID NO.2、SEQ ID NO.186和SEQ ID NO.4所示。
较佳地，所述的人源化抗EGFRvIII抗体还包括人源抗体重链恒定区和/或人源抗 体轻链恒定区。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人源化抗体全长蛋白。其中所述的人源化抗体重链恒定区为本领域常规，可以包含衍生自人恒定区的恒定区，其进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区；所述的人源化抗体轻链恒定区为本领域常规，可以包含衍生自人恒定区的恒定区，其进一步包含人源κ、λ链或其变体的轻链恒定区。
本发明的人源化抗EGFRvIII抗体以使用各种方法中的任何一种进行制备，包括互补决定区(CDRs)的镶饰、移植、缩短的CDRs的移植、特异性决定区(SDRs)的移植、和Frankenstein装配。其中，特异性决定区(SDRs)是与抗原直接相互作用的CDRs内的残基。SDRs对应高变残基。参见Padlan等人(1995)FASEB J.9:133-139)。
本发明的代表性人源化抗EGFRvIII抗体的制备在实施例1中得到描述。
本发明中所述的人源抗体广义上也是一类嵌合抗体，其中负责抗原结合的可变区残基，包括衍生自非人物种的互补决定区、缩短的互补决定区、或参与抗原结合的任何其它残基；而其余可变区残基如，构架区的残基和恒定区至少部分衍生自人抗体序列。人源化抗体的构架区残基和恒定区残基的一个子集可以衍生自非人来源。人源化抗体的可变区也描述为人源化的轻链可变区和/或重链可变区。非人物种一般是用于由抗原免疫接种的物种，例如小鼠、大鼠、兔、非人灵长类、或其他非人哺乳动物物种。人源化抗体一般比传统嵌合抗体的免疫原性小，并且在给人施用后显示出改善的稳定性。
所述的人源化抗体还包括超人源化抗体，它是一种人源化抗体的制备方法，此方法不依赖于将人构架序列作为分析点，而是依赖于比较非人抗体的规范CDR结构类型和人抗体的CDR结构类型，尤其是人胚系序列所编码的人抗体，从中识别出可以得到适宜的人构架序列的候选的人抗体序列。例如，人残基可以置换CDRs中的非人残基，其中一个或多个变化已引入CDRs中。镶饰(veneering)的一个前提是鼠源抗体可变区的免疫原性起源于它的表面残基，且残基的运动性和溶剂的可及性是其成为抗原决定簇的基本条件。根据对现有的抗体晶体结构数据的分析结果统计，在序列配对位置上，人和鼠的抗体可变区残基的相对溶剂可及性分布的保真度达98％，这说明在异种间诱导免疫反应的残基是由其余的种特异性溶剂可及表面残基引起的。因此将鼠特异性表面残基换成人源性的，就可以模拟人源抗体的表面轮廓，逃避人体免疫系统的识别，达到人源化的目的。简单来说，镶饰基于通过用人氨基酸序列重建抗体的溶剂可及的表面来减少在啮齿类动物或其他非人抗体中的潜在免疫原性的氨基酸序列的概念。参见Padlan(1991)Mol.Immunol.28:489-980.通过鉴定非人抗体中暴露在表面的溶剂可及性残基的外部构架区残基(所述残基不同于人抗体的构架区中相同位置上的那些残基)，并用占据人抗体 中的那些相同位置的氨基酸替换所鉴定的残基，以进行镶饰，即镶饰的抗体，其表面残基主要是人源序列，而包裹在内部的残基主要是最初的鼠源序列。CDRs的移植通过用供体抗体(如，非人抗体)的CDRs替换受体抗体(例如，包含所需构架残基的人抗体或其他抗体)的一个或多个CDRs来进行。受体抗体可以基于在候选受体抗体和供体抗体之间的构架残基的相似性进行选择。例如，根据Frankenstein方法，鉴定与相关的非人抗体的各构架区具有实质上的序列同源性的人构架区，并且将非人抗体的CDRs移植到这些不同的人构架区的复合物上。可以结合上述方法，以产生任何所需序列的抗EGFRvIII抗体。
本发明中所述的EGFRvIII抗体较佳地为抗体全长蛋白、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(single chain antibody fragment，scFv)、单域抗体(single domain antibody，sdAb)和单区抗体(Signle-domain antibody)中的一种或多种，以及上述抗体所制得的单克隆抗体或多克隆抗体。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。所述的蛋白质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。
本发明的所述EGFRvIII抗体还包括超人源化抗体、双抗体(diabody)等。
其中，所述EGFRvIII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：从重组表达该蛋白质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蛋白质的表达转化体中分离获得优选如下方法：将编码所述EGFRvIII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EGFRvIII抗体。
本发明还提供一种核酸，其编码上述的EGFRvIII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.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、SEQ ID No.119、SEQ ID No.121、SEQ ID No.123、SEQ ID No.125、SEQ ID No.127、SEQ ID No.129、SEQ ID No.185、SEQ ID No.154、SEQ ID No.155、SEQ ID No.156、SEQ ID No.157、SEQ ID No.162、SEQ ID No.163、SEQ ID No.164、SEQ ID No.165、SEQ ID No.166、SEQ ID No.167或SEQ ID No.168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.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、SEQ ID No.120、SEQ ID No.122、SEQ ID No.124、SEQ ID No.126、SEQ ID No.128、SEQ ID No.130、SEQ ID No.158、SEQ ID No.159、SEQ ID No.160、SEQ ID No.161、SEQ ID No.169、SEQ ID No.170、SEQ ID No.171、SEQ ID No.172、SEQ ID No.173或SEQ ID No.174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SEQ ID No.118所示；编码所述重链可变区的核酸的核苷酸序列如序列表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SEQ ID No.120所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.121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SEQ ID No.122所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.123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SEQ  ID No.124所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.125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SEQ ID No126.所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.127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SEQ ID No.128所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.129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SEQ ID No.130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.185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SEQ ID No.154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SEQ ID No.158所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.154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SEQ ID No.159所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.154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SEQ ID No.160所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.154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SEQ ID No.161所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.155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SEQ ID No.158所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.155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SEQ ID No.159所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.155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SEQ ID No.160所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.155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SEQ ID No.161所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.156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SEQ ID No.158所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.156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SEQ ID No.159所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.156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SEQ ID No.160所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.156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SEQ ID No.161所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.157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SEQ ID No.158所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.157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SEQ ID No.159所示； 编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.157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SEQ ID No.160所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.157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SEQ ID No.161所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.164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SEQ ID No.170所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.165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SEQ ID No.170所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.166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SEQ ID No.170所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.167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SEQ ID No.170所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.168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SEQ ID No.170所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.164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SEQ ID No.169所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.164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SEQ ID No.171所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.164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SEQ ID No.172所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.164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SEQ ID No.173所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.164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SEQ ID No.174所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.162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SEQ ID No.170所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.162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SEQ ID No.171所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.162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SEQ ID No.172所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.162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SEQ ID No.173所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.162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SEQ ID No.174所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.163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SEQ ID No.170所示；编码所述重 链可变区的核酸的核苷酸序列如序列表SEQ ID No.163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SEQ ID No.171所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.163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SEQ ID No.172所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.163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SEQ ID No.173所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.163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SEQ ID No.174所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.166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SEQ ID No.169所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.167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SEQ ID No.169所示；或者，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.168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SEQ ID No.169所示。
综上所述，上述核苷酸序列的编号如表1-2和表2所示。
表1-2.EGFRvIII抗体基因序列编号
Figure PCTCN2018120959-appb-000004
Figure PCTCN2018120959-appb-000005
其中，表1-2中的数字即为序列表中序列号，如h75G7C6-1的重链蛋白可变区的核苷酸序列为SEQ ID No.154，而h75G7C6-1的轻链蛋白可变区的核苷酸序列为SEQ ID No.158。
所述核酸的制备方法为本领域常规的制备方法，较佳地，包括以下的步骤：通过基因克隆技术获得编码上述人源化抗EGFRvIII抗体的核酸分子，或者通过人工全序列合成的方法得到编码上述人源化抗EGFRvIII抗体的核酸分子。
本领域技术人员知晓，编码上述人源化抗EGFRvIII抗体的核酸可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该人源化抗EGFRvIII抗体的核酸的一个或多个核苷酸在保持抗体活性范围内进行替换、缺失或增加来制得。
表2 EGFRvIII抗体基因序列编号
克隆号 重链蛋白可变区 轻链蛋白可变区
75G7C6 105 106
63A10A7 107 108
64F1F8 109 110
7E5-2A9 111 112
43H6A1 113 114
46C4A6 115 116
49G12G5 117 118
51G7C2 119 120
53D8H4 121 122
54D2G10 123 124
56F10G2 125 126
59G3F12 127 128
64B11F2 129 130
其中，表2中的数字即为序列表中序列号，如编码75G7C6的重链蛋白可变区的核苷酸序列为SEQ ID No.105。
本发明中所述的互补决定区(CDRs)是参与抗原结合的抗体可变区的残基。用于标识CDRs的几种编号系统是常用的，包括例如Kabat定义、Chothia定义和AbM定义。概括地说，Kabat定义是基于序列的变异性、Chothia定义是基于结构环区的位置，AbM定义是Kabat和Chothia方法之间的折中。根据Kabat、Chothia或AbM算法，轻链可变区有三个CDR区，其CDR1位于由第24-34位氨基酸(CDR1-L)、CDR2位于第50-56位氨基酸(CDR2-L)、CDR3位于第89-97位氨基酸(CDR3-L)。由于可变区的长度变化，在不同的中枢或不同的亚群中，第27位可有1-6个氨基酸，第95位也可有1-6个氨基酸，它们在原编号的基础上加上英文字母进行编位，如：27A、27B、95A、95B等。根据Kabat定义，重链可变区的CDRs由在第31和35B位(CDR1-H)、第50和65位(CDR2-H)、以及第95和102位(CDR3-H)的残基界定(根据Kabat编号)。根据Chothia定义，重链可变区的CDRs由第26和32位(CDR1-H)、第52和56位(CDR2-H)、以及第95和102位(CDR3-H)上的残基界定(根据Chothia编号)。根据AbM定义，重链可变区的CDRs由26和35B位(CDR1-H)、第50和58位(CDR2-H)、以及第95和102位(CDR3-H)的残基界定(根据Kabat编号)。与轻链可变区类似，在第35位、52位、82位及100位也可有多个氨基酸，以A、B、C……等编号。参见Martin等人(1989)Proc.Nat l.Acad.Sci.USA 86:9268-9272；Martin等人(1991)Methods Enzymol.203:121-153；Pedersen等人(1992)lmmunomethods 1:126；Protein Structure Prediction,Oxford University Press,Oxford,第141-172页。本发明中以下鼠源抗体的CDR编号系统为Chothia，后续的人源化抗体采用了Kabat编号系统。
其中，编码75G7C6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75G7C6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75G7C6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339位；
编码75G7C6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105位；
编码75G7C6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151位至第171位；
编码75G7C6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268位至第294位；
编码63A10A7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63A10A7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c75G7C6-1的重链蛋白可变区的CDR2的核苷酸序列如序列表中SEQ ID No.185中的第154位至第171位所示；
编码c75G7C6-2的重链蛋白可变区的CDR2的核苷酸序列如序列表中SEQ ID No.187中的第154位至第171位所示。
编码63A10A7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318位；
编码63A10A7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102位；
编码63A10A7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148位至第168位；
编码63A10A7的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.108中的第265位至第291位。
编码64F1F8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64F1F8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64F1F8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64F1F8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99位；
编码64F1F8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145位至第165位；
编码64F1F8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262位至第288位。
编码7E5-2A9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7E5-2A9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7E5-2A9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309位；
编码7E5-2A9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120位；
编码7E5-2A9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166位至第186位；
编码7E5-2A9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283位至第309位。
编码43H6A1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127位至第150位；
编码43H6A1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208位至第222位；
编码43H6A1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346位至第366位；
编码43H6A1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102位；
编码43H6A1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148位至第168位；
编码43H6A1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265位至第291位；
编码46C4A6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46C4A6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46C4A6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306位；
编码46C4A6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46C4A6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46C4A6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49G12G5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49G12G5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49G12G5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318位；
编码49G12G5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105位；
编码49G12G5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151位至第171位；
编码49G12G5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268位至第294位。
编码51G7C2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51G7C2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51G7C2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318位；
编码51G7C2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105位；
编码51G7C2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151位至第171位；
编码51G7C2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268位至第294位。
编码53D8H4的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.121中的第76位至第96位；
编码53D8H4的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.121中的第154位至第171位；
编码53D8H4的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.121中的第295位至第318位；
编码53D8H4的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.122中的第70位至第102位；
编码53D8H4的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.122中的第148位至第168位；
编码53D8H4的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.122中的第265位至第291位。
编码54D2G10的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.123中的第76位至第96位；
编码54D2G10的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.123中的第154位至第171位；
编码54D2G10的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.123中的第295位至第315位；
编码54D2G10的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.124中的第70位至第99位；
编码54D2G10的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.124中的第145位至第165位；
编码54D2G10的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.124中的第262位至第288位。
编码56F10G2的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.125中的第76位至第96位；
编码56F10G2的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.125中的第154位至第171位；
编码56F10G2的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.125中的第295位至第315位；
编码56F10G2的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.126中的第70位至第99位；
编码56F10G2的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.126中的第145位至第165位；
编码56F10G2的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.126中的第262位至第288位。
编码59G3F12的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.127中的第76位至第99位；
编码59G3F12的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.127中的第157位至第171位；
编码59G3F12的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.127中的第295位至第318位；
编码59G3F12的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.128中的第70位至第99位；
编码59G3F12的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.128中的第145位至第165位；
编码59G3F12的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.128中的第262位至第288位。
编码64B11F2的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.129中的第76位至第96位；
编码64B11F2的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.129中的第154位至第171位；
编码64B11F2的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.129中的第295位至第315位；
编码64B11F2的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.130中的第70位至第102位；
编码64B11F2的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.130中的第148位至第168位；
编码64B11F2的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.130中的第265位至第291位。
所述核酸的制备方法为本领域常规的制备方法，较佳地，包括以下的步骤：通过基因克隆技术获得编码上述蛋白质的核酸分子，或者通过人工全序列合成的方法得到编码上述蛋白质的核酸分子。
本领域技术人员知晓，编码上述蛋白质的氨基酸序列的碱基序列可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该蛋白序列基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。
本发明还提供一种包含所述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。
本发明还提供一种所述EGFRvIII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，从培养物中获得所述EGFRvIII抗体。
本发明还提供一种免疫偶联物，其包括共价附着至细胞毒剂的上述EGFRvIII抗体。
较佳地，所述的免疫偶联物中，上述的1当量的蛋白质通过x当量接头与y当量的 细胞毒剂相连，具有式1的结构，
Ab-(L) x-(D) y
式1
其中，Ab为上述的EGFRvIII抗体；L为接头；D为细胞毒剂；所述x为本领域常规的交联度，x为自然数，优选为1～20；y为大于的自然数，优选为1～20的整数；x和y各自独立地优选为2w，w为1～5的整数、进一步优选为3～4；x和y的比例优选为1:1。
所述L是本领域常规的接头(或称交联剂或偶联剂)。所述L包含2个官能团，即与抗体反应的基团，和与药物反应的基团(例如，醛或酮)。
药物经由接头分子与上述的EGFRvIII抗体偶联。所述L进入细胞后释放，其包括但不限于如下的官能团，活性酯、碳酸盐类、氨基甲酸酯类、亚胺磷酸酯、肟类、腙类、缩醛类、原酸酯类、氨基类、小肽段或核苷酸片段，又例如马来酰亚胺基己酰(maleimidocaproyl，MC)、马来酰亚胺基己酰-L-缬氨酸-L-瓜氨酸对氨基苄醇(MC-VC-PAB)或4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯(SMCC)。
较佳地，所述L主要含有式2的结构，其为L中离去基团离去后对应的剩余部分：
(CO-Alk 1-Sp 1-Ar-Sp 2-Alk 2-C(Z 1)＝Q-Sp)
式2
其中，Alk 1和Alk 2独立地是键或分支的或不分支的(C 1-C 10)亚烷基链；Sp 1是-S-、-O-、-CONH-、-NHCO-、-NR’-、-N(CH 2CH 2) 2N-、或-X-Ar’-Y-(CH 2) n-Z，其中X、Y和Z是独立的键、-NR’-、-S-或-O-，条件是当n＝0时，Y和Z中的至少一个必须是键，且Ar’是由(C 1-C 5)烷基、(C 1-C 4)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-(CH 2) nCOOR’、S(CH 2) nCOOR’、-O(CH 2) nCONHR’或-S(CH 2) nCONHR’的1、2或3个基团任选取代的1，2-、1，3-或1，4-亚苯基，n是0-5的整数，条件是当Alk 1是键时，Sp 1是键；R’是由-OH、(C 1-C 4)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、(C 1-C 3)二烷基氨基、或(C 1-C 3)三烷基铵-A的一个或2个基团任选取代的分支的或不分支的(C 1-C 5)链，其中A是完成盐的药学上可接受的阴离子；Ar是由(C 1-C 6)烷基、(C 1-C 5)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH 2) nCOOR’、-S(CH 2) nCOOR’、-O(CH 2) nCONHR”或-S(CH 2) nCONHR’的1、2或3个基团任选取代的1,2-、1,3-或1,4-亚苯基，其中n和R’如上述的定义，或Ar是1,2-、1,3-、1,4-、1,5-、1,6-、1,7-、1,8-、2,3-、2,6-或2,7-亚萘基，其中亚萘基或吩噻嗪各任选地由(C 1-C 6)烷基、(C 1-C 5)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH 2) nCOOR’、-S(CH 2) nCOOR’、或-S(CH 2) nCONHR’的1、2、3或4个基团取 代，其中n和R’如上文定义，条件是当Ar是吩噻嗪时，Sp 1是仅与氮连接的键；
Sp 2是键、-S-或-O-，条件是当Alk 2是键时，Sp 2是键；
Z 1是H、(C 1-C 5)烷基、或由(C 1-C 5)烷基、(C 1-C 5)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH 2) nCOOR’、-S(CH 2) nCOOR’、-O(CH 2) nCONHR’或-S(CH 2) nCONHR’的1、2、或3个基团任选取代的苯基，其中n和R’如上文定义；
Sp是直链或支链二价或三价(C 1-C 18)基团，二价或三价芳基或杂芳基基团，二价或三价(C 3-C 18)环烷基或杂环烷基基团，二价或三价芳基或杂芳基-芳基(C 1-C 18)基团，二价或三价环烷基或杂环烷基-烷基(C 1-C 18)基团，或二价或三价(C 2-C 18)不饱和的烷基基团，其中杂芳基优选是呋喃基、噻吩基，N-甲基吡咯基、吡啶基、N-甲基咪唑基、噁唑基、嘧啶基。喹啉基、异喹啉基、N-甲基咔唑基、氨基豆素基、或吩嗪基、并且其中如果Sp是三价基团，那么Sp还可以由低级(C 1-C 5)二烷基氨基、低级(C 1-C 5)烷氧基、羟基、或低级(C 1-C 5)烷硫基任选取代；且，Q是＝NHNCO-、＝NHNCS-、＝NHNCONH-、＝NHNCSNH-或＝NHO-。
优选地，Alk 1是分支或不分支的(C 1-C 5)亚烷基链，Sp 1是键、-S-、-O-、-CONH-、-NHCO-或-NR’,其中R’如上文定义，条件是当Alk 1是键时，Sp 1是键；
Ar是由(C 1-C 6)烷基、(C 1-C 5)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH 2) nCOOR’、-S(CH 2) nCOOR’、-O(CH 2) nCONHR’或-S(CH 2) nCONHR’的1、2或3个基团任选取代的1,2-、1,3-或1,4-亚苯基，其中n和R’如上文定义，或Ar是各自由C 1-C 6)烷基、(C 1-C 5)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH 2) nCOOR’、-S(CH 2) nCOOR’、-O(CH 2) nCONHR’或-S(CH 2) nCONHR’的1、2、3或4个基团任选取代的1,2-、1,3-、1,4-、1,5-、1,6-、1,7-、1,8-、2,3-、2,6-或2,7-亚萘基。
Z 1是(C 1-C 5)烷基、或由(C 1-C 5)烷基、(C 1-C 4)烷氧基、(C 1-C 4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH 2) nCOOR’、-S(CH 2) nCOOR’、-O(CH 2) nCONHR’或-S(CH 2) nCONHR’的1、2、或3个基团任选取代的苯基；Alk 2和Sp 2均为键；且Sp和Q如仅在上文中所定义的。上述的键的含义为共价键。
所述L为马来酰亚胺基己酰(maleimidocaproyl，MC)。
所述D可为本领域常规的细胞毒剂，较佳地选自细胞毒素、化学治疗剂、放射性同位素、治疗性核酸、免疫调节剂、抗血管生成剂、抗增殖促凋亡剂或细胞溶解酶。
其中，所述细胞毒素为本领域常规的细胞毒素，一般指抑制或阻止细胞功能和/或导 致细胞破坏的活性剂。较佳地选自抗生素、微管蛋白聚合的抑制剂、烷化剂、蛋白合成抑制剂、蛋白激酶抑制剂、磷酸酶抑制剂、拓扑异构酶抑制剂、蛋白激酶、磷酸酶、拓扑异构酶或细胞周期蛋白。更佳地选自多柔比星、柔红霉素、依达比星、阿柔比星、佐柔比星、米托蒽醌、表柔比星、卡柔比星、诺加霉素、美诺立尔、吡柔比星、戊柔比星、阿糖胞苷、吉西他滨、曲氟尿苷、安西他滨、依诺他滨、阿扎胞苷、去氧氟尿苷、喷司他丁、溴尿苷、卡培他滨、克拉屈滨、地西他滨、氟尿苷、氟达拉滨、谷氏菌素、嘌呤霉素、替加氟、噻唑羧胺核苷、阿霉素、顺铂、卡铂、环磷酰胺、达卡巴嗪、长春碱、长春新碱、博来霉素、氮芥、强的松、甲基苄肼、氨甲喋呤、氟尿嘧啶、依托泊苷、泰素、泰素类似物、铂类(如顺铂和卡铂)、丝裂霉素、噻替派、紫杉烷、道诺红菌素、放线菌素、安曲霉素、氮丝氨酸、它莫西芬、多拉司他汀、奥瑞他汀及其衍生物、哈米特林、埃斯波霉素或美登素类化合物，进一步更佳地选自甲基奥瑞他汀E(MMAE)、甲基奥瑞他汀F(MMAF)或N2’-脱乙酰-N2’-3-巯基-1氧代丙基-美登素(DM1)。最佳地为甲基奥瑞他汀F(MMAF)或者甲基奥瑞他汀E(MMAE)。
其中，所述化学治疗剂为本领域常规的化学治疗剂，较佳地选自烷化剂、烷基磺酸酯类化学治疗剂、氮丙啶类化学治疗剂、乙烯酰胺类和甲基密胺类化学治疗剂、氮芥、硝基脲类化学治疗剂、抗生素、抗代谢物、叶酸类化学治疗剂、嘌呤类似物、嘧啶类似物、雄激素、抗肾上腺素、叶酸补充剂、美登醇、多糖复合物、紫杉烷、铂类似物或类视黄醇，或者，其在药学上可接受的盐、酸和衍生物。
所述的烷化剂为本领域常规的烷化剂，较佳地选自噻替派或环磷酰胺。所述的烷基磺酸酯类化学治疗剂为本领域常规的烷基磺酸酯类化学治疗剂，较佳地选自白消安、英丙舒凡或哌泊舒凡。所述的氮丙啶类化学治疗剂为本领域常规的氮丙啶类化学治疗剂，较佳地选自氮丙唳如、卡巴醌、美妥替哌或乌瑞替派。所述乙烯酰胺类和甲基密胺类化学治疗剂为本领域常规的乙烯酰胺类和甲基密胺类化学治疗剂，较佳地选自六甲蜜胺、三乙撑蜜胺、三亚乙基磷酰胺，三亚乙基硫代磷酰胺或三羟甲蜜胺。所述的氮芥为本领域常规的氮芥，较佳地选自苯丁酸氮芥、萘氮芥、雌氮芥(estramustine)、异环磷酰胺、氮芥、氧氮芥盐酸盐、苯丙氨酸氮芥、新氮芥、苯芥胆甾醇、泼尼氮芥、曲磷胺或尿嘧啶氮芥。所述硝基脲类化学治疗剂为本领域常规的硝基脲类化学治疗剂，较佳地选自卡莫司汀、氯脲菌素、福莫司汀、洛莫司汀、尼莫司汀或雷莫司汀。所述抗生素为本领域常规的抗生素，较佳地选自阿克拉霉素、放线菌素、安曲霉素、氮丝氨酸、博来霉素、放线菌素c、加力车霉素、卡柔比星、洋红霉素、嗜癌素、色霉素、更生霉素、柔红霉素、地托比星、6-重氮基-5-氧代-L-正亮氨酸、多柔比星、表柔比星、依索比星、依达比星、发波 霉素、丝裂霉素、霉酚酸、诺加霉素、橄榄霉素、培洛霉素、紫菜霉素、嘌呤霉素、三铁阿霉素、罗多比星、链黑菌素、链脲菌素、杀结核菌素、乌苯美司、静司他丁或佐柔比星。所述的抗代谢物为本领域常规的抗代谢物，较佳地选自氨甲喋呤或5-氟尿嘧啶(5-FU)。所述的叶酸类化学治疗剂为本领域常规的叶酸类化学治疗剂，较佳地选自二甲叶酸、蝶罗呤或三甲曲沙。所述的嘌呤类似物为本领域常规的嘌呤类似物，较佳地选自氟达拉滨、6-巯嘌呤、硫咪嘌呤或硫鸟嘌呤。所述的嘧啶类似物为本领域常规的嘧啶类似物，较佳地选自安西他滨、阿扎胞苷、6-阿扎尿苷、卡莫氟、阿糖胞苷、二脱氧尿苷、去氧氟尿苷、依诺他滨、氟尿苷或5-EU。所述的雄激素为本领域常规的雄激素，较佳地选自卡普睾酮、丙酸甲雄烷酮、环硫雄醇、美雄烷或睾内酯。所述的抗肾上腺素为本领域常规的抗肾上腺素，较佳地选自安鲁米特、米托坦或曲洛司坦。所述的叶酸补充剂为本领域常规的叶酸补充剂，较佳地选自亚叶酸、醋葡全内酯、醛磷酰胺糖苷、氨基酮戊酸、安吖啶、阿莫司汀、比生群、依达曲沙、地磷酰胺、秋水仙胺、地吖醌、依氟鸟氨酸、依利醋铵、埃坡西龙、依托格鲁、硝酸镓、羟基脲、香菇多糖或氯尼达明。所述的美登醇为本领域常规的美登醇，较佳地选自美登素、安丝菌素、米托胍腙、米托蒽醌、莫哌达醇、二胺硝吖啶、喷司他丁、蛋氨氮芥、吡柔比星、洛索蒽醌、鬼臼酸、2-乙基酰肼或丙卡巴肼。所述的多糖复合物为本领域常规的多糖复合物，较佳地选自雷佐生、根霉素、西佐喃、锗螺胺、细交链孢菌酮酸、三亚胺醌2，2′，2″-三氯三乙胺、单端孢霉烯族毒素、乌拉坦、长春地辛、达卡巴嗪、甘露莫司汀、二溴甘露醇、二溴卫矛醇、哌泊溴烷、gacytosine、阿糖胞苷、环磷酰胺或噻替派。更佳地选自T-2毒素、疣孢菌素A、杆孢菌素A或anguidine。所述紫杉烷为本领域常规的紫杉烷，较佳地选自紫杉醇、无氢化蓖麻油、紫杉醇的白蛋白工程化纳米颗粒制剂(American Pharmaceutical Partners，Schaumberg，Illinois)、多西他赛、苯丁酸氮芥、吉西他滨、6-硫代鸟嘌呤、巯嘌呤或甲氨蝶呤。所述的铂类似物为本领域常规的铂类似物，较佳地选自顺铂、卡铂、长春碱、依托泊苷、异环磷酰胺、米托蒽醌、长春新碱、诺安托、替尼泊苷、依达曲沙、道诺霉素、氨基蝶呤、卡培他滨伊班膦酸盐、CPT-11、拓扑异构酶抑制剂RFS 2000或二氟甲基鸟氨酸。所述的类视黄醇为本领域的类视黄醇，较佳地为视黄酸。
其中，所述放射性同位素为本领域常规的放射性同位素，较佳地，其与上述EGFRvIII抗体直接结合，或者通过螯合剂与上述EGFRvIII抗体结合。更佳地，其与所述EGFRvIII抗体的半胱氨酸残基直接结合。较佳地，所述放射性同位素选自适于放射治疗的α-发射体、β-发射体和俄歇电子以及适于诊断的正电子发射体或γ-发射体。更佳地，所述放射性同位素选自 18氟、 64铜、 65铜。 67镓、 68镓、 77溴、 80m溴、 95钌、 97钌、 103钌、 105钌、 99m 锝、 107汞、 203汞、 123碘、 124碘、 125碘、 126碘、 131碘、 133碘、 111铟、 113铟、 99m铼、 105铼、 101铼、 186铼、 188铼、 121m碲、 99锝、 122m碲、 125m碲、 165铥、 167铥、 168铥、 90钇、 213铋、 213铅或 225锕，或者其衍生的氮化物或氧化物。
其中，所述治疗性核酸为本领域常规的核酸，较佳地为编码免疫调节剂、抗血管生成剂、抗增殖剂或促凋亡剂的基因。所述治疗剂包括所述治疗剂、其衍生物和所述治疗剂在药学上可接受的盐、酸及衍生物。
其中，所述的免疫调节剂为本领域常规的免疫调节剂，即引发免疫应答，包括体液免疫应答(例如抗原特异性抗体的产生)和细胞介导的免疫应答(例如淋巴细胞增殖)的试剂。较佳地选自细胞因子、生长因子、激素、抗激素药、免疫抑制剂或皮质类固醇。所述细胞因子为本领域常规的细胞因子，较佳地选自黄嘌呤、白介素或干扰素。所述的生长因子为本领域常规的生长因子，较佳地选自TNF、CSF、GM-CSF或G-CSF。所述的激素为本领域常规的激素，较佳地选自雌激素、雄激素或孕激素。更佳地，所述的雌激素为已烯雌酚或雌二醇。更佳地，所述的雄激素为睾酮或氟甲睾酮。更佳地，所述的孕激素为乙酸甲地孕酮或乙酸甲羟孕酮。所述的皮质类固醇为本领域常规的皮质类固醇，较佳地选自强的松、地塞米松或化可的松。所述抗激素药为本领域常规的抗激素药，其能阻断激素对肿瘤的作用，抑制细胞因子生产，下调自身抗原表达、或掩蔽MHC抗原的免疫抑制剂。较佳地选自抗雌激素药、抗雄激素药或抗肾上腺素药。更佳地，所述抗雌激素药选自它莫西芬、雷洛昔芬、芳香酶抑制性4(5)-咪唑类、4-羟基它莫西芬、曲沃昔芬或托瑞米芬。所述抗雄激素药选自氟他胺、尼鲁米特、比卡鲁胺、亮丙瑞林或戈舍瑞林。所述免疫抑制剂为本领域常规的免疫抑制剂，较佳地选自2-氨基-6芳基-5取代的嘧啶类、硫唑嘌呤、环磷酰胺、溴隐亭、达那唑、氨苯砜、戊二醛、针对MHC抗原和MHC片段的抗独特型抗体、环孢菌素A、类固醇例如糖皮质类固醇、链激酶、TGFb、雷帕霉素、T细胞受体、T细胞受体片段、细胞因子受体拮抗剂或T细胞受体抗体。更佳地，所述细胞因子受体拮抗剂选自抗干扰素抗体、抗IL10抗体、抗TNFa抗体或抗IL2抗体。
其中，所述的抗血管生成剂为本领域常规的抗血管生成剂，较佳地选自法尼基转移酶抑制剂、COX-2抑制剂、VEGF抑制剂、bFGF抑制剂、类固醇硫酸酯酶抑制剂、白介素-24、凝血栓蛋白、metallospondin蛋白质、I类干扰素、白介素12、鱼精蛋白、血管他丁、层粘连蛋白、内皮他丁或催乳激素片段。更佳地为2-甲氧基雌二醇二氨基磺酸酯(2-MeOE2bisMATE)。
其中，所述抗增殖促凋亡剂为本领域常规的抗增殖促凋亡剂，较佳地选自PPAR-γ激活剂、类视黄醇、三萜类化合物、EGF受体抑制剂、端粒末端转移酶抑制剂、铁螯合 剂、凋亡蛋白、Bcl-2和Bcl-X(L)的抑制剂、TNF-α/FAS配体/TNF相关的凋亡诱导配体及其信号传导的激活物或PI3K-Akt存活途径信号抑制剂。所述PPAR-γ激活剂为本领域常规的PPAR-γ激活剂，较佳地为环戊烯酮前列腺素(cyPGs)。所述三萜类化合物为本领域常规的三萜类化合物，较佳地选自环菠萝蜜烷、羽扇豆烷、乌苏烷、齐敦果烷、木栓烷、达玛烷、葫芦素、柠檬苦素类似物或三萜类化合物。所述EGF受体抑制剂为本领域常规的EGF受体抑制剂，较佳地选自HER4、雷帕霉素或1，25-二羟基胆钙化醇(维生素D)。所述的铁螯合物为本领域常规的铁螯合物，较佳地为3-氨基吡啶-2-甲醛硫代缩氨基脲。所述的凋亡蛋白为本领域常规的凋亡蛋白，较佳地为鸡贫血病病毒的病毒蛋白质3-VP3。所述PI3K-Akt存活途径信号抑制剂为本领域常规的PI3K-Akt存活途径信号抑制剂，较佳地为UCN-01或格尔德霉素。
其中，所述细胞溶解酶为本领域常规的细胞溶解酶，较佳地为RNA酶。
本发明优选地，式1中x＝y＝n；所述D为微管蛋白合成酶抑制剂——甲基奥瑞他汀F，(MMAF)，且所述接头L为马来酰亚胺基己酰(maleimidocaproyl，MC)，所述免疫偶联物的结构如式3-1或3-2所示，
Figure PCTCN2018120959-appb-000006
其中，mAb为上述的EGFRvIII抗体。其中，n为自然数，优选为1～20的整数，更优选为2w，w为1～5的整数、进一步优选为3～4；
Figure PCTCN2018120959-appb-000007
式3-2中m为1～10，优选m为5，L为马来酰亚胺基己酰-L-缬氨酸-L-瓜氨酸对氨基苄醇；D为甲基奥瑞他汀E(MMAE)；其中，n为自然数，优选为1～20的整数，更优选为2w，w为1～5的整数、进一步优选为3～4。
所述的免疫偶联物的制备方法为本领域常规，较佳地采用Doronina，2006，Bioconjugate Chem.17,114-124所记载的制备方法。较佳地，所述的制备方法产生具有最低限度的低偶联级分(LCF)小于10％的免疫偶联物。
在本发明一较佳实施例中，所述的制备方法包括以下的步骤：将上述EGFRvIII抗体经过pH 6.5～8.5的硼酸钠缓冲液透析后，加入三(2-羧乙基)膦(TCEP)，其中TCEP与上述EGFRvIII抗体的摩尔比比率为2～10，室温下还原2～4小时，经过G25脱盐填料去除多余的TCEP，加入一定比例的MC-MMAF(药物/抗体比率为5-20)反应4小时。再加入半胱氨酸用以中和多余的药物，并通过G25除去多余的小分子。得到纯化的抗体药物偶联物(偶联方法参见Doronina，2006，Bioconjugate Chem.17,114-124)。
所述的免疫偶联物能够以本领域所知的任何物理形态而存在，较佳地为澄清溶液。
本发明还提供一种药物组合物，其包括上述的EGFRvIII抗体或者免疫偶联物，以及药学可接受的载体。
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EGFRvIII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、阴道内、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EGFRvIII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抗体、上述偶联物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EGFRvIII蛋白的细胞的方法，包括如下的步骤：将上述的EGFRvIII抗体与待检样品在体外接触，检测所述的EGFRvIII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EGFRvIII蛋白的细胞的组合物，其包括上述的EGFRvIII抗体作为活性成分。
本发明还提供一种上述抗体、免疫偶联物或者药物组合物在制备预防或治疗与EGFRvIII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EGFRvIII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、前列腺癌或者肾癌。
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：本发明所述的蛋白质是一种EGFRvIII抗体，其与EGFRvIII蛋白具有高度亲和力，能够在蛋白水平和细胞水平结合EGFRvIII蛋白受体的 胞外区。所述的EGFRvIII抗体与MC-MMAF等小分子化合物偶联后得到的抗体交联药，能够有效的对EGFRvIII阳性细胞进行细胞毒杀伤作用。通过EGFRvIII抗体把小分子毒素，如MMAF，通过内吞作用带入细胞，并在细胞内降解释放小分子化合物，从而起到细胞毒的作用。因此所述的EGFRvIII抗体能够用于抗体交联药的制备、有效杀伤肿瘤细胞或，能够运用于治疗肿瘤的药物的制备中。
附图说明
图1为人EGFRvIII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CHO-K1细胞FACS筛选检测结果。
图3为人EGFRvIII蛋白转染的U87MG细胞FACS筛选检测结果。
图4为人EGFRvIII蛋白转染的293F细胞FACS筛选检测结果。
图5A和5B为ELISA检测EGFRvIII蛋白免疫后小鼠血清抗体效价情况。
图6为ELISA检测EGFRvIII抗体与人EGFRvIII-hFc蛋白的结合反应。
图7A为FACS检测EGFRvIII抗体与U87MG-hEGFRvIII的结合反应；图7B为FACS检测EGFRvIII抗体与A431的结合反应；图7C为FACS检测EGFRvIII抗体与U87MG的结合反应；图7D为FACS检测EGFRvIII抗体与HEB的结合反应。
图8A为EGFRvIII抗体-MMAF抗体偶联药对U87MG-EGFRvIII的细胞杀伤作用；图8B为EGFRvIII抗体-MMAF抗体偶联药对A431的细胞杀伤作用；图8C为EGFRvIII抗体-MMAF抗体偶联药对U87MG的细胞杀伤作用；图8D为EGFRvIII抗体-MMAF抗体偶联药对HEB的细胞杀伤作用。
图9A为FACS检测EGFRvIII抗体与人类正常组织HFF-1细胞系的结合反应；图9B为FACS检测EGFRvIII抗体与人类正常组织HFL-I细胞系的结合反应；图9C为FACS检测EGFRvIII抗体与人类正常组织QSG-7701细胞系的结合反应；图9D为FACS检测EGFRvIII抗体与人类正常组织HEEC细胞系的结合反应；图9E为FACS检测EGFRvIII抗体与人类正常组织HEB细胞系的结合反应；图9F为FACS检测EGFRvIII抗体与人类正常组织WPMY-1细胞系的结合反应；图9G为FACS检测EGFRvIII抗体与人类正常组织MCF-10A细胞系的结合反应。
图10A为EGFR抗体(鼠抗80E11)对人类脑胶质瘤组织芯片免疫组化染色结果；图10B为EGFR抗体(鼠抗80E11)对人类正常组织芯片免疫组化染色结果；图10C为EGFRvIII抗体(鼠抗63A10)对人类脑胶质瘤组织芯片免疫组化染色结果；图10D为EGFRvIII抗体(鼠抗63A10)对人类正常组织芯片免疫组化染色结果；图10E为EGFRvIII 抗体(鼠抗75G7)对人类脑胶质瘤组织芯片免疫组化染色结果；图10F为EGFRvIII抗体(鼠抗75G7)对人类正常组织芯片免疫组化染色结果。
图11为酶联免疫吸附实验(ELISA)检测嵌合抗体与EGFRvIII蛋白的结合。
图12A为FACS检测EGFRvIII嵌合抗体与U87MG-hEGFRvIII的结合反应；图12B为FACS检测EGFRvIII嵌合抗体与A431的结合反应；图12C为FACS检测EGFRvIII嵌合抗体与U87MG的结合反应；图12D为FACS检测EGFRvIII嵌合抗体与HEB的结合反应。
图13A为EGFRvIII抗体-MMAF抗体偶联药对U87MG-EGFRvIII的细胞杀伤作用；图13B为EGFRvIII抗体-MMAF抗体偶联药对A431的细胞杀伤作用；图13C为EGFRvIII抗体-MMAF抗体偶联药对U87MG的细胞杀伤作用；图13D为EGFRvIII抗体-MMAF抗体偶联药对HEB的细胞杀伤作用。
图14A为U87MG-EGFRvIII异种移植荷瘤小鼠肿瘤体积变化；图14B为U87MG-EGFRvIII异种移植荷瘤小鼠体重变化。
图15为75G7C6抗体重链可变区CDR2的NG突变后的抗体的ELISA结合活性鉴定。
图16为75G7C6抗体重链可变区CDR3的NG突变后的抗体的ELISA结合活性鉴定。
图17A为FACS检测75G7C6抗体重链可变区CDR2的NG突变后的抗体与CHOK-EGFRvIII细胞结合活性；图17B为FACS检测75G7C6抗体重链可变区CDR2的NG突变后的抗体与CHOK-EGFR细胞结合活性。
图18A为FACS检测75G7C6抗体重链可变区CDR3的DG突变成SG、EG或DA后的突变抗体与CHOK-EGFRvIII细胞结合活性；图18B为FACS检测75G7C6抗体重链可变区CDR3的DG突变成SG、EG或DA后的突变抗体与CHOK-EGFR细胞结合活性。
图19为人源化抗EGFRvIII抗体h75G7C6重链可变区h75G7C6.VH及其变体与嵌合抗体c75G7C6.VH及人种系VH外显子IGHV1-46*01/JH6b的序列比较，方框处为CDR。
图20为人源化抗EGFRvIII抗体h75G7C6轻链可变区h75G7C6.VL及其变体与嵌合抗体c75G7C6.VL及人种系VL外显子IGKV3-11*01/JK4的序列比较，方框处为CDR。
图21为人源化抗EGFRvIII抗体h63A10A7重链可变区h63A10A7.VH及其变体与嵌合抗体c63A10A7.VH及人种系VH外显子IGHV1-46*01/JH4的序列比较，方框处为CDR。
图22为人源化抗EGFRvIII抗体h63A10A7轻链可变区h63A10A7.VL及其变体与嵌合抗体c63A10A7.VL及人种系VL外显子IGKV1-39*01/JK2的序列比较，方框处为CDR。
图23A-C为ELISA检测人源化抗体h75G7C6变体与人EGFRvIII-hFc蛋白的结合反应。
图24A-E为ELISA检测人源化抗体h63A10A7变体与人EGFRvIII-hFc蛋白的结合反应。
图25A-C为FACS检测人源化抗体h75G7C6变体与U87MG-EGFRvIII细胞的结合反应。
图26A-E为FACS检测人源化抗体h63A10A7变体与U87MG-EGFRvIII细胞的结合反应。
图27为FACS检测人源化h75G7C6变体与表面表达EGFR的肿瘤细胞A431和正常人原代肝细胞的结合。
图28A-B为FACS检测人源化h63A10A7变体与表面表达EGFR的肿瘤细胞A431和正常人原代肝细胞的结合。
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。
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。
实施例中所述的室温为本领域常规的室温，一般为10～30℃。
实施例1 EGFRvIII抗体的制备
(一)、免疫原A的制备
将含有编码人源EGFRvIII蛋白胞外区氨基酸序列(NCBI：NP_005219.2，缺失30-297位氨基酸)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EGFRvIII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EGFRvIII蛋白(EGFRvIII-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EGFRvIII全长氨基酸序列(NCBI：NP_005219.2，缺失30-297位氨基酸)和编码人源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、U87MG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EGFRvIII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3和图1所示，IgG亚型对照为小鼠IgG对照。表3说明，已经制得一系列EGFRvIII阳性表达的CHO-K1，U87MG以及293F细胞系，以及EGFR阳性表达的CHO-K1细胞系。图1～4中，横坐标为细胞荧光强度，纵坐标为细胞数。图1，2，3，4的结果说明，CHO-K1-hEGFRvIII 1G10、CHO-K1-hEGFR 3G2、U87MG-hEGFRvIII 2G2以及293F-hEGFRvIII 1C10为EGFRvIII高水平表达细胞株；CHO-K1-hEGFR 3G2为EGFR高水平表达细胞株。EGFR抗体也是通过相同的免疫途径得到，主要是为了作为对照，证明EGFRvIII抗体相比较EGFR抗体具有更好的组织特异性。
表3人源EGFRvIII蛋白的CHO-K1稳转细胞系FACS检测结果
Figure PCTCN2018120959-appb-000008
Figure PCTCN2018120959-appb-000009
表4人源EGFR蛋白的CHO-K1稳转细胞系FACS检测结果
Figure PCTCN2018120959-appb-000010
表5人源EGFRvIII蛋白的HEK293F稳转细胞系FACS检测结果
Figure PCTCN2018120959-appb-000011
表6人源EGFRvIII蛋白的U87MG稳转细胞系FACS检测结果
Figure PCTCN2018120959-appb-000012
(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5和表7-8所示。表7-8说明，经人源EGFRvIII-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7 ELISA检测EGFRvIII蛋白免疫后Balb/c小鼠血清抗体效价
Figure PCTCN2018120959-appb-000013
表8 ELISA检测EGFRvIII蛋白免疫后SJL小鼠血清抗体效价
Figure PCTCN2018120959-appb-000014
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EGFRvIII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EGFRvIII的HEK293稳定细胞系293F-hEGFRvIII 1C10(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(针对免疫原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EGFRvIII蛋白和EGFRvIII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EGFRvIII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EGFRvIII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EGFRvIII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EGFRvIII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EGFRvIII抗体。
将纯化的EGFRvIII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9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9纯化的EGFRvIII抗体质量控制
抗体名称 克隆号 抗体纯度 蛋白浓度(毫克/毫升) 内毒素(EU/毫克)
鼠抗75G7 75G7C6 >95％ 0.44 <1.0
鼠抗63A10 63A10A7 >95％ 1.26 <1.0
鼠抗64F1 64F1F8 >95％ 1.52 <1.0
鼠抗7E5 7E5-2A9 >95％ 0.81 <1.0
鼠抗43H6 43H6A1 >95％ 1.08 <1.0
鼠抗46C4 46C4A6 >95％ 1.43 <1.0
鼠抗49G12 49G12G5 >95％ 1.16 <1.0
鼠抗51G7 51G7C2 >95％ 1.07 <1.0
鼠抗53D8 53D8H4 >95％ 0.93 <1.0
鼠抗54D2 54D2G10 >95％ 1.29 <1.0
鼠抗56F10 56F10G2 >95％ 0.92 <1.0
鼠抗59G3 59G3F12 >95％ 1.29 <1.0
鼠抗64B11 64B11F2 >95％ 1.11 <1.0
实施例3先导抗体的检定
A、酶联免疫吸附实验(ELISA)检测抗体与EGFRvIII蛋白的结合
对实施例2所得的纯化的EGFRvIII抗体进行与人EGFRvIII-hFc蛋白进行反应。
将实施例1获得的纯化的免疫原A(EGFRvIII-hFc)(其制备方法参见实施例1步骤(一)用PBS稀释到终浓度1.0μg/mL，然后以100μl每孔加到96孔ELISA板。用塑料膜封好4℃孵育过夜，第二天用洗板液[PBS+0.01％(v/v)Tween20]洗板2次，加入封闭液[PBS+0.01％(v/v)Tween20+1％(w/w)BSA]室温封闭2小时。倒掉封闭液，加入实施例2所得的纯化的EGFRvIII抗体100μl每孔。37℃孵育2小时后，用洗板液[PBS+0.01％(v/v)Tween20]洗板3次。加入HRP(辣根过氧化物酶)标记的二抗(购自Sigma)，37℃孵育2小时后，用洗板液[PBS+0.01％(v/v)Tween20]洗板3次。加入TMB底物100μl每孔，室温孵育30分钟后，加入终止液(1.0N HCl)100μl每孔。用ELISA读板机(SpectraMax 384plus，购自Molecular Device)读取A450nm数值，结果如图6和表10所示，表10说明，纯化后的抗体与EGFRvIII重组蛋白在ELISA水平结合。其中IgG对照为小鼠IgG，表中的数据为OD 450nm值。
表10 ELISA检测EGFRvIII抗体与人EGFRvIII-hFc蛋白的结合反应
Figure PCTCN2018120959-appb-000015
Figure PCTCN2018120959-appb-000016
B、流式细胞实验(FACS)检测抗体与EGFRvIII表达细胞的结合
将所需细胞在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用HBSS缓冲液(Hanks Balanced Salt Solution)(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HBSS缓冲液洗涤细胞2次，进行细胞计数后将细胞用HBSS缓冲液稀释至2╳10 6细胞每毫升，加入1％山羊血清封闭液，所述百分比为质量百分比，冰上孵育30分钟，然后用HBSS缓冲液离心洗涤2次。将收集的细胞用FACS缓冲液(HBSS+1％BSA，所述百分比为质量百分比)悬浮至2╳10 6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EGFRvIII抗体待测样品每孔100微升，冰上孵育2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)，冰上孵育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。通过软件(CellQuest)进行数据分析，的到细胞的平均荧光密度(MFI)。再通过软件(GraphPad Prism5)分析，进行数据拟合，计算EC50。分析结果如表11以及图7所示，待测抗体均可结合U87MG-hEGFRvIII细胞表面的EGFRvIII蛋白(图7A)。A431细胞(人表皮细胞癌细胞系)表面有大量野生型EGFR蛋白的表达，从图7B来看，鼠抗75G7，63A10和64F1可以与A431细胞表面的野生型EGFR蛋白弱结合，而鼠抗7E5(抗体克隆号7E5-2A9)不识别野生型EGFR蛋白，只能特异性识别突变型EGFRvIII蛋白。U87MG细胞(人脑胶质瘤细胞系)表面有少量野生型EGFR蛋白的表达，而HEB细胞(人脑胶质细胞系)表面有较高水平的野生型EGFR蛋白的表达。待测抗体与U87MG和HEB细胞均无结合反应，说明鼠抗75G7，63A10和64F1具有较好的特异性，只识别肿瘤组织中的突变型EGFRvIII蛋白以及超表达的野生型EGFR蛋白，不识别正常组织中的野生型EGFR蛋白(图7C、D)。
表11 FACS检测EGFRvIII抗体与U87MG-hEGFRvIII、A431、U87MG和HEB细胞的结合反应
Figure PCTCN2018120959-appb-000017
Figure PCTCN2018120959-appb-000018
实施例4EGFRvIII抗体药物偶联物的细胞杀伤活性实验
抗体经过Ph6.5-8.5的硼酸钠缓冲液透析后，加入一定比例的TCEP还原(TCEP/抗体比率为2-10)，室温或37度进行还原2-4小时，经过G25脱盐填料去除多余的TCEP，加入一定比例的MC-MMAF(药物/抗体比率为5-20)反应4小时。再加入半胱氨酸用以中和多余的药物，并通过G25除去多余的小分子。得到纯化的抗体药物偶联物。(偶联方法参见Doronina，2006，Bioconjugate Chem.17,114-124)。通过HIC分析药物的交联率，纯度等参数后，进行细胞毒活性的分析。为了便于比较，所有抗体偶联物的药物交联率为8。
得到的抗体偶联物，分别用完全培养基进行梯度稀释，96孔细胞培养板以1000细胞/孔加入90微升U87MG-EGFRvIII细胞悬液，每孔再分别加入10微升不同浓度的抗体药物偶联物的稀释液，继续培养5天后，用CellTiter-Glo试剂盒(购自Promega，使用方法参照产品说明书)检测细胞活力。
结果如表12以及图8所示，其中表13-1的IC50指药物作用后，细胞的活性受到抑制的半数有效量，能够通过检测细胞的活性从而反映细胞杀伤活性。其中，图8为抗体药物偶联物对EGFRvIII阳性的重组肿瘤细胞系U87MG-EGFRvIII的细胞杀伤活性检测，结果说明，在体外实验中，待测抗体均可对表面有突变型EGFRvIII蛋白表达的U87MG-hEGFRvIII细胞产生良好的杀伤作用(图8A)。A431细胞(人表皮细胞癌细胞系)表面 有大量野生型EGFR蛋白的表达，从图8B来看，鼠抗75G7，63A10和64F1对A431有较弱的杀伤作用。U87MG细胞(人脑胶质瘤细胞系)表面有少量野生型EGFR蛋白的表达，而HEB细胞(人脑胶质细胞系)表面有较高水平的野生型EGFR蛋白的表达。鼠抗75G7，63A10和64F1对U87MG和HEB细胞杀伤作用很弱或者没有杀伤作用，见图8C、图8D。说明鼠抗75G7，63A10和64F1具有较好的特异性，可以特异性杀伤表达突变型EGFRvIII蛋白以及超表达野生型EGFR蛋白的肿瘤组织，而对表达野生型EGFR蛋白的正常组织没有杀伤作用。
表12细胞杀伤实验检测抗体偶联物对EGFRvIII阳性细胞的特异性杀伤作用
Figure PCTCN2018120959-appb-000019
实施例5 EGFRvIII抗体的抗原表位分析
A、竞争性ELISA法分析抗原表位(Epitope Binning)
为了鉴定抗体对抗原的结合位点，采用竞争ELISA的方法对EGFRvIII抗体进行分组。
纯化的待测抗体用PBS稀释至1μg/mL，以50μL/孔包被96孔高吸附酶标板，4℃过夜包被后用250微升封闭液[含有0.01％(v/v)Tween20和1％(w/w)BSA的PBS]进行室温一小时封闭，每孔加入0.05μg/mL的生物素标记的重组EGFRvIII蛋白。同时加入5μg/mL的竞争抗体，即实施例2所得的纯化的EGFRvIII抗体，并于25-37℃孵育1～2小时。用洗板液[含有0.01％(v/v)Tween20的PBS]洗板3次，加入HRP(辣根过氧化物 酶)标记的链亲和素(购自Sigma)。37℃孵育0.5小时后，用洗板液[含有0.01％(v/v)Tween20的PBS]洗板3次。加入TMB底物100μL每孔，室温孵育30分钟后，加入终止液(1.0N HCl)100μL每孔。用ELISA读板机(SpectraMax 384plus，购自Molecular Device)读取A450nm数值，结果如图6所示。根据A450nm数值，计算出抗体相互之间的竞争率，结果如表13-1所示。竞争率的数值越高，表示两个抗体的抗原表面越是接近。
表13-1竞争性ELISA法分析抗原表位(Epitope Binning)
Figure PCTCN2018120959-appb-000020
竞争性ELISA的结果显示，对照抗体02和鼠抗7E5，63A10和64F1的抗原表位具有竞争性，应该是相同或者相似表位。而鼠抗75G7与对照抗体02没有竞争性，因此鼠抗75G7的抗原表位应该与对照抗体02不同；其中对照抗体01是AMG-595(购自Amgen)，对照抗体02是ABT-414(购自Abbvie)。
B、多肽点阵ELISA法分析抗原表位(Epitope Mapping)
为了确定本发明的4个候选抗体特定的抗体结合表位，我们使用多肽点阵ELISA法来分析抗体的抗原结合表位。以EGFRvIII蛋白的1～50位氨基酸为模板，从N末端开始，以15个氨基酸重叠的方法进行移动，合成16个氨基酸长度的多肽。多肽C末端添加生物素标记。多肽合成由吉尔生化完成，共合成生物素标记多肽35条，详细序列见表13-2。
表13-2多肽点阵ELISA法分析抗原表位
Figure PCTCN2018120959-appb-000021
Figure PCTCN2018120959-appb-000022
多肽点阵ELISA的结果显示，对照抗体01和鼠抗7E5，鼠抗64F1，鼠抗63A10具有相同或者相似的抗原表位，即CGADSYEMEEDGVRKC。而鼠抗75G7与这35条多肽 均不结合，因此鼠抗75G7的抗原表位应该与对照抗体01和对照抗体02均不同。其中对照抗体01是AMG-595(购自Amgen)，对照抗体02是ABT-414(购自Abbvie)。因此鼠抗75G7的抗原表位有待进一步的实验验证。
实施例6EGFRvIII抗体的结合特异性分析
所述EGFRvIII抗体能够特异性识别激活状态的EGFR，即肿瘤组织中的突变体EGFRvIII以及过度表达的野生型EGFR，但是不识别正常组织中的野生型EGFR。因此该抗体具有优秀的靶点特异性，可以有效避免抗体及其偶联物对人体正常组织的伤害，使抗体及其偶联物有效的聚集在肿瘤位置，从而精准地实现对肿瘤组织的有效杀伤以及大幅度降低药物对其他正常器官的毒副作用。
A、FACS检测EGFRvIII抗体对人类正常组织细胞系结合的特异性
选取了7个人类正常组织的细胞系，分别来自不同的组织，详细信息见表14。FACS检测分析了EGFRvIII抗体对这7个人类正常组织的细胞系的结合情况，结果如图9A～G所示，4个EGFRvIII嵌合抗体与人类正常组织的细胞系均无结合，仅嵌合抗体75G7与HEB和WPMY-1有较弱结合。说明这4个EGFRvIII嵌合抗体均具有良好的组织特异性，不识别正常组织中的野生型EGFR。
表14来源于7种不同人类正常组织的细胞系
细胞系名称 组织来源
HFF-1 皮肤
HFL-I 肺
QSG-7701 肝
HEEC 食道
HEB 脑
WPMY-1 前列腺
MCF-10A 乳腺
B、免疫组化染色(IHC)检测EGFRvIII抗体的结合特异性
人类脑胶质瘤组织芯片(货号：GL805d)和人类多器官正常组织芯片(货号：FDA999n)均购于西安艾丽娜生物科技有限公司。
免疫组化染色的实验流程如下：
1)烤片，60℃，2小时。烤片的目的是将带有蜡的组织切片牢固地黏在载玻片上，以免染色过程中切片脱落。切片在56～60℃的恒温箱中至少放置1小时才能达到此目的，但是，通常的烤片温度为；8～60℃，时间为2～6小时。由于高温干燥可加速组织中抗原的氧化，因此高温烤片对抗原有破坏作用。
2)脱蜡和水化。分别在二甲苯Ⅰ、Ⅱ中浸泡15～30分钟，以脱掉组织中的蜡。但是，进人组织中的二甲苯不能与水溶性染色液相溶，故需进一步通过下行梯度乙醇把组织中的二甲苯逐步替代出来。切片在100％乙醇Ⅰ、Ⅱ中分别浸泡5分钟，95％、90％、80％和70％乙醇中分别浸泡2分钟，PBS漂洗3次，每次3分钟，置蒸馏水中待用。
3)抗原修复，将组织芯片放入pH9.0的EDTA溶液中，高温修复5分钟。
4)封闭内源性过氧化物酶，将组织芯片放入3％H 2O 2中，室温放置5分钟。
5)非免疫血清封闭，将组织芯片放入5％FBS中，室温放置15分钟。抗体能被组织切片中富有电荷的胶原和结缔组织成分吸附。从而导致背景着色，为了防止这种现象发生，最好在特异性抗体处理切片之前，选择与二抗种属相同的非免疫血清封闭电荷，阻止一抗与之结合，抑制非特异性背景着色。常用方法是用2％～10％羊血清或2％～5％牛血清白蛋白在室温下作用10～30分钟。
6)第一抗体孵育，室温放置1小时。
7)第二抗体孵育，室温放置0.5小时。
8)DAB显色，室温放置5分钟。
9)苏木精复染细胞核，室温放置10秒。
10)封片检测。
免疫组化染色的结果见图10A～F。鼠抗80E11是针对野生型EGFR的抗体，从图10A～F来看，野生型EGFR在人类脑胶质瘤组织和人类多器官正常组织中均有表达，且在肿瘤组织中的表达量高于正常组织。如表15所示，鼠抗7E5、鼠抗64F1与鼠抗63A10在人类脑胶质瘤组织芯片中的阳性率为34％，与人类多器官正常组织芯片反应基本为阴性，只有少数样本呈弱阳性。鼠抗75G7与人类脑胶质瘤组织芯片和人类多器官正常组织芯片反应均为阴性。因为鼠抗75G7的抗原结合表位与其他抗体不同，因此可能是因为芯片处理过程中破坏了鼠抗75G7的抗原结合表位。本次实验中所用组织芯片均为石蜡包埋，可能这种方法不适用于检测鼠抗75G7抗体。后续我们会尝试冷冻切片IHC。
表15 EGFR抗体和EGFRvIII抗体对人类脑胶质瘤组织芯片的反应率
Figure PCTCN2018120959-appb-000023
通过FACS和IHC实验证明了所述EGFRvIII抗体能够特异性识别肿瘤组织中的EGFR，但是不识别正常组织中的EGFR。因此该抗体具有优秀的靶点特异性，可以有效避免抗体及其偶联物对人体正常组织的伤害，使抗体及其偶联物有效的聚集在肿瘤位置，从而精准地实现对肿瘤组织的有效杀伤以及大幅度降低药物对其他正常器官的毒副作用。
实施例7轻重链可变区氨基酸序列测定
总RNA分离：通过离心搜集实施例1所得的杂交瘤细胞5×10 7个，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10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％(v/v)乙醇，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H 2O溶解(55℃水浴促进溶解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50μL PCR体系，包括1μL cDNA、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，95℃预变性3分钟，95℃变性30秒，55℃退火30秒，72℃延伸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 PCTCN2018120959-appb-000024
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16～17所示。
表16 EGFRvIII抗体蛋白序列编号
Figure PCTCN2018120959-appb-000025
其中，表17中的数字即为序列表中序列号，如43H6A1的重链蛋白可变区的氨基酸序列为SEQ ID No.1，而43H6A1的重链蛋白可变区中CDR1的氨基酸序列为SEQ ID No.2。
表17 EGFRvIII抗体基因序列编号
克隆号 重链蛋白可变区 轻链蛋白可变区
75G7C6 105 106
63A10A7 107 108
64F1F8 109 110
7E5-2A9 111 112
43H6A1 113 114
46C4A6 115 116
49G12G5 117 118
51G7C2 119 120
53D8H4 121 122
54D2G10 123 124
56F10G2 125 126
59G3F12 127 128
64B11F2 129 130
其中，表18中的数字即为序列表中序列号，如编码75G7C6的重链蛋白可变区的核苷酸序列为SEQ ID No.105。
实施例8EGFRvIII人鼠嵌合抗体的生产和纯化
1)质粒构建与准备：将上述鼠源先导抗体的重链可变区序列重组到包含信号肽和人源重链抗体IgG1恒定区的表达载体(其中表达载体购买自Invitrogen，重组步骤也由上海睿智化学完成)中，将EGFRvIII抗体的轻链可变区序列重组到包含信号肽和人源抗体轻链kappa恒定区的表达载体(其中表达载体购买自Invitrogen，重组步骤也由上海睿智化学完成)当中，得重组质粒(上述质粒重组的实验原理及步骤的出处见《分子克隆实验指南(第三版)》，(美)J.萨姆布鲁克等著)并经测序验证。使用碱裂解法试剂盒(购自MACHEREY-NAGEL)中量抽提高纯度的重组质粒，质量为500μg以上，经0.22μm滤膜(购自Millopore)过滤，供转染使用。
2)细胞转染：在培养基Freestyle 293 expression medium(购自Invitrogen)培养293E细胞(购自Invitrogen)。摇床设置为37℃、130RPM和8％CO 2(v/v)。Freestyle 293 expression medium在转染时添加10％(v/v)F68(购自Invitrogen)至F68终浓度为0.1％(v/v)，得含0.1％(v/v)F68的Freestyle 293表达培养基，即培养基A。取5mL培养基A和200μg/mL PEI(购自Sigma)混匀，得培养基B。取5mL培养基A和100μg步骤1)所得的重组质粒(此处为上述重链重组质粒和轻链重组质粒按常规等比例混合的混合重组质粒)混匀，得培养基C。5分钟后将培养基B和培养基C合并混匀，静置15分钟，得混合液D。将10mL混合液D缓缓加入100mL含293E细胞的培养基Freestyle 293 expression medium中至293E的细胞密度为1.5×10 6个/mL，边加边振荡，避免PEI过度集中，放入摇床培养。第二天加入蛋白胨至终浓度为0.5％(w/v)。第5～7天，测培养液抗体效价。第6～7天，离心(3500RPM，30分钟)收集上清，经0.22μm滤膜过滤，得滤好的细胞上清液，以供纯化。
3)抗体纯化：对于连续生产的无内毒素的层析柱和蛋白A填料，使用0.1M NaOH处理30min或者5个柱体积0.5M NaOH冲洗；对于长期未使用的柱料和层析柱至少使用1M NaOH浸泡1h，用无内毒的水冲洗至中性，用10倍柱体积的1％Triton X100对柱料清洗。使用5个柱体积的PBS进行平衡，将过滤好的细胞上清上柱，必要时收集流穿液。上柱完成后，使用5倍柱体积PBS清洗。用5倍柱体积的0.1M pH3.0的Glycine-HCl进行洗脱，收集洗脱液，并用1/10体积的pH8.5的1M Tris-HCl(1.5M NaCl)中和。收获抗体后，在1×PBS中透析过夜，避免内毒素污染。透析结束后，使用分光光度或试剂盒测定浓度，使用HPLC-SEC测定抗体纯度，使用内毒素检测试剂盒(购自Lonza)检 测抗体内毒素含量。
分别获得纯化的EGFRvIII嵌合抗体75G7,63A10,64F1和7E5。
将纯化的EGFRvIII嵌合抗体进行蛋白浓度(A280/1.4)、纯度、内毒(Lonza试剂盒)等检测分析，结果如表18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18纯化的EGFRvIII嵌合抗体质量控制
Figure PCTCN2018120959-appb-000026
实施例9EGFRvIII嵌合抗体的检定
A、酶联免疫吸附实验(ELISA)检测抗体与EGFRvIII蛋白的结合
对实施例2所得的纯化的EGFRvIII嵌合抗体进行与人EGFRvIII-hFc蛋白进行反应。
将实施例1获得的纯化的免疫原A(EGFRvIII-hFc)(其制备方法参见实施例1步骤(一)用PBS稀释到终浓度1.0μg/mL，然后以100μl每孔加到96孔ELISA板。用塑料膜封好4℃孵育过夜，第二天用洗板液[PBS+0.01％(v/v)Tween20]洗板2次，加入封闭液[PBS+0.01％(v/v)Tween20+1％(w/w)BSA]室温封闭2小时。倒掉封闭液，加入实施例2所得的纯化的EGFRvIII嵌合抗体100μl每孔。37℃孵育2小时后，用洗板液[PBS+0.01％(v/v)Tween20]洗板3次。加入HRP(辣根过氧化物酶)标记的二抗(购自Sigma)，37℃孵育2小时后，用洗板液[PBS+0.01％(v/v)Tween20]洗板3次。加入TMB底物100μl每孔，室温孵育30分钟后，加入终止液(1.0N HCl)100μl每孔。用ELISA读板机(SpectraMax 384plus，购自Molecular Device)读取A450nm数值，结果如图11和表19所示，表19说明，纯化后的抗体与EGFRvIII重组蛋白在ELISA水平结合。其中IgG对照为小鼠IgG，表中的数据为OD 450nm值。
表19 ELISA检测EGFRvIII嵌合抗体与人EGFRvIII-hFc蛋白的结合反应
Figure PCTCN2018120959-appb-000027
B、流式细胞实验(FACS)检测抗体与EGFRvIII表达细胞的结合
将所需细胞在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用HBSS缓冲液(Hanks Balanced Salt Solution)(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HBSS缓冲液洗涤细胞2次，进行细胞计数后将细胞用HBSS缓冲液稀释至2╳10 6细胞每毫升，加入1％山羊血清封闭液，所述百分比为质量百分比，冰上孵育30分钟，然后用HBSS缓冲液离心洗涤2次。将收集的细胞用FACS缓冲液(HBSS+1％BSA，所述百分比为质量百分比)悬浮至2╳10 6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EGFRvIII抗体待测样品每孔100微升，冰上孵育2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)，冰上孵育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。通过软件(CellQuest)进行数据分析，的到细胞的平均荧光密度(MFI)。再通过软件(GraphPad Prism5)分析，进行数据拟合，计算EC50。分析结果如表20以及图12A～D所示，待测嵌合抗体均可结合U87MG-hEGFRvIII细胞表面的EGFRvIII蛋白(图12A)。A431细胞(人表皮细胞癌细胞系)表面有大量野生型EGFR蛋白的表达，从图12B来看，嵌合抗体75G7、嵌合抗体63A10和嵌合抗体64F1可以与A431细胞表面的野生型EGFR蛋白弱结合，而嵌合抗体7E5不识别野生型EGFR蛋白，只能特异性识别突变型EGFRvIII蛋白。U87MG细胞(人脑胶质瘤细胞系)表面有少量野生型EGFR蛋白的表达，而HEB细胞(人脑胶质细胞系) 表面有较高水平的野生型EGFR蛋白的表达。待测抗体与U87MG和HEB细胞均无结合反应，说明嵌合抗体75G7、嵌合抗体63A10和嵌合抗体64F1具有较好的特异性，只识别肿瘤组织中的突变型EGFRvIII蛋白以及超表达的野生型EGFR蛋白，不识别正常组织中的野生型EGFR蛋白(图12C、D)。
表20 FACS检测EGFRvIII嵌合抗体与U87MG-hEGFRvIII、A431、U87MG和HEB细胞的结合反应
Figure PCTCN2018120959-appb-000028
实施例10EGFRvIII嵌合抗体药物偶联物的细胞杀伤活性实验
抗体经过Ph6.5～8.5的硼酸钠缓冲液透析后，加入一定比例的TCEP还原(TCEP/抗体比率为2-10)，室温或37度进行还原2-4小时，经过G25脱盐填料去除多余的TCEP，加入一定比例的MC-MMAF(药物/抗体摩尔比率为4.5)反应4小时。再加入半胱氨酸用以中和多余的药物，并通过G25除去多余的小分子。得到纯化的抗体药物偶联物。(偶联方法参见Doronina，2006，Bioconjugate Chem.17,114-124)。通过HIC分析药物的交联率，纯度等参数后，进行细胞毒活性的分析。为了便于比较，所有抗体偶联物的药物交联率为8[通过实验调整DAR(毒物抗体偶联比率)达到预设的数值，后期对确切的DAR进行检测确定]。
得到的抗体偶联物，分别用完全培养基进行梯度稀释，96孔细胞培养板以1000细胞/孔加入90微升U87MG-EGFRvIII细胞悬液，每孔再分别加入10微升不同浓度的抗体药物偶联物的稀释液，继续培养5天后，用CellTiter-Glo试剂盒(购自Promega，使用方法参照产品说明书)检测细胞活力。
结果如表21以及图13A～D所示，其中表23的IC50指药物作用后，细胞的活性受到抑制的半数有效量，能够通过检测细胞的活性从而反映细胞杀伤活性。其中，图13为抗体药物偶联物对EGFRvIII阳性的重组肿瘤细胞系U87MG-EGFRvIII的细胞杀伤活性检测，结果说明，在体外实验中，待测抗体均可对表面有突变型EGFRvIII蛋白表达的U87MG-hEGFRvIII细胞产生良好的杀伤作用(图13A)。A431细胞(人表皮细胞癌细胞系)表面有大量野生型EGFR蛋白的表达，从图13B来看，嵌合抗体75G7,63A10和64F1 对A431有较弱的杀伤作用。U87MG细胞(人脑胶质瘤细胞系)表面有少量野生型EGFR蛋白的表达，而HEB细胞(人脑胶质细胞系)表面有较高水平的野生型EGFR蛋白的表达。嵌合抗体75G7,63A10和64F1对U87MG和HEB细胞杀伤作用很弱或者没有杀伤作用，见图13C，图13D。说明嵌合抗体75G7、63A10和64F1具有较好的特异性，可以特异性杀伤表达突变型EGFRvIII蛋白以及超表达野生型EGFR蛋白的肿瘤组织，而对表达野生型EGFR蛋白的正常组织没有杀伤作用。
表21细胞杀伤实验检测嵌合抗体偶联物对EGFRvIII阳性细胞的杀伤作用
Figure PCTCN2018120959-appb-000029
实施例11EGFRvIII嵌合抗体药物偶联物的体内药效学研究
由于EGFRvIII基因在肿瘤细胞体外培养的过程中会逐渐丧失表达，因此没有稳定表达EGFRvIII基因的内源性肿瘤细胞系。因此我们构建了U87MG-EGFRvIII重组细胞系，建立了U87MG-EGFRvIII皮下异种移植肿瘤模型，用来测试嵌合抗体药物偶联物的体内药效。
U87MG-EGFRvIII重组细胞系的构建方法见实施例1。将U87MG-EGFRvIII细胞悬液200μl(1×10 6个)接种于Balb/c nude小鼠右后背，待7天后肿瘤长至150-250mm 3后，挑选合适肿瘤体积的荷瘤鼠，按肿瘤体积将小鼠随机分组，然后按照既定计划给药。具体研究计划及给药剂量见表22。
实验指标是考察肿瘤生长是否可以被抑制、延缓或治愈。隔天用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5×a×b 2，a和b分别表示肿瘤的长径和短径。
化合物的抑瘤疗效(TGI)用TGI(％)评价。TGI％＝1-(TRTVn-TRTV1)/(CRTVn-CRTV1)×100％(TRTVn：治疗组最后一天的RTV；CRTV：阴性对照组RTV)。相对肿瘤体积RTV的计算公式为RTV＝Vt/V0。其中V0为分笼给药时(即Day0)测量所得肿瘤 体积，Vt为每一次测量时的肿瘤体积。T/C(％)的百分比值反映肿瘤生长抑制率，根据药审中心抗肿瘤药物的指导原则TGI％≤58％，认为此药有效。
表22.EGFRvIII嵌合抗体药物偶联物的体内药效学研究
Figure PCTCN2018120959-appb-000030
结果见图14A和图14B：所有受试小鼠体重未见明显变化，说明所有测试的抗体药物偶联物在U87MG-EGFRvIII异种移植荷瘤小鼠中具有良好的耐受性。与未治疗的溶剂对照组相比，所有抗体药物偶联物给药组的小鼠肿瘤体积都明显受到抑制，且高剂量组(3mg/kg)与低剂量组(1mg/kg)相比，具有明显的剂量依赖性。其中高剂量组(3mg/kg)在给药18天(每4天给药一次，共给药4次)后，完全抑制了肿瘤生长(TGI％>100％)。 而低剂量组(1mg/kg)也表现出了较好的抗肿瘤活性(TGI％>58％)。测试的4个嵌合抗体药物偶联物63A10-MMAE，75G7-MMAE，64F1-MMAE，7E5-MMAE均表现出优于对照抗体药物偶联物对照抗体02-MMAE的抗肿瘤活性，且63A10-MMAE最佳。
实施例12抑制脱酰胺化、异构化、水解反应的突变的导入
通过对75G7C6和63A10A7的抗体序列分析，发现75G7C6抗体重链可变区(SEQ ID No.1)CDR2(SEQ ID No.3)的第54和55位的NG存在脱酰胺化反应的可能，位于CDR3(SEQ ID No.4)的第98和99位DG存在异构化的可能，为了抑制脱酰胺化、异构化和水解，经过计算和分析，将位于75G7C6抗体CDR2的NG突变成NA，突变后75G7C6抗体重链可变区氨基酸序列如SEQ ID No.179所示；或将NG突变成QG，突变后75G7C6抗体重链可变区氨基酸序列如SEQ ID No.180所示。同时将位于CDR3的DG突变成SG，突变后75G7C6抗体重链可变区氨基酸序列如SEQ ID No.181所示(所对应的核酸序列如SEQ ID No.188所示)；或将DG突变成EG，突变后75G7C6抗体重链可变区氨基酸序列如SEQ ID No.182所示(所对应的核酸序列如SEQ ID No.131所示)；或将DG突变成DA，突变后75G7C6抗体重链可变区氨基酸序列如SEQ ID No.183所示(所对应的核酸序列如SEQ ID No.132所示)。拟通过上述定点突变实施去除作为接受脱酰胺化的位点的天冬酰胺残基和天冬酰残基的氨基酸修饰。
嵌合75G7C6抗体重链可变区定点突变序列经基因合成后，按实施例8所述进行质粒构建、细胞转染和抗体纯化，纯化抗体经过如实施例9所述的ELISA和FACS进行突变后的嵌合抗体结合活性鉴定，ELISA鉴定结果如图15和图16及表23所示，其中图15是75G7C6抗体重链可变区CDR2的NG突变后的抗体的ELISA结合活性鉴定，图16是75G7C6抗体重链可变区CDR3的NG突变后的抗体的ELISA结合活性鉴定，表23汇总了c75G7C6野生型抗体及突变体抗体与EGFRvIII-hFc蛋白结合的EC50值，从表23可看出，位于重链CDR2的NG突变成NA或QG后的抗体突变体c75G7C6-1和c75G7C6-2与EGFRvIII-hFc的结合活性与野生型抗体c75G7C6接近，说明NG突变成NA或QG不影响抗体与EGFRvIII-hFc的结合。但位于CDR3的DG不管是突变成EG、SG或DA均影响了对EGFRvIII-hFc的结合活性，其中DG突变成EG后的突变抗体c75G7C6-3与EGFRvIII-hFc的结合能力比野生型嵌合抗体c75G7C6下降22倍，而DG突变成SG或DA后的突变抗体c75G7C6-4和c75G7C6-5与EGFRvIII-hFc没有结合，说明DG突变成EG、SG或DA均严重影响了抗体与EGFRvIII-hFc的结合。
表23.ELISA检测c75G7C6嵌合抗体突变体与人EGFRvIII-hFc蛋白的结合反应
Figure PCTCN2018120959-appb-000031
Figure PCTCN2018120959-appb-000032
FACS鉴定结果如图17A-B和图28A-B及表24和表25所示，其中图17A是75G7C6抗体重链可变区CDR2的NG突变成NA或QG后的突变抗体c75G7C6-1和c75G7C6-2及野生型抗体c75G7C6与CHOK-EGFRvIII细胞结合活性检测，图17B是75G7C6抗体重链可变区CDR2的NG突变成NA或QG后的突变抗体c75G7C6-1和c75G7C6-2及野生型抗体c75G7C6与CHOK-EGFR细胞结合活性检测；图18A是75G7C6抗体重链可变区CDR3的DG突变成SG、EG或DA后的突变抗体c75G7C6-3、c75G7C6-4和c75G7C6-5及野生型抗体c75G7C6与CHOK-EGFRvIII细胞结合活性检测，图18B是75G7C6抗体重链可变区CDR3的DG突变成SG、EG或DA后的突变抗体c75G7C6-3、c75G7C6-4和c75G7C6-5及野生型抗体c75G7C6与CHOK-EGFR细胞结合活性检测。表24总结了中c75G7C6野生型抗体及突变抗体在CHOK-EGFRvIII细胞中的结合活性，表25总结了c75G7C6野生型抗体及突变抗体在CHOK-EGFR细胞中的结合活性。
表24.FACS检测c75G7C6嵌合抗体突变体与CHOk1-EGFRvIII细胞的结合反应
抗体名称 点突变 EC50(单位：nM) 最大平均荧光强度
c75G7C6 野生型(NG&DG) 10.5 111769
c75G7C6-1 N G/N A 7.452 109947
c75G7C6-2 NG/ QG 11.64 111041
c75G7C6-3 DG/ EG 24.82 94071
c75G7C6-4 DG/ SG 24 52584
c75G7C6-5 D G/D A 无 296
阴性对照hIgG 无 无 132.6
表25.FACS检测c75G7C6嵌合抗体突变体与CHOk1-EGFR细胞的结合反应
Figure PCTCN2018120959-appb-000033
从表24和图17A、图18A来看，位于c75G7C6重链可变区CDR2的NG突变成NA 或QG的突变抗体c75G7C6-1和c75G7C6-2与CHOk1-EGFRvIII细胞的结合活性与野生型抗体c75G7C6接近，说明NG突变成NA或QG不影响抗体与CHOK-EGFRvIII细胞的结合。而位于CDR3的DG不管是突变成EG、SG或DA均影响了抗体与对CHOK-EGFRvIII的结合活性，其中DG突变成EG或SG后的突变抗体c75G7C6-3和c75G7C6-4与CHOK-EGFRvIII的结合能力比野生型嵌合抗体c75G7C6下降约2.5倍，且最大平均荧光强度分别下降16％和53％，而DG突变成DA后的突变抗体c75G7C6-5与CHOK-EGFRvIII没有结合，说明DG突变成EG、SG或DA均严重影响了抗体与CHOK-EGFRvIII细胞的结合。
从表25和图17B、图18B来看，位于c75G7C6重链可变区CDR2的NG突变成NA或QG的突变抗体c75G7C6-1和c75G7C6-2与CHOk1-EGFR细胞的结合活性与野生型抗体c75G7C6接近，说明NG突变成NA或QG不影响抗体与CHOK-EGFR细胞的结合。而位于CDR3的DG不管是突变成EG、SG或DA均影响了抗体与对CHOK-EGFR的结合活性，其中DG突变成EG后的突变抗体c75G7C6-3与CHOK-EGFR的结合能力比野生型嵌合抗体c75G7C6下降约3倍，且最大平均荧光强度下降38％，而DG突变成SG或DA后的突变抗体c75G7C6-4和c75G7C6-5与CHOK-EGFR弱结合或没有结合，说明DG突变成EG、SG或DA均严重影响了抗体与CHOK-EGFR细胞的结合。
从上述ELISA和FACS结果来看，位于c75G7C6重链可变区CDR2的NG可突变成NA或QG，突变后抗体与抗原的结合活性不受影响，但位于CDR3的DG不管是突变成EG、SG或DA均影响了抗体与抗原的结合，不宜进行突变。因此，后续将选用CDR2的NG突变成NA后的抗体c75G7C6-1进行后续研究。
实施例13人源化EGFRvIII抗体的制备
在Germline数据库中选取与上述嵌合抗体63A10A7和75G7C6的非CDR区匹配最好的人种系抗体重链和轻链可变区模板。人源化EGFRvIII抗体的人接受序列选自人种系外显子V H、J H、V k和J k序列。其中63A10A7抗体重链可变区的模板为人种系抗体重链V H外显子的IGHV1-46*01，J H外显子的J H-4，轻链可变区的模板为人种系抗体轻链V K外显子的IGKV1-39*01，J K外显子的/J K-2。其中75G7C6抗体重链可变区的模板为人种系抗体重链V H外显子的IGHV1-46*01，J H外显子的J H-6b，轻链可变区的模板为人种系抗体轻链V K外显子的IGKV3-11*01，J K外显子的J K-4。
根据Kabat定义确定的嵌合抗体63A10A7和75G7C6的重链和轻链CDR分别移植到所选人种系模板中，替换人种系模板的CDR区，得到人源化的抗体。然后，以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH 的构象有重要影响的构架区的残基进行回复突变，得到人源化之后的抗体。简要说，产生跨越人源化V H或V L结构域的合成重叠寡核苷酸、并利用PCR重叠延伸来组装各结构域。利用掺入PCR产物的限制性位点将V H结构域定向克隆到包含信号肽和人源抗体重链IgG1恒定区的表达载体，将V L结构域定向克隆到包含信号肽和人源抗体轻链kappa恒定区的表达载体中，得到的重组质粒经过测序验证，使用碱裂解法试剂盒(购自MACHEREY-NAGEL)中量抽提高纯度的重组质粒，质量为500μg以上，经0.22μm滤膜(购自Millopore)过滤，供转染使用。
人源化抗EGFRVIII抗体变体的重链和轻链可变区与人种系重链和轻链可变区及嵌合抗体的重链和轻链可变区的序列比对如图19-22所示，其中图19为人源化抗EGFRvIII抗体h75G7C6重链可变区h75G7C6.VH及其变体与嵌合抗体c75G7C6.VH及人种系VH外显子IGHV1-46*01/JH6b的序列比较，图20为人源化抗EGFRvIII抗体h75G7C6轻链可变区h75G7C6.VL及其变体与嵌合抗体c75G7C6.VL及人种系VL外显子IGHV3-11*01/JK4的序列比较，图21为人源化抗EGFRvIII抗体h63A10A7重链可变区h63A10A7.VH及其变体与嵌合抗体c63A10A7.VH及人种系VH外显子IGHV1-46*01/JH4的序列比较，图22为人源化抗EGFRvIII抗体h63A10A7轻链可变区h63A10A7.VL及其变体与嵌合抗体c63A10A7.VL及人种系VL外显子IGKV1-39*01/JK2的序列比较，方框处为CDR。
人种系重链可变区模板IGHV1-46*01/JH4
IGHV1-46*01：
Figure PCTCN2018120959-appb-000034
JH4：YFDYWGQGTLVTVSS(SEQ ID NO.189)。
人种系重链可变区模板IGHV1-46*01/JH6b
IGHV1-46*01：
Figure PCTCN2018120959-appb-000035
JH6b：YYYYYGMDVWGQGTTVTVSS(SEQ ID NO.190)
人种系轻链可变区模板IGKV3-11*01/JK4
IGKV3-11*01：
Figure PCTCN2018120959-appb-000036
JK4：LTFGGGTKVEIK(SEQ ID NO.191)。
人种系轻链可变区模板IGKV1-39*01/JK2
IGKV1-39*01：
Figure PCTCN2018120959-appb-000037
JK2：YTFGQGTKLEIK(SEQ ID NO.192)。
选择数个框架位置以重新引入小鼠供体残基。可通过在VH结构域中掺入小鼠框架供体残基或在VL结构域中掺入人CDR残基的不同组合产生数种人源化75G7C6和63A10A7变体。下表26和表27小结了这些变体。其中，表26和表27显示的是这些变体的可变区，不包括恒定区。表26、24中，c开头表示嵌合抗体，h开头表示人源化抗体；其中供体构架残基及回复突变栏中显示的、例如各人源化抗EGFRvIII抗体75G7C6重链可变区h75G7C6.VH及其变体中“Q1E”表明如图19中所示的第1位氨基酸由“Q”谷氨酰胺突变为“E”谷氨酸，回复突变的位点位于构架区，以此为例，不一一说明。
表26
Figure PCTCN2018120959-appb-000038
Figure PCTCN2018120959-appb-000039
Figure PCTCN2018120959-appb-000040
表27
Figure PCTCN2018120959-appb-000041
Figure PCTCN2018120959-appb-000042
Figure PCTCN2018120959-appb-000043
Figure PCTCN2018120959-appb-000044
Figure PCTCN2018120959-appb-000045
注：表中的“/”代表的含义是“和”，为并列关系。
根据各人源化抗体轻链可变区和重链可变区的氨基酸序列合成cDNA(即分别为序列表中的SEQ ID NO.154，155，156，157，158，159，160，161，162，163，,164，165，166，167，168，169，170，171，172，173，174所示序列)，重链cDNA用FspAI和AfeI消化，轻链cDNA用FspAI和BsiwI后，将cDNA片段通过FspAI/AfeI或FspAI/BsiwI酶切位点分别插入到包含信号肽和人源重链抗体IgG1恒定区的表达载体及包含信号肽和人源抗体轻链kappa恒定区的表达载体当中(其中表达载体购买自Invitrogen，重组步骤也由上海睿智化学完成)中，重组质粒并经测序验证，使用碱裂解法试剂盒(购自MACHEREY-NAGEL)中量抽提高纯度的重组质粒，质量为500μg以上，经0.22μm滤膜(购自Millopore)过滤，供转染使用。
转染前，用培养基Freestyle 293 expression medium(购自Invitrogen)培养293E细胞(购自Invitrogen)。转染时在Freestyle 293 expression medium中添加10％(v/v)F68(购自Invitrogen)至F68终浓度为0.1％(v/v)，得含0.1％(v/v)F68的Freestyle 293表达培养基，即培养基A。取5mL培养基A和200μg/mL PEI(购自Sigma)混匀，得培养基B。取5mL培养基A和100μg重链和轻链重组质粒(重链重组质粒与轻链重组质粒的质量比为1:1-1:3范围)混匀，得培养基C。5分钟后将培养基B和培养基C合并混匀，静置15分钟，得混合液D。将10mL混合液D缓缓加入100mL含293E细胞的培养基 Freestyle 293 expression medium中至293E的细胞密度为1.5×10 6个/mL，边加边振荡，避免PEI过度集中，放入摇床培养，摇床设置为37℃、130RPM和8％CO 2(v/v)。第二天加入蛋白胨至终浓度为0.5％(w/v)。第5～7天，测培养液抗体效价。第6～7天，离心(3500RPM，30分钟)收集上清，经0.22μm滤膜过滤，得滤好的细胞上清液，以供纯化。
抗体纯化时，对于连续生产的无内毒素的层析柱和蛋白A填料(购自GE)，使用5个柱体积的0.5M NaOH冲洗。然后用5个柱体积的PBS(PBS缓冲液，pH7.4)进行平衡至中性后，将过滤好的细胞上清液上柱，必要时收集流穿液。上柱完成后，使用5倍柱体积的PBS清洗。用5倍柱体积的0.1M pH 3.0的Glycine-HCl进行洗脱，收集洗脱液，并立刻在洗脱液中加入0.1倍体积的pH 8.5的1MTris-HCl(1.5M NaCl)中和EGFRvIII抗体。上述所用溶液均需要新鲜配制。收获EGFRvIII抗体后，在1×PBS中透析4小时，避免内毒素污染。透析结束后，使用分光光度或试剂盒测定浓度，使用HPLC-SEC测定抗体纯度，使用内毒素检测试剂盒(购自Lonza)检测抗体内毒素含量。并对所获EGFRvIII抗体进行特性鉴定(操作步骤如下述实施例13所述)。
实施例14人源化抗EGFRvIII抗体的鉴定
A、酶联免疫吸附实验(ELISA)检测抗体与EGFRvIII蛋白的结合
对实施例12所得的纯化的人源化抗EGFRvIII抗体进行与人EGFRvIII-hFc蛋白进行反应。
将实施例1获得的纯化的免疫原A(EGFRvIII-hFc)(其制备方法参见实施例1步骤(一)用PBS稀释到终浓度1.0μg/mL，然后以100μl每孔加到96孔ELISA板。用塑料膜封好4℃孵育过夜，第二天用洗板液[PBS+0.01％(v/v)Tween20]洗板2次，加入封闭液[PBS+0.01％(v/v)Tween20+1％(w/w)BSA]室温封闭2小时。倒掉封闭液，加入实施例2所得的纯化的EGFRvIII嵌合抗体100μl每孔。37℃孵育2小时后，用洗板液[PBS+0.01％(v/v)Tween20]洗板3次。加入HRP(辣根过氧化物酶)标记的二抗(购自Sigma)，37℃孵育2小时后，用洗板液[PBS+0.01％(v/v)Tween20]洗板3次。加入TMB底物100μl每孔，室温孵育30分钟后，加入终止液(1.0N HCl)100μl每孔。用ELISA读板机(SpectraMax 384plus，购自Molecular Device)读取A450nm数值，结果如图23A-C和图24A-E及表28和表29所示，其中，图23A-C为纯化的人源化h75G7C6变体与人源EGFRVIII-hFc蛋白的结合反应，图24A-E为纯化的人源化h63A10A7变体与人源EGFRVIII-hFc蛋白的结合反应。表28和表29分别为h75G7C6变体和h63A10A7变体的根据OD 450nm值计算出来的EC50值，说明纯化的人源化EGFRvIII抗体变体与EGFRvIII 重组蛋白在ELISA水平有较好的结合。
表28 ELISA检测人源化h75G7C6抗体变体与人EGFRvIII-hFc蛋白的结合反应
Figure PCTCN2018120959-appb-000046
表29 ELISA检测人源化h63A10A7抗体变体与人EGFRvIII-hFc蛋白的结合反应
Figure PCTCN2018120959-appb-000047
Figure PCTCN2018120959-appb-000048
B、流式细胞实验(FACS)检测抗体与EGFRvIII表达细胞的结合
FACS检测所需的U87MG-EGFRvIII制备如实施例1免疫原B的制备所述，正常人原代肝细胞购自BioreclamationIVT，肿瘤细胞A431购自ATCC。肝细胞复苏后直接用于FACS检测，U87MG-EGFRvIII和A431细胞复苏后在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用HBSS缓冲液(Hanks Balanced Salt Solution)(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HBSS缓冲液洗涤细胞2次，进行细胞计数后将细胞用HBSS缓冲液稀释至2╳10 6细胞每毫升，加入1％山羊血清封闭液，所述百分比为质量百分比，冰上孵育30分钟，然后用HBSS缓冲液离心洗涤2次。将收集的细胞用FACS缓冲液(HBSS+2％FBS，所述百分比为体积百分比)重悬至2╳10 6细胞/mL，按每孔100微升加入到96孔FACS反应板中，加入实施例12所得的纯化的EGFRvIII抗体待测样品每孔100微升，冰上孵育2小时。用FACS缓冲液离心洗涤2次，加入每孔100微升1:1000稀释的荧光(Alexa 488)标记的二抗(购自Invitrogen)，冰上孵育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]重悬细胞，10分钟后用FACS缓冲液离心洗涤2次。用100微升FACS缓冲液重悬细胞，用FACS(FACS Calibur，购自BD公司)检测和分析结果。通过软件(CellQuest)进行数据分析，的到细胞的平均荧光密度(MFI)。再通过软件(GraphPad Prism5)分析，进行数据拟合，计算EC50。
分析结果如表30和表31，以及图25A-C和图26A-E所示，待测人源化抗体均可与U87MG-hEGFRvIII细胞表面的EGFRvIII蛋白特异结合，图25A-C是人源化h75G7C6抗体变体与U87MG-hEGFRvIII细胞表面的EGFRvIII的结合，图26A-E人源化h63A10A7抗体变体与U87MG-hEGFRvIII细胞表面的EGFRvIII的结合。
表30.FACS检测人源化h75G7C6抗体变体与U87MG-hEGFRvIII细胞表面的EGFRvIII的结合反应
Figure PCTCN2018120959-appb-000049
Figure PCTCN2018120959-appb-000050
表31.FACS检测人源化h63A10A7抗体变体与U87MG-hEGFRvIII细胞表面的EGFRvIII的结合反应
Figure PCTCN2018120959-appb-000051
Figure PCTCN2018120959-appb-000052
此外，人源化75G7C6和63A10A7抗体变体还能与野生型EGFR弱结合。A431细胞(人表皮细胞癌细胞系)表面过表达大量野生型EGFR蛋白，正常人原代肝细胞表面也有一定量的野生型EGFR蛋白表达，如图27和图28A-B所示，相对于抗EGFR对照抗体Cetuximab，人源化75G7C6抗体变体及人源化63A10A7抗体变体均可以与肿瘤细胞A431过表达的EGFR，但与正常人原代肝细胞表面的野生型EGFR蛋白弱结合，这可能与人源化75G7C6和63A10A7抗体的抗原决定簇所处的空间位置有关。但人源化75G7C6抗体的各变体与A431和正常人原代肝细胞表面EGFR蛋白结合能力不同，如表32所示，待测抗体与正常人原代肝细胞弱结合或无结合，平均荧光强度MFI值在250左右，与阴性对照hIgG的结合能力相近(MFI值219)，但与肿瘤细胞A431表面的EGFR有较高水平结合，平均荧光强度MFI在10000-13000，显示出人源化75G7C6抗体具有选择性结合肿瘤细胞表面过表达的EGFR蛋白，而不结合或弱结合正常细胞表达的EGFR蛋白，选择性窗口在35-47倍。
类似地，人源化63A10A7抗体的各变体与A431和正常人原代肝细胞表面EGFR蛋白结合能力不同，如图28A-B和表33所示，部分待测抗体，如人源化抗体h63A10A7-17～26与正常人原代肝细胞的结合能力与抗EGFR抗体接近(MFI 433)，平均荧光强度MFI范围在433-677，因此认为此部分待测抗体对表达EGFR的正常细胞不具有选择性；但部分待测抗体表现出很好的对表达EGFR的肿瘤细胞和正常细胞的选择性，如人源化抗体h63A10A7‐37～39，与正常人原代肝细胞弱结合或无结合，平均荧光强度MFI值在260左右，与阴性对照hIgG的结合能力相近(MFI值219)，但与肿瘤细胞A431表面的EGFR有一定水平的结合，平均荧光强度MFI在1000-2000，显示出部分人源化63A10A7抗体变体具有选择性结合肿瘤细胞表面过表达的EGFR蛋白，而不结合或弱结合正常细胞表达的EGFR蛋白，选择性窗口在5～7.5倍。
表32.FACS检测人源化h75G7C6抗体变体与肿瘤细胞A431和正常人原代肝细胞表面的EGFR的结合反应
Figure PCTCN2018120959-appb-000053
Figure PCTCN2018120959-appb-000054
表33.FACS检测人源化h63A10A7抗体变体与肿瘤细胞A431和正常人原代肝细胞表面的EGFR的结合反应
Figure PCTCN2018120959-appb-000055
C.人源化抗EGFRvIII抗体的结合亲和力检测
为了评估人源化抗EGFRvIII抗体与EGFRvIII和EGFR蛋白的结合特异性和亲和 力，在OctetRED(购自Pall)仪器上，使用抗人Fc的生物传感器AHC(购自ForteBio)结合待检测人源化h75G7C6抗体变体，然后再与EGFRvIII-his(购自Sino Biological)或EGFR-his(购自Sino Biological)蛋白结合，通过生物膜层光学干涉(BLI)技术进行检测。BLI技术通过检测当传感器上固定的分子与溶液中的分子发生相互作用时，生物膜厚度增加，干涉光谱曲线向波长增加的方向移动，光波相位移动由工作站实时探测，对光波的相位移动进行分析便可定量得出传感器表面分子数量变化及相关浓度和动力学数据。人源化h75G7C6抗体变体与EGFRvIII和EGFR蛋白的结合速率(K a)、解离速率(K dis)及结合亲和力(K D)如表34和表35所示，其中嵌合抗体c75G7C6作为对照。
表34.人源化h75G7C6抗体变体与EGFRvIII蛋白的结合亲和力
抗体名称 Ka(1/Ms) Kdis(1/s) KD(M)
嵌合抗体c75G7C6 9.14E+04 1.32E-04 1.45E-09
人源化抗体h75G7C6-1 8.28E+04 1.41E-04 1.71E-09
人源化抗体h75G7C6-2 9.06E+04 1.40E-04 1.54E-09
人源化抗体h75G7C6-9 9.89E+04 1.75E-04 1.77E-09
人源化抗体h75G7C6-13 1.25E+05 1.81E-04 1.45E-09
表35.人源化h75G7C6抗体变体与EGFR蛋白的结合亲和力
抗体名称 Ka(1/Ms) Kdis(1/s) KD(M)
嵌合抗体c75G7C6 1.65E+04 3.62E-03 2.20E-07
人源化抗体h75G7C6-1 1.72E+04 3.53E-03 2.05E-07
人源化抗体h75G7C6-2 2.03E+04 4.05E-03 2.00E-07
人源化抗体h75G7C6-9 2.02E+04 4.35E-03 2.15E-07
人源化抗体h75G7C6-13 2.25E+04 4.76E-03 2.11E-07
OctetRED结果显示，人源化h75G7C6抗体变体与EGFRvIII和EGFR蛋白的结合亲和力与嵌合抗体c75G7C6非常接近，这验证了人源化h75G7C6抗体与嵌合抗体相比，没有显著降低抗原结合活性。且人源化h75G7C6抗体变体与EGFRvIII蛋白的结合亲和力约为1.5nM，与EGFR蛋白的结合亲和力约为0.2uM。
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 (23)

  1. 一种EGFRvIII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、SEQ ID No.90或SEQ ID No.98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、SEQ ID No.91、SEQ ID No.99、SEQ ID NO.184或者SEQ ID NO.186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、SEQ ID No.92或SEQ ID No.100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、SEQ ID No.94或SEQ ID No.102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、SEQ ID No.95或SEQ ID No.103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、SEQ ID No.96或SEQ ID No.104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或SEQ ID No.98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、SEQ ID No.99、SEQ ID NO.184或者SEQ ID NO.186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或SEQ ID No.100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或SEQ ID No.102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或SEQ ID No.103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或SEQ ID No.104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EGFRvIII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；所述重链CDR1的氨基酸序列如序列表中SEQ ID No.98所示，所述重链CDR2的氨基酸序列如序列表中SEQ ID No.99所示，且所述重链CDR3的氨基酸序列如序列表中SEQ ID No.100所示；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.184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；或者，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.186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；或所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.102所示，所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.103所示，且所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.104所示。
  3. 如权利要求1或2所述的EGFRvIII抗体，其特征在于，其还包括抗体的构架区，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，所述轻链构架区为人抗体轻链构架区，优选为人种系抗体轻链IGKV1-39*01或IGKV3-11*01的FR1、FR2、FR3区，和JK-2或JK-4的FR4区的人抗体轻链构架区的组合；且所述重链构架区为人抗体重链构架区，优选为人种系抗体重链IGHV1-46*01的FR1、FR2、FR3区，和JH-4或JH-6b的FR4区的人抗体重链构架区的组合。
  4. 如权利要求3所述的EGFRvIII抗体，其特征在于，所述EGFRvIII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、SEQ ID No.89、SEQ ID No.97、SEQ ID No.179、SEQ ID No.180、SEQ ID NO.133、SEQ ID NO.134、SEQ ID NO.135、SEQ ID NO.136、SEQ ID NO.141、SEQ ID NO.142、SEQ ID NO.143、SEQ ID NO.144、SEQ ID NO.145、SEQ ID NO.146或SEQ ID NO.147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、SEQ ID No.93、SEQ ID No.101、SEQ ID NO.137、SEQ ID NO.138、 SEQ ID NO.139、SEQ ID NO.140、SEQ ID NO.148、SEQ ID NO.149、SEQ ID NO.150、SEQ ID NO.151、SEQ ID NO.152或SEQ ID NO.153所示；
    或者，所述的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、SEQ ID No.89、SEQ ID No.97、SEQ ID No.179、SEQ ID No.180、SEQ ID NO.133、SEQ ID NO.134、SEQ ID NO.135、SEQ ID NO.136、SEQ ID NO.141、SEQ ID NO.142、SEQ ID NO.143、SEQ ID NO.144、SEQ ID NO.145、SEQ ID NO.146或SEQ ID NO.147所示的氨基酸序列至少有80％序列同源的氨基酸序列所示；所述的轻链可变区序列如序列表中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、SEQ ID No.93、SEQ ID No.101、SEQ ID NO.137、SEQ ID NO.138、SEQ ID NO.139、SEQ ID NO.140、SEQ ID NO.148、SEQ ID NO.149、SEQ ID NO.150、SEQ ID NO.151、SEQ ID NO.152或SEQ ID NO.153所示的氨基酸序列至少有80％序列同源的氨基酸序列所示。
  5. 如权利要求4所述的EGFRvIII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；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；所述重链可变区的氨基酸序列如序列表中SEQ ID No.97所示，且所述轻链可变区的氨基酸序列如序列表中SEQ ID No.101所示；所述重链可变区的氨基酸序列如序列表中SEQ ID No.179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.180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SEQ ID No.133所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.137所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.133所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.138所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.133所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.139所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.133所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.140所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.134所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.137所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.134所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.138所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.134所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.139所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.134所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.140所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.135所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.137所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.135所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.138所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.135所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.139所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.135所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.140所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.136所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.137所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.136所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.138所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.136所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.139所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.136所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID  No.140所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.143所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.149所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.144所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.149所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.145所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.149所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.146所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.149所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.147所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.149所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.143所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.148所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.143所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.150所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.143所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.151所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.143所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.152所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.143所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.153所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.141所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.149所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.141所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.150所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.141所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.151所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.141所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.152所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.141所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.153所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.142所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.149所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.142所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.150所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.142所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.151所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.142所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.152所示的序列；所述重链可变区的氨基酸序列 如序列表SEQ ID No.142所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.153所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.145所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.148所示的序列；所述重链可变区的氨基酸序列如序列表SEQ ID No.146所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.148所示的序列；或者，所述重链可变区的氨基酸序列如序列表SEQ ID No.147所示的序列，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.148所示的序列。
  6. 如权利要求1～5任一项所述的EGFRvIII抗体，其特征在于，所述的EGFRvIII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EGFRvIII抗体，其特征在于，所述的EGFRvIII抗体是EGFRvIII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EGFRvIII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.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、SEQ ID No.119、SEQ ID No.121、SEQ ID No.123、SEQ ID No.125、SEQ ID No.127、SEQ ID No.129、SEQ ID No.185、SEQ ID No.154、SEQ ID No.155、SEQ ID No.156、SEQ ID No.157、SEQ ID No.162、SEQ ID No.163、SEQ ID No.164、SEQ ID No.165、SEQ ID No.166、SEQ ID No.167或SEQ ID No.168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.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、SEQ ID No.120、SEQ ID No.122、SEQ ID No.124、SEQ ID No.126、SEQ ID No.128、SEQ ID No.130、SEQ ID No.158、SEQ ID No.159、SEQ ID No.160、SEQ ID No.161、SEQ ID No.169、SEQ ID No.170、SEQ ID No.171、SEQ ID No.172、SEQ ID No.173或SEQ ID No.174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.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；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；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.121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.122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.123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.124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.125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 No126.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.127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.128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.129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.130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.185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SEQ ID No.154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SEQ ID No.158所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.154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SEQ ID No.159所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.154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SEQ ID No.160所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.154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SEQ ID No.161所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.155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SEQ ID No.158所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.155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SEQ ID No.159所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID  No.155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SEQ ID No.160所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.155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SEQ ID No.161所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.156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SEQ ID No.158所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.156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SEQ ID No.159所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.156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SEQ ID No.160所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.156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SEQ ID No.161所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.157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SEQ ID No.158所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.157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SEQ ID No.159所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.157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SEQ ID No.160所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.157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SEQ ID No.161所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.164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SEQ ID No.170所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.165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SEQ ID No.170所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.166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SEQ ID No.170所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.167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SEQ ID No.170所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.168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SEQ ID No.170所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.164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SEQ ID No.169所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.164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SEQ ID No.171所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.164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SEQ ID No.172所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.164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SEQ ID No.173所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.164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SEQ ID No.174所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.162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SEQ ID No.170所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.162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SEQ ID No.171所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.162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SEQ ID No.172所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.162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SEQ ID No.173所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.162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SEQ ID No.174所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.163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SEQ ID No.170所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.163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SEQ ID No.171所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.163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SEQ ID No.172所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.163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SEQ ID No.173所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.163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SEQ ID No.174所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.166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SEQ ID No.169所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.167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SEQ ID No.169所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.168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SEQ ID No.169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EGFRvIII抗体的制备方法，其包括如下步骤：培养权利要求12所述的重组表达转化体，从培养物中获得EGFRvIII抗体。
  14. 一种免疫偶联物，其特征在于，其包括共价附着至细胞毒剂的如权利要求1～7任一项所述的EGFRvIII抗体。
  15. 如权利要求14所述的免疫偶联物，其特征在于，1当量如权利要求1～7任一项所述的EGFRvIII抗体通过x当量接头与y当量细胞毒剂相连，其具有式1所示结构，
    Ab-(L) x-(D) y
    式1
    其中，Ab为如权利要求1～7中任一项所述的EGFRvIII抗体；L为接头；D为细胞毒剂；x为自然数，优选为1～20；y为大于0的自然数，优选为1～20；x和y各自独立地更优选为2w，w为1～5的整数、进一步优选为3～4；x和y的比例优选为1:1。
  16. 如权利要求15所述的免疫偶联物，其特征在于，所述接头L含有式2的结构，其为L中离去基团离去后对应的剩余部分：
    (CO-Alk 1-Sp 1-Ar-Sp 2-Alk 2-C(Z 1)＝Q-Sp)
    式2
    较佳地，所述的接头L为马来酰亚胺基己酰(MC)或马来酰亚胺基己酰-L-缬氨酸-L-瓜氨酸对氨基苄醇(MC-VC-PAB)；和/或，所述D为甲基奥瑞他汀F(MMAF)或者甲基奥瑞他汀E(MMAE)。
  17. 如权利要求14～16任一项所述的免疫偶联物，其特征在于，所述式1中x＝y＝n，所述免疫偶联物的结构如式3-1或3-2所示，
    Figure PCTCN2018120959-appb-100001
    式3-1中m为1～10，优选m为5，L为马来酰亚胺基己酰；D为甲基奥瑞他汀F(MMAF)；其中，n为自然数，优选为1～20的整数，更优选为2w，w为1～5的整数、进一步优选为3～4；
    Figure PCTCN2018120959-appb-100002
    式3-2中m为1～10，优选m为5，L为马来酰亚胺基己酰-L-缬氨酸-L-瓜氨酸对氨基苄醇；D为甲基奥瑞他汀E(MMAE)；其中，n为自然数，优选为1～20的整数，更优选为2w，w为1～5的整数、进一步优选为3～4。
  18. 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EGFRvIII抗体或者权利要求14～17任一项所述的免疫偶联物，以及药学上可接受的载体。
  19. 如权利要求18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EGFRvIII抗体或者如权利要求14～17任一项所述的免疫偶联物，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。
  20. 一种如权利要求1～7任一项所述的EGFRvIII抗体、或者如权利要求14～17任一项所述的免疫偶联物、或者如权利要求18～19所述的药物组合物在制备抗肿瘤药物中的应用。
  21. 一种检测过表达EGFRvIII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EGFRvIII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EGFRvIII抗体与所述待检样品的结合即可。
  22. 一种检测过表达EGFRvIII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EGFRvIII抗体作为活性成分。
  23. 一种如权利要求1～7中任一项所述的EGFRvIII抗体、或者如权利要求14～17任一项所述的免疫偶联物、或者如权利要求18～19所述的药物组合物在制备预防或治疗与EGFRvIII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EGFRvIII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、前列腺癌或者肾癌。
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