WO2013149526A1 - 针对表皮生长因子受体隐蔽表位和t细胞抗原的多功能抗体多肽 - Google Patents

针对表皮生长因子受体隐蔽表位和t细胞抗原的多功能抗体多肽 Download PDF

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WO2013149526A1
WO2013149526A1 PCT/CN2013/072098 CN2013072098W WO2013149526A1 WO 2013149526 A1 WO2013149526 A1 WO 2013149526A1 CN 2013072098 W CN2013072098 W CN 2013072098W WO 2013149526 A1 WO2013149526 A1 WO 2013149526A1
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antibody
polypeptide
seq
egfr
cells
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PCT/CN2013/072098
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French (fr)
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李宗海
蒋华
石必枝
王华茂
孔娟
高慧萍
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上海益杰生物技术有限公司
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Priority to EP13772058.7A priority Critical patent/EP2835379B1/en
Priority to US14/389,665 priority patent/US10023639B2/en
Publication of WO2013149526A1 publication Critical patent/WO2013149526A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Definitions

  • the invention relates to the field of biomedicine. More specifically, the present invention relates to a multifunctional antibody polypeptide capable of recognizing and binding to a cryptic epitope of the epidermal growth factor receptor (EGFR) and a sputum cell antigen.
  • the present invention also relates to a nucleotide sequence encoding the antibody polypeptide, a vector containing the nucleotide sequence, a host cell containing the vector, and the like.
  • the invention further relates to the use of the multifunctional antibody polypeptide in the preparation of an anti-tumor drug and in the preparation of a kit for the diagnosis and/or prevention of tumors. Background technique
  • EGFR epidermal growth factor receptor
  • EGFR When EGFR is activated, overexpressed, or mutated, it may result in exposure of the cryptic epitope (287CGADSYEMEEDGVRKC302) and binding to an antibody against the epitope, such as mAb806 (Garrett TP et al. Antibodies specifically targeting a locally misfolded region of Tumor associated EGFR. Proc Natl Acad Sci US A. 2009; 106(13):5082-7. These antibodies showed some anti-tumor effects in animal experiments and showed better tumor specificity than previous anti-EGFR antibodies.
  • the human murine chimeric antibody ch806 derived from the antibody mAb806 antibody showed strong tumor targeting in clinical phase I trials, and no significant dermal toxicity was observed (Scott AM, Lee FT et al, A phase I clinical trial) With monoclonal antibody ch806 targeting transitional state and mutant epidermal growth factor receptors. ProcNatlAcadSci US A. 2007 Mar 6; 104(10):4071 -6. At a dose of 5 mg/m 2 , ch806 showed an absorption at the tumor site. Previously, other anti-EGFR antibodies have been shown to require approximately 10 to 20 times the absorption at the tumor site (Divgi CR et al.
  • bifunctional antibodies that specifically recognize EGFR and CD3 antigens have been described in the prior art, with a portion of the domain specific for EGFR and another portion of the domain specific for the CD3 antigen of T cells.
  • Bifunctional antibodies constructed with Cetuximab or P anitumumab antibodies and anti-CD 3 antibodies showed good antitumor activity, but in primate experiments, these bifunctional antibodies were found to be stronger against normal cells expressing EGFR or Tissue toxic effects (Lutterbuese R, Kunststoff T et. al T cell-engaging BiTE antibodies specific for EGFR potently eliminate KRAS- and B RAF -mutated colorectal cancer cells. ProcNatl AcadSci USA 2010;107(28): 12605-10.).
  • a first aspect of the present application relates to a multifunctional antibody polypeptide comprising
  • a second aspect of the application relates to a nucleotide sequence encoding the polypeptide.
  • a third aspect of the present application relates to a vector comprising the nucleotide sequence.
  • a fourth aspect of the present application relates to a eukaryotic host cell or prokaryotic host cell comprising the vector.
  • a fifth aspect of the present application relates to the use of the polypeptide for the preparation of a medicament for the diagnosis, treatment and/or prevention of a tumor.
  • the degree of "specific recognition” and specificity can be judged by classical immunological techniques including, but not limited to, immunoblotting, immunoaffinity chromatography, flow cytometry, and the like.
  • specific recognition is preferably determined by flow cytometry, and the criteria for specific recognition in specific cases can be judged by one of ordinary skill in the art based on the common knowledge in the art.
  • “Functional domain” refers to an antibody or antibody fragment that specifically recognizes an antigen, including intact antibodies, Chain antibody (scFV), Fd fragment, Fab fragment, F(ab') 2 fragment, single domain antibody fragment, isolated CDR fragment, and derivatives thereof.
  • an “intact antibody” consists of two identical heavy and light chains, each of which contains a variable region (V region) and one or more constant regions (C region).
  • the variable region is responsible for binding to the antigen, while the constant region is primarily responsible for binding the effector molecule.
  • There are three highly diverse flexible loops in each variable region termed complementarity determining regions (CDRs), which are primarily responsible for the recognition of antigens.
  • CDRs complementarity determining regions
  • the other part of the variable region contains rigid beta sheets and supports so-called frame regions (FRs).
  • the CDRs and FRs are spaced apart to form a sandwich structure.
  • Single-chain antibody (scFV) fragment refers to an antibody fragment constructed by genetic engineering, which is a heavy chain variable region ( VH ) and a light chain variable region (VJ recombinant protein) linked by a linker.
  • the linker associates the two domains to form an antigen binding site.
  • the size of the ScFV is typically 1/6 of that of an intact antibody.
  • Fd fragment refers to an antibody fragment consisting of heavy chain VH and CH1 .
  • Fab fragment refers to a heterodimer formed by an Fd fragment (composed of heavy chains VH and CH1) and an entire light chain formed by an interchain disulfide bond.
  • the “Fab antibody” is 1/3 the size of an intact antibody and contains only one antigen binding site.
  • F(ab') 2 fragment refers to a bivalent fragment comprising two linked Fab fragments.
  • a "single domain antibody” consists of a heavy chain variable region or a light chain variable region. Since the antibody fragment consists of only one domain, it is named. The size of this fragment is 1/12 of an intact antibody.
  • Derivatives of antibodies includes, for example, when a derivative of the antibody is obtained by phage display technology, surface plasmon resonance techniques such as those used in the BIAcore system can be used to increase the efficiency of phage antibodies that bind to EGFR or CD3 epitopes ( Schier, Human Antibody Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).
  • the antibody or fragment thereof used in the present invention may be further modified, either singly or in combination, using conventional techniques known in the art, such as amino acid deletion, insertion, substitution, addition, and/or recombination, and/or other modification methods.
  • Methods for introducing such modifications into their DNA sequences based on the amino acid sequence of an antibody are well known to those skilled in the art; see, for example, Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.
  • the indicated modifications are preferably carried out at the nucleic acid level.
  • the antibodies or antibody fragments used in the invention may be humanized, chimeric or murine.
  • Figure 1 shows the structure of the pH-806/CD3 expression vector in the upper part and the enlarged view of the inserted gene fragment in the lower part.
  • FIG. 1 Schematic diagram of pH-7B3/CD3 expression vector
  • M is the molecular weight marker (SDS-polyacrylamide gel electrophoresis small molecular weight standard protein provided by Shanghai Shengzheng Biotechnology Co., Ltd.), the first column is 806/CD3, the second column is 7B3/CD3 .
  • Figure 3B Protein hybridization assay for purified bifunctional antibody polypeptides
  • the first column is 806/CD3 and the second column is 7B3/CD3.
  • Figure 4A Determination of specific binding of three bifunctional single-chain antibodies (NGR/CD3, 7B 3/CD3 and 806/CD3 ) to U87 MG tumor cells by fluorescence activated cell sorter (FACS)
  • Figure 4B Determination of specific binding of three bifunctional single-chain antibodies (NGR/CD3, 7B3/CD3 and 806/CD3) to U87 MG-EGFRvIII tumor cells by fluorescence activated cell sorter (FACS)
  • FIG. 4C Determination of specific binding of three bifunctional single-chain antibodies (NGR/CD3, 7B3/CD3 and 806/CD3) to A431 tumor cells by fluorescence activated cell sorter (FACS)
  • Figure 4D Determination of specific binding of three bifunctional single-chain antibodies (NGR/CD3, 7B3/CD3 and 806/CD3) to U87 MG-de4 EGFR tumor cells by fluorescence activated cell sorter (FACS)
  • FIG. 4E Determination of specific binding of three bifunctional single-chain antibodies (NGR/CD3, 7B3/CD3 and 806/CD3) to NCI-H1650 tumor cells by fluorescence activated cell sorter (FACS)
  • FIG. 4F Determination of specific binding of three bifunctional single-chain antibodies (NGR/CD3, 7B3/CD3 and 806/CD3) to NCI-H1975 tumor cells by fluorescence activated cell sorter (FACS)
  • Figure 4G Determination of specific binding of three bifunctional single-chain antibodies (NGR/CD3, 7B 3/CD3 and 806/CD3 ) to Jurkat tumor cells by fluorescence activated cell sorter (FACS)
  • Figure 6A is a series of gradient dilutions of three bifunctional single-chain antibodies (NGR/CD3, 7B3/CD3 and 806/CD3) that induce T cell killing against U87 MG tumor cells.
  • Figure 6B is a series of gradient dilutions of three bifunctional single-chain antibodies (NGR/CD3, 7B3/CD3 and 806/CD3)-induced T cell killing against U87 MG-EGFRvIII tumor cells.
  • Figure 6C series of serial dilutions of three bifunctional single-chain antibodies (NGR/CD3, 7B3/CD3 and 806/CD3) induced T cell killing rates against A431 tumor cells.
  • Figure 6D series of serial dilutions of three bifunctional single-chain antibodies (NGR/CD3, 7B3/CD3 and 806/CD3) induced T cell killing rates against U87 MG-de4 EGFR tumor cells.
  • Figure 6E is a series of gradient dilutions of three bifunctional single-chain antibodies (NGR/CD3, 7B3/CD3 and 806/CD3)-induced T cell killing ratios for NCI-H 1650 tumor cells.
  • Figure 6F is a series of gradient dilutions of three bifunctional single-chain antibodies (NGR/CD3, 7B3/CD3 and 806/CD3)-induced T cell killing rates for NCI-H1975 tumor cells.
  • Figure 7 Anti-tumor activity assay of different concentrations of bifunctional antibodies (7B3/CD3 and 806/CD3) in the NOD/SCID tumor-bearing (U87 MG-EGFRvIII) mouse model
  • Figure 10A-B Gel filtration chromatography curve of 806/CD3 bifunctional antibody expressed by genetic engineering
  • the present invention provides a multifunctional antibody against a series of tumors, including a tumor comprising an amplified EGFR gene and a EGFR expressing a variant, such as a tumor expressing de2-7 EGFR lacking the exon 2-7 exon, including But not limited to lung cancer, colon cancer, breast cancer, stomach cancer, brain cancer, bladder cancer, head and neck cancer, ovarian cancer, kidney cancer and prostate cancer.
  • the multifunctional antibody comprises a covert epitope formed by specifically recognizing amino acid sequences 287-302 of a tumor-expressing EGFR comprising an amplified EGFR gene or a mutated EGFR gene, as set forth in SEQ ID NO: And a second domain that recognizes human T cell surface antigens.
  • the multifunctional antibody of the present invention is capable of inducing T cell killing toxicity against tumor cells in vitro and in vivo at a very low concentration, such as 100 pg/mL - Ing/mL. Even when a lower effector cell (E): target cell ratio, such as 10:1, is used, specific lysis of the relevant tumor cell line can be observed without any other form of T cell pre-excitation.
  • the relevant tumor cells to which the present invention is directed include EGFR expressing a mutant such as de2-7 EGFR, and the above-mentioned tumor cells expressing the amplified EGFR can be obtained from commercial sources, such as NCI-1650, NCI-1975, A431, obtained from the American Collection Culture Center.
  • U87 MG-EGFRvIII which is a U87 MG cell line stably expressing EGFRvIII, and its construction method is described in the literature (Jiang H, J Biol. Chem., 2011, 286(7): 5913 -20), U87 MG can also be obtained from ATCC.
  • the multifunctional antibody of the present invention hardly binds to cells in which EGFR is not amplified or mutated (e.g., U87 MG).
  • the antitumor drug prepared from the multifunctional antibody of the present invention has improved tumor targeting in vivo and reduces killing of normal tissues.
  • the first domain of the invention specifically recognizes a hidden epitope formed by the amino acid sequence set forth in SEQ ID NO: 1.
  • Antibodies that specifically recognize the above-described occult epitopes, such as the epitopes contained in amino acids 287-302 of human wild-type EGFR, are disclosed, for example, in US Patent Application No.
  • the first domain of the invention is capable of specifically binding to tumors that express multiple copies of EGFR and tumors that express variants of EGFR, such as de2-7 EGFR.
  • the second domain of the invention includes antibodies and antibody fragments that specifically recognize human T cell antigens.
  • the T cell surface antigens include, but are not limited to, CD3, CD16, CD28.
  • the T cell surface antigen is CD3.
  • CD3 is an antigen expressed by T cells. It is part of a multicellular T cell receptor complex (TCR) containing three different chains, CD3 e, CD3 ⁇ , and CD3 ⁇ .
  • TCR multicellular T cell receptor complex
  • Clustering CD3 on sputum cells e. g., by immobilized anti-CD3 antibodies
  • anti-CD3 antibodies recognize the CD3 epsilon chain.
  • This bifunctional antibody kills tumor cells primarily by stimulating the immune system and is not restricted by major histocompatibility antigens (MHCs).
  • MHCs major histocompatibility antigens
  • the first domain comprises at least one complementational decision of an anti-EGFR antibody heavy chain variable region selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID N0.4 District (CDR).
  • the first functional domain is the entire heavy chain variable region comprising the above three CDRs in sequence.
  • the first domain comprises at least one complement of an anti-EGFR antibody light chain variable region selected from the group consisting of SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID N0.7 Decision Area (CDR).
  • the first functional domain is a light chain variable region comprising the above three CDRs in sequence.
  • the first functional domain is a single-chain anti-EGFR monomer comprising the entire heavy chain variable region and the entire light chain variable region, which are ligated in sequence.
  • the second domain is a single chain anti-CD3 antibody.
  • the two domains of the multifunctional antibody of the invention may comprise two different single chain antibodies, and thus the antibody is also referred to as a single chain bifunctional antibody.
  • the bifunctional antibody polypeptide has the amino acid sequence set forth in SEQ ID NO.
  • the bifunctional antibody polypeptide has the amino acid sequence set forth in SEQ ID NO.
  • the polypeptide linker preferably comprises several hydrophilic peptide-bonded amino acids of sufficient length to span the C-terminus of the functional domain containing the binding site and another N of the functional domain containing the binding site The distance between the ends, so that when the multifunctional antibody of the present invention is placed in an aqueous solution, it exhibits a conformation suitable for binding.
  • the polypeptide linker comprises a plurality of glycine, alanine and/or serine residues.
  • the polypeptide linker has an amino acid sequence (GlyGlyGlyGlySer) n, wherein n is an integer from 1 to 5, preferably n is from 1 to 3, more preferably n is 3.
  • variable regions are joined by the above-described polypeptide linker.
  • the first and second domains of the antibodies of the invention may be a pair of VH-VL, VH-VH, or VL-VL domains of the same or different antibodies.
  • the order of the VH and VL functional domains is not certain to the present invention, and when the order is reversed, there is usually no loss of function. Importantly, the arrangement of the VH and VL domains allows the antigen binding site to fold correctly, resulting in a multifunctional antibody that specifically recognizes and binds to multiple antigens.
  • the functional domains are arranged in the order of VL E GFR-VHEGFR-HcD3 - VLcD3.
  • nucleotide sequence of the polypeptide described above is contemplated.
  • the aspect relates to a nucleotide sequence of SEQ ID NO. 10 encoding the amino acid sequence set forth in SEQ ID NO.
  • the present aspect relates to a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO. 9 SEQ ID NO.
  • a vector comprising a nucleotide sequence encoding the above polypeptide.
  • the vector may be a eukaryotic cell vector or a prokaryotic cell vector, as long as the vector satisfies: (a) the coding sequence comprises a replication initiation sequence, such that the vector is capable of being replicated in a host cell, and (b) A gene sequence encoding a marker that encodes a protein that is required for survival and growth of the host cell in a particular selection medium. In the case where the host cell is not transfected or transformed into a vector comprising the gene, the host cell cannot survive in a particular selection medium.
  • Typical screening marker genes encode proteins comprising proteins that are tolerant to antibiotics or toxins, including, for example, ampicillin, kanamycin, tetracycline, neomycin, hygromycin, methotrexate, and the like; A protein that compensates for auxotrophs and supplies key nutrients that are not present in the culture medium, such as the D-alanine racemase gene.
  • resistance screening include obtaining a host cell containing a drug neomycin or G418 by transfecting an exogenous vector containing a neomycin resistance gene. In the case of raising the foundation, continue to grow and grow.
  • DHFR dihydrofolate reductase screening markers
  • mammalian cells such as Chinese hamster ovary cells (CHO).
  • Mammalian cell host cells refer to DHFR-deficient cells and do not contain the dihydrofolate reductase gene. , can not synthesize nucleic acids, must be grown in the medium containing HT.
  • a positive clone containing the exogenous vector containing the gene of interest and the DHFR gene can be selected by selection of the above culture conditions.
  • the expression vector may also include other constituent sequences, including a signal peptide sequence, a transcription termination sequence, an enhancer sequence, etc.
  • the vector of the present invention is a eukaryotic cell vector.
  • the vector of the present invention is a pH vector derived from eukaryotic expression of an antibody, which comprises a promoter of CMV, an internal ribosome entry site (IRES), a DHFR screening marker and the like.
  • IRS internal ribosome entry site
  • DHFR screening marker a DHFR screening marker and the like.
  • Methotrexate (MTX) is an inhibitor of DHFR that can hinder its action.
  • the cell culture medium contains MTX
  • DHFR is inhibited, and the gene is self-amplified by feedback regulation, and the upstream and downstream genes are amplified, and the target gene is also amplified, thereby increasing the expression level of the target protein.
  • a host cell comprising the vector is used for expression of a desired multi-functional antibody polypeptide.
  • the host cell of the present invention may be any prokaryotic host cell or eukaryotic host cell, in accordance with the vector used.
  • Eukaryotic host cells including yeast, insect cells, plant cells, mammalian cells, etc., may be preferred because eukaryotic cells have complex post-translational modifications (eg, glycosylation) of the protein of interest, and are increasingly used. For large-scale cultivation.
  • Common host cell lines include monkey kidney cells (COS-7 ATCC CRL 1651), human embryonic kidney cells 293 and their subcloned cell lines, baby hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells (CHO), and the like.
  • the eukaryotic host cell of the invention is a Chinese hamster ovary cell.
  • VH and VL sequences of amino acid cryptic epitopes 287-302 of human EGFR The single-chain antibody against the occult epitope contained in amino acids 287-302 of human EGFR may be 1) 806
  • the VH and VL of the antibody have nucleotide sequences such as SEQ ID NO. 1 and SEQ ID NO. 3 in US7589180B2, respectively. Shown, or 2) the VH and VL of the 7B3 antibody, the nucleotide sequences of which are shown in SEQ ID NO: 0.13 and SEQ ID NO.
  • VL and VH genes of the 806 antibody were obtained by PCR, respectively.
  • the VL gene was using primers 5 ⁇ 806-2 and 3'L806 ; the VH gene was using primers 5 ⁇ 806 and 3 ⁇ 806.
  • VL or VH genes of the 7 ⁇ 3 antibody were obtained by PCR, respectively.
  • the VL gene was using primers 5'L7B3-2 and 3'L7B3; the VH gene was using primers 5 ⁇ 7 ⁇ 3 and 3 ⁇ 7 ⁇ 3.
  • 3'L806 ccgccagagccacctccgcctgaaccgcctccaccacgtttgatttccagcttgg (SEQ ID NO. 16) Amplification of the primer used for the VH region of the 806 antibody
  • 3 ⁇ 806 ggatccaccacctcctgcagagacagtgac (SEQ ID NO. 18) Primer used to amplify the VL region of the 7B3 antibody
  • 5'L7B3-2 gttgctttggtttccaggtgcaagatgtgatattcagatgacc (SEQ ID NO. 19)
  • the nucleotide sequences of the VH and VL genes of the murine anti-human CD3 antibody were obtained by PCR amplification of the sequences SEQ ID NO. 9 (847-1203) and SEQ ID NO. 9 (1258-1575) shown in US71 12324B1.
  • the nucleotide sequences of the VL and VH domains of the anti-human CD3 antibody were used as follows.
  • 3'HCD3 cacttccaccagaacctccacttccaccttcgactgaggagactgtgag (SEQ ID NO. 25) Primers used to amplify the VL region of an anti-human CD3 antibody
  • 5'LCD3 ctggtggaagtggaggttcaggtggagtcgacgacattcagc (SEQ ID NO. 26)
  • the nucleotide sequence of the VH and VL regions of the 806 antibody obtained by the above PCR amplification and the nucleotide sequence encoding the linker 1 amino acid (GlyGlyGlyGlySer) 3 and the coding linker 2 amino acid (GlyGlyGlyGlySer) were amplified by fusion-PCR.
  • VL806-Linker 1-VH806-Linker 2 was obtained; and the nucleotide sequence of the above-mentioned PCR-amplified and anti-human CD3 antibody VH and VL regions and the nucleic acid sequence encoding the linker 3 amino acid VE(GGS)4GG were subjected to fusion PCR (fusion -PCR) The VHCD3-linker 3-VLCD3 was amplified separately.
  • the amplified product is then amplified by fusion PCR to obtain a nucleotide sequence of a single-chain bifunctional antibody, and the joining sequence is as follows:
  • the sequence after ligation of the above linker ([VL 8 . 6 -linker 1-VH 8 . 6 -linker 2-VH CD3 -linker 3-VL CD3 ]) was then subjected to a third round of amplification by the following primers to A signal peptide sequence was added to the end and a site for introduction of the restriction endonuclease Nhel, and a histidine tag was added at the C-terminus and a restriction endonuclease Notl site was introduced.
  • L806-1 ctagctagccaccatggtgtccacagctcagttccttgcattcttgttgctttggtttc (SEQ ID NO. 28)
  • 3'LCD3 ctatgcggccgcctaatgatgatggtgatgatgtttcagctcca (SEQ ID NO. 27) Amplified sequence SEQ ID NO: 10 was simultaneously digested with restriction endonuclease Nhel/Notl-HF Double digestion in buffer 2 according to the reaction conditions recommended by the enzyme supplier (New England Biolabs, NEB).
  • the nucleotide sequence of the VH and VL regions of the 7B3 antibody obtained by the above PCR amplification and the nucleotide sequence encoding the amino acid (GlyGlyGlyGlySer) 3 and the amino acid (GlyGlyGlyGlySer) encoding the linker 2 are amplified by fusion-PCR.
  • VL 7B3 - linker 1 -VH 7B3 - linker 2 is obtained; and the nucleotide sequence of the above-mentioned PCR-amplified and anti-human CD3 antibody VH and VL regions is fused to the nucleic acid sequence encoding the linker 3 amino acid VE(GGS) 4 GG
  • the VH CD3 -linker 3-VL CD3 was amplified by PCR (fusion-PCR).
  • the above-described ligated sequence [VL7B3-linker 1-VH7B3-linker 2] is then further amplified by primer pairs as shown in SEQ ID NOS: 20 and 29 to add a signal peptide sequence and a Nhel site at the N-terminus, and The BmHI site was introduced at the C-terminus.
  • This further amplified sequence SEQ ID NO: 12 was digested with Nhel and BamHI and carried out in buffer 2 according to the reaction conditions recommended by the enzyme supplier (NEB).
  • 5'L7B3-1 ctagctagccaccatggtgtccacagctcagttccttgcattcttgttgctttggtttc(SEQ ID NO. 20) 3 ⁇ 7 ⁇ 3-2: tcttgccagttcagcccctgactgctgcagtttgatatcggatccaccacctccg
  • SEQ ID NO. 29 The vector pH-806/CD3 constructed in the above Example 2 was digested by the same Nhel and BamHI. The long fragment obtained by digestion is ligated to SEQ ID NO: 12, whereby the nucleotide sequence of SEQ ID NO: 11 encoding the 7B3/CD3 single-chain difunctional antibody polypeptide is cloned into a vector, and the new vector is named pH- 7B3/CD3, its detailed structure is shown in Figure 2
  • the expression vectors PH-806/CD3 and pH-7B3/CD3 were transfected into Chinese hamster ovary (CHO) cells according to the instructions of FreeStyle MAX Reagent Transfection Reagent (from Invitrogen), respectively, and then according to the OptiCHOTM Protein Expression Kit (from Invitrogen) Screening for stable clones.
  • Stable clones of CHO cells transfected with one of the above expression vectors, respectively were cultured in shake flasks at 37 ° C, 130 rpm for 7 days, and the medium used was CD OptiCHO (from Gibco ). The culture supernatant was obtained by centrifugation and then stored at -20 °C.
  • Protein purification was performed using a histidine affinity column (His Trap HP column, from GE Healthcare) according to the method steps provided by the manufacturer. Specifically, the column was equilibrated with buffer A (20 mM sodium phosphate pH 7.4, 0.4 M NaCl), and then the cell culture supernatant (500 mL supernatant) was added to the column (1 mL) after dialysis against PBS. , the flow rate is 3ml/min. The column was then washed with 5 volumes of buffer A and 10 volumes of buffer A containing 50 mM imidazole to remove the heteroprotein. The bound protein of interest was eluted with the same buffer A supplemented with 250 mM imidazole. All purification steps were performed at 4 °C.
  • the purified 806/CD3 and 7B3/CD3 proteins were detected by reducing SDS-PAGE.
  • the molecular weights of the two single-chain bifunctional antibody molecules were all around 60 kD as shown in Fig. 3A, which was consistent with the calculation of the amino acid sequence of 806/.
  • the purified protein is subjected to Western blotting by an anti-histidine antibody, and the results are as follows.
  • Figure 3B shows that the obtained proteins all have a histidine tag and have a molecular weight of about 60 kD.
  • the concentration of 806/CD3 and 7B3/CD3 in the supernatant of transfected CHO cells was approximately 3 mg/L by ELISA.
  • the purified protein concentration was detected at a wavelength of 280 nm of 0.5 mg/L.
  • the single-chain bifunctional antibody obtained by one-step purification of the histidine affinity chromatography column was further subjected to gel filtration chromatography for separation of monomers and aggregates.
  • the prepacked column superdex 200 10/300 GL (from GE Healthcare) was equilibrated with 2 column volumes of PBS buffer, and the sample was loaded with 500 ⁇ l via the loading loop at a flow rate of 0.4 ml/min and then with 1 column.
  • the volume of PBS was eluted.
  • Fig. 10A the dimer protein peaked at 13 ml
  • the monomer protein peaked at 15 ml.
  • the purity analysis of the monomeric protein by gel filtration chromatography was carried out in the same manner as above, and the results are shown in Fig. 10B as having a purity of more than 95%.
  • FACS fluorescence activated cell sorter
  • each tumor cell as listed in Table 1 was inoculated into a 6 cm dish, and the inoculated cell density was about 90%, and cultured overnight at 37 °C.
  • NBS PBS phosphate buffered saline
  • the two experimental groups were added with the test antibodies 7B3/CD3 and 806/CD3, respectively, while one control group was added with antibody NGR/CD3 as a negative control, and the other control group was a non-antibody PB S blank control.
  • the final concentration of each antibody was 20 ⁇ ⁇ / ⁇ 1, and 100 ul was added to each tube. Ice bath, 45 minutes.
  • the fluorescent peaks of the bifunctional antibody 7B3/CD3 shown by green in the present invention and the fluorescent peak of the bifunctional antibody 806/CD 3 shown in blue, and the negative control (NGR/CD3) and The blank control (PB S ) showed significant differences compared to the ability to efficiently bind to U87 MG-EGFRvIII and A431 cells.
  • the two bifunctional antibodies of the invention also bind to U87 MG-de4 EGFR, NCI-1650 and NCI-1975, but are less efficient than U87 MG-EGFRvIII or A431.
  • both antibodies (7B3/CD3 and 806/CD3) were almost incompatible with U87 MG cells. These results indicate that 7B3/CD3 and 806/CD3 can specifically bind to tumor cells of mutant human EGFR and overexpressed EGFR, and hardly bind to tissues that normally express EGFR.
  • Figure 4G shows that the bifunctional antibodies of the invention (7B3/CD3 and 806/CD3) and the negative control antibody (NGR/CD3) bind efficiently to Jurkat cells (human peripheral blood leukemia T cells) expressing CD3 at substantially the same level.
  • Figure 4H shows that the bifunctional antibodies (7B3/CD3 and 806/CD3) and the negative control antibody (NGR/CD3) of the present invention bind efficiently to human peripheral blood mononuclear cells (PBMC) at similar levels.
  • Figures 4G and 4H show that the 7B3/CD3 and 806/CD3 bifunctional antibodies of the present invention can specifically bind to CD3 antigens on the surface of T cells.
  • Figures 4A-4H show the ability of the bifunctional antibodies of the invention (7B3/CD3 and 806/CD3) to specifically bind to tumor cells expressing mutant human EGFR and overexpressed E GFR, while capable of Specifically binds to CD3 expressing effector cells (T cells).
  • the 806 monoclonal antibody (mAb 806) can bind to the EGFR concealed epitope CC16 polypeptide ( 287 CKGYEDSRVMEAGDEC 3 . 2 ) (Johns TG, et al" J. Biol. Chem. 2004; 279(29): 30375-84. ) Binding. It is generally believed that the conversion of a monoclonal antibody into a single-chain antibody should not alter its antigen-binding epitope specificity. To further verify that the bifunctional antibody of the present invention can bind to the hidden epitope, the experimental use contains The two recombinant proteins of this epitope were used as antigens for ELISA detection.
  • Protein coating three antigens rEGFRvIIIex (extracellular domain protein of EGFRvIII, its preparation method see patent WO/2011/035465), N12-CC16 recombinant protein (domain of M13 phage pill protein)
  • the fusion polypeptide of N1N2 and CC16 is prepared by Jiang H, et al., J Biol. Chem., 2011, 286(7): 5913-20) and BSA (purchased from Shanghai Bioengineering Co., Ltd.) control protein, respectively. It was used to coat each well of a 96-well plate in an amount of 50 ng/well (lng/ ⁇ , 50 ⁇ l/well), and incubated at 37 ° C for 2 h.
  • Blocking Wash 3 times with 0.1 M phosphate buffer (PBS), then add 5% PBS skim milk powder (Guangming Dairy Co., Ltd.) for 2 h at 37 °C.
  • PBS phosphate buffer
  • the antibody to be tested 806/CD3, 783/ 03 was diluted to 2 ng with 5% ? 88 skim milk powder, 50 ⁇ l per well, and incubated at 37 ° C for 1 h.
  • PB ST was washed 3 times, and goat anti-mouse IgG-HRP (purchased from Santa Cruz) diluted 1:2000 was added and incubated at 37 ° C for 1 h.
  • Color development Wash PBST 5 times, add ABTS color solution 100 ⁇ 7 well, and develop color at 37°C for 10min.
  • Detection Absorbance values were measured at a wavelength of 405 nm using a Bio-Rad Model 680 microplate reader.
  • Bifunctional antibodies 806/CD3 and 7B3/CD3 can specifically bind to Nl 2-CC 16 (CC16 polypeptide fused at the carboxy terminus of the N1N2 domain of M13 phage pill protein) and rEGFRvIIIex, respectively. .
  • the binding strength of these two antibodies to the above two antigens is significantly different from the non-specific binding of BSA.
  • the bispecific antibody 806/CD3 and 7B3/CD3 binding epitopes are all CC16 polypeptides, ie ( 287 CKGYEDSRVMEAGDEC 302 ).
  • PBMC Peripheral blood mononuclear cells
  • the mixed cell suspension was added to a 96-well plate at a volume of 75 ⁇ L/well. Then add 25 ⁇ L of the following ten-fold serial dilutions of the following reagents from 1000 ng/mL to 0.1 ng/mL for each well:
  • NGR/CD3 single-chain bifunctional antibody (negative control, NGR is a neovascular targeting peptide, which has no cross-binding site with EGFR. It is prepared according to conventional methods)
  • the CytoTox 96® non-radioactive cytotoxicity assay is based on a colorimetric assay that replaces the 51 Cr release method.
  • the CytoTox 96® assay quantitatively measures lactate dehydrogenase (LDH).
  • LDH lactate dehydrogenase
  • LDH is a stable cytoplasmic enzyme that is released when cells are lysed and released in much the same way as 51Cr is released in radioactive analysis.
  • the released LDH medium supernatant can be detected by a 30-minute coupled enzyme reaction in which LDH converts a tetrazolium salt (INT) into red formazan (formazan).
  • INT tetrazolium salt
  • formazan red formazan
  • EGFR-associated tumor cells listed in Table 1 below were used to separately analyze the two bifunctional antibodies 7B3/CD3 and 806/CD3 of the present invention, and the EGFR-unrelated NGR/CD3 single-chain dual function as a control. Antibody-mediated T cell tumor killing ability.
  • the killing rate of tumor cells (i.e., % cytotoxicity) is calculated according to the following formula provided by the CytoTox96® non-radioactive cytotoxicity test G1780 product instruction manual: Experimental-effector cell spontaneous-target cell spontaneous
  • Cytotoxicity 0 / 0 X 100 Target cell max - target cell spontaneous
  • Experiment refers to the release of LDH from the experimental wells to which antibody/effector cells/target cells are added.
  • Effective cells spontaneously refers to the spontaneous release of LDH by effector cells.
  • target cell spontaneous refers to the release of LDH produced when cells are not treated by other factors.
  • Maximum target cells is the LDH release produced by complete lysis of target cells after treatment with 0.8% Triton X-100.
  • Target cell maximal - target cell spontaneous represents the release of LDH from complete lysis of cells after external treatment.
  • the minimum specific cytotoxicity was 23.3, and the maximum was 75.2; the smallest specific cytotoxicity in the above-mentioned tumor cell group treated with 806/CD3
  • the % is 28.7 and the maximum is 97.9.
  • the above-mentioned bifunctional specific antibody 7B3/CD3 or 806/CD3 has very low cytotoxicity to tumor cells expressing low levels of endogenous normal EGFR, such as U87 MG, which are 1.3 and 9.4, respectively, which are significantly lower than the above-mentioned expression mutations. % cytotoxicity of EGFR and/or tumor cells overexpressing EGFR.
  • 7B3 / CD3 EC 5 bifunctional single-chain antibody.
  • the value is 0.29 ng/ml.
  • 7B3 / CD3 EC 5 bifunctional single-chain antibody. Value of 53.6ng / ml, and 806 / CD3 single chain diabodies EC 5. The value is 1000 ng/ml.
  • the suspension was mixed with a cell concentration of 1 ⁇ 10 6 by U1 MG-EGFRvIII or NCI-H1975 tumor cells and cell concentration.
  • mice were intravenously administered with 0.4 mg/kg/d and 0.04 mg/kg/d of 7B3/CD3 and 0.04 mg/kg 806/CD3, respectively. For 5 consecutive days.
  • mice were intravenously administered with 0.4 mg/kg/d and 0.04 mg/kg/d of 7B 3/CD3, respectively, and the administration was repeated for 10 consecutive days.
  • the control group consisted of two groups administered with PBS medium, namely, a control group 1, a group in which only tumor cells were injected; and a control group 2, a group in which tumor cells and PBMC were injected, to evaluate the non-specific killing effect induced by PBMC effector cells.
  • the size of the tumor was measured at a designated daily caliper, and the tumor volume was calculated according to the following formula:
  • control group 2 ie, mice injected only with human peripheral blood lymphocytes and tumor cells, but no bispecific antibody
  • control group 1 i.e., the group in which only tumor cells were injected
  • the 806/CD3 antibody showed strong concentration at a concentration of 0.04 mg/kg/d and 7B3/CD3 at a concentration of 0.4 mg/kg/d.
  • the ability to inhibit the growth of U87 MG-EGFRvIII was 74% and 80%, respectively, on day 23 after cell seeding (p ⁇ 0.05 compared with control 2).
  • the tumor growth inhibition rate was 35.3% (compared to control 2).
  • the 7B3/CD3 treated mice exhibited a dose-dependent anti-tumor growth effect compared to the control groups 1 and 2.
  • One of the 6 mice receiving the 0.4 mg/kg/d dose group had no tumor formation on the 30th day after cell seeding, and all of the 0.04 mg/kg/d dose group mice were tumorigenic.
  • the tumor suppression effects of the two groups were: 0.4 mg/kg/d dose group, 87% (p ⁇ 0.05) and 0.04 mg/kg/d dose group, 35% (p ⁇ 0.05). .
  • This experiment consists of two experimental groups, three tumor cells treated with SP 806/CD3 and three tumor cells treated with ch806.
  • the relevant experimental materials involved are basically prepared as described in Example 6, except for (1) humanized singles.
  • the cloned antibody ch806 was prepared as follows: A nucleic acid encoding the heavy and light chain variable regions of the ch806 antibody was artificially synthesized according to the sequence disclosed in CN 102405235 A, and then Nhel and Apal cleavage sites were added at both ends of the heavy chain coding sequence, The EcoRV and Bsiwl restriction sites are added to both ends of the strand coding sequence, and then the above-mentioned heavy chain variable region and light chain variable region are separately loaded into the expression vector pH and pK, as described in, for example, CN101602808B, in particular, in Example 7, pH-ch806 and pK-ch806 were obtained.
  • PH-ch806 and pK-ch806 were co-transfected into CHO-DG44 cells (Invitrogen) according to the lipofection method. After MTX screening, positive clones with high expression of antibodies were selected, and cells were domesticated to gradually adapt the cell lines. Serum-free culture. The CHO-ch806 cells successfully obtained by domestication were serum-free cultured, and the serum-free culture supernatant was collected for affinity purification of protein A (Code No. 7-5280-02, GE Healthcare Life Sciences) to obtain the purified ch806 antibody protein. .
  • U87 MG EGFR cells which are U87 MG cell lines overexpressing EGFR, the construction method of which is described in the literature (Wang H, Neoplasia, 2011, 13(5): 461-471.), U87 MG can be obtained from ATCC.
  • the mixed tumor cell suspension prepared as described in the first and second paragraphs of Example 6 was added to the 96-well plate in a volume of 75 ⁇ ?7 well. Then, each well was added with 25 ⁇ L of a ten-fold serial gradient of 20 nM to 0.0002 nM to dilute the following reagents: bifunctional antibody 806/CD3 and humanized antibody ch806.
  • Bifunctional antibodies were calculated using the GraphPad Prism 5 software (GraphPad Software inc., San Diego, USA) analysis program according to the cytotoxicity % data of Tables 8-10 and 9A-9C above and the concentration of the bifunctional antibody used.
  • the 806/CD3 single-chain bifunctional antibody had an EC50 value of 0.136 nM, while the ch806 monoclonal antibody had an EC50 value of 40.79 nM.

Abstract

一种多功能抗体多肽，包括：（a）第一功能域，其特异性识别EGFR的第287-302位氨基酸序列形成的隐蔽表位，如SEQ ID NO:1所示，（b）第二功能域，其特异性识别人T细胞表面抗原。

Description

针对表皮生长因子受体隐蔽表位和 T细胞抗原的多功能抗体多肽
技术领域
本发明涉及生物医学领域。 更具体地， 本发明涉及能识别并结合表皮生长因子 受体 (EGFR)的隐蔽表位和 Τ细胞抗原的多功能抗体多肽。本发明还涉及编码该抗体多 肽的核苷酸序列， 含有该核苷酸序列的载体， 含有所述载体的宿主细胞等。 本发明 还涉及所述多功能抗体多肽在制备抗肿瘤药物和制备诊断治疗和 /或预防肿瘤的试 剂盒中的应用。 背景技术
已有文献表明， 表皮生长因子受体（EGFR)在多种类型的人类实体瘤中过表达。 这些肿瘤包括肺癌、 结肠癌、 乳腺癌、 胃癌、 脑癌、 膀胱癌、 头颈部肿瘤、 卵巢癌、 食管癌、 肝癌、 肾癌和前列腺癌。 开发针对表皮生长因子受体家族的抗体药物为治 疗这些肿瘤提供了机会。
有至少两个针对 E GFR的抗体药物已经用于临床肿瘤治疗中， 例如爱必妥 ®(Erbitux， 又称 Cetuximab)和帕尼单抗（Panitumumab)。但是这些抗体的应用受到一 些限制。 这是因为一方面 EGFR在很多人体实体脏器如皮肤和肝脏中均有表达， 导致 上述抗体药物可能在人体内给药后被这些脏器所吸收（Baselga J et al.Phase I studies of anti-epidermal growth factor receptor chimeric antibody C225 alone and in combination with cisplatin.J.Clin.Oncol. 2000 Feb; 18(4): 904- 14.和 Faillot T et al. A phase I study of an anti-epidermal growth factor receptor monoclonal antibody for the treatment of malignant gliomas. Neurosurgery.1996 Sep;39(3) :478-83. ) 。 另一方面， 这些抗体对正常表达 EFGR的组织的非特异性作用， 可能导致在使用抗体药物如 Erbitux时， 产生 all作用如皮疫 ( Agero AL, et al, Dermatologic side effects associated with the epidermal growth factor receptor inhibitors. J Am Acad Dermatol. 2006 Oct;55(4):657-70. ) 等， 严重的可导致患者不得不停用药物。
为了减少现有 E G F R抗体由于与正常组织的 E G F R结合造成的副作用， 人们筛选了 多种针对肿瘤特异性 EGFR表位的单克隆抗体，如针对 de2-7EGFR (又称为 EGFRvI I I ) 由于缺失了 EGFR的第 2-7个外显子的 267个氨基酸所产生的铰链部位 （junction ) LEEKKGNY的抗体， 参见专利 PCT/US2004/020295公开的抗体 131 ; 又如针对 EGFR的隐 蔽表位 （ cryptic epitope ) 的抗体 mAb806, CH12等， 参见美国专利申请 US201 1 /0076232A1和 W0/201 1 /035465。 当 EGFR被激活、 过表达、 或突变时， 可能导 致该隐蔽表位（287CGADSYEMEEDGVRKC302 )的暴露并与针对该表位的抗体，如 mAb806， 结合 ( Garrett TP et al. Antibodies specifically targeting a locally misfolded region of tumor associated EGFR. Proc Natl Acad Sci U S A. 2009; 106(13):5082-7. ) 。 这些抗体 在动物实验中都显出一定的抗肿瘤效果， 并且显示出比之前的抗 EGFR抗体更好的肿 瘤特异性。抗体 mAb806抗体所衍生的人鼠嵌合抗体 ch806在临床 I期试验中表现出很 强的肿瘤靶向性， 而且没有观察到明显的皮肤毒性（ Scott AM, Lee FT et al, A phase I clinical trial with monoclonal antibody ch806 targeting transitional state and mutant epidermal growth factor receptors. ProcNatlAcadSci U S A. 2007 Mar 6; 104( 10) :4071 -6. ) 。 在 5 mg/m2的剂量下 ch806就可以显示出在肿瘤部位的吸收。 而 之前的其它抗 EGFR抗体要显示出在肿瘤部位的吸收大概需要 10到 20倍的剂量（Divgi CR et al. Phase I and imaging trial of indium 1 1 1 -labeled anti-epidermal growth factor receptor monoclonal antibody 225 in patients with squamous cell lung carcinomaJ Natl Cancer Inst. 1991 Jan 16;83(2) :97- 104. ) 。 这表明 mAb806的衍生抗体由于不与正常组 织 EGFR结合，所以有更好的药物学分布。但这并不代表它的用药最小剂量是 5mg/m2。 事实上， 806抗体在动物药效学研宄时， 较有效的剂量是 lmg/小鼠（该剂量约相当于 50mg/kg) ( RushikaM. Perera, et al. Treatment of Human Tumor XenograftswithMonoclonal Antibody 806 in Combinationwith a Prototypical Epidermal Growth Factor Receptor ASpecific Antibody Generates Enhanced Antitumor Activity. Clin Cancer Res 2005; 1 1 (17) 6390-9 ) 。 此外针对上述表位的这些抗体， 如 CH 12， 对 另外一些形式的表达 EGFR (比如 T790M突变的 EGFR ) 的肿瘤却没有太明显效果。 上 述 T790M突变经常出现在一种与 EGFR相关的肺腺癌使用小分子酪氨酸酶抑制剂治疗 一段时间后 ( Xu Y et.al, Acquired resistance of lung adenocarcinoma to EGFR-tyrosine kinase inhibitors gefitinib and erlotinib. Cancer Biol Ther. 2010 Apr;9(8): 572-82. Epub 2010 Apr 26 ) 。
如何改造这些抗体， 提高其抗肿瘤活性 （即降低最小作用剂量） ， 并扩展其抗 肿瘤作用的范围是有价值的。 一种感兴趣的提高抗体的抗肿瘤活性的方式是构建双功能抗体。 能特异性识别 EGFR和 CD3抗原的双功能抗体在现有技术中已有描述， 其一部分功能域特异性针 对 EGFR , 另一部分功能域特异性针对 T细胞的 CD3抗原。 采用 Cetuximab或 P anitumumab抗体与抗 CD 3抗体所构建的双功能抗体显示出很好的抗肿瘤活性， 但 是在灵长类动物实验中， 发现这些双功能抗体较强的针对表达 EGFR的正常细胞或 组织的毒性作用 (Lutterbuese R, Raum T et.al T cell-engaging BiTE antibodies specific for EGFR potently eliminate KRAS- and B RAF -mutated colorectal cancer cells. ProcNatlAcadSci U.S.A. 2010;107(28): 12605-10.)。
双功能抗体的制备技术尽管已经存在， 但对制成的双功能抗体的各个功能域是 否能保留原有的抗原结合特异性， 以及更进一步是否形成肿瘤杀伤活性是不确定的， 这是由于生物学实验的复杂性的本质所决定的。
本领域还需要针对 EGFR相关肿瘤的既具有提高的肿瘤杀伤生物学活性， 也具 有提高的肿瘤识别特异性的双功能抗体。 本发明实现了这一目的。 发明内容
本申请的第一方面涉及一种多功能抗体多肽， 其包括
( a) 第一功能域， 其特异性识别 EGFR的第 287-302位氨基酸序列形成的隐 蔽表位， 如 SEQ ID N0: 1所示，
( b ) 第二功能域， 其特异性识别人 T细胞表面抗原。
本申请的第二方面涉及一种编码所述多肽的核苷酸序列。
本申请的第三方面涉及一种包含所述核苷酸序列的载体。
本申请的第四方面涉及包含所述载体的真核宿主细胞或原核宿主细胞。
本申请的第五方面涉及所述的多肽在制备诊断，治疗和 /或预防肿瘤的药物中的 应用。
本发明中所使用的术语的含义如下：
"特异性识别"和特异性的程度可以通过经典的免疫学技术来判断， 包括但不 限于免疫印迹， 免疫亲和层析， 流式细胞分析等。 在本发明中， 特异性识别优选通 过流式细胞技术来确定， 而具体情况下特异性识别的标准可由本领域一般技术人员 根据其掌握的本领域常识来判断。
"功能域"指的是能够特异性识别抗原的抗体或抗体片段， 包括完整抗体， 单 链抗体（scFV) ， Fd片段， Fab片段， F(ab')2片段， 单结构域抗体片段， 分离的 CDR 片段， 及其衍生物。
"完整抗体" 由两个同样的重链和轻链组成， 各条链分别包含一个可变区 （V 区） 和一个或多个恒定区 （C区） 。 可变区负责与抗原结合， 而恒定区主要负责结 合效应分子。 在各可变区有三个具有高度多样性的柔性的环， 称作互补决定区 ( CDR)，其主要负责识别抗原。可变区的其他部分包含刚性的 β片层并支持所谓的 框架区 （FRs ) 。 CDR和 FR间隔排列形成三明治结构。
"单链抗体 （scFV) 片段"指的是通过基因工程构建的抗体片段， 其是有通过 接头 （linker) 连接的重链可变区 （VH) 和轻链可变区 (VJ的重组蛋白， 接头使得 这两个结构域相关联以形成抗原结合位点。 ScFV的大小一般是一个完整抗体的 1/6。
" Fd片段" 指的是由重链 VH和 CH1组成抗体片段。
" Fab片段" 指的是由 Fd片段 （由重链 VH和 CH1组成） 和整条轻链通过链间二 硫键形成的异二聚物。 " Fab抗体" 的大小是完整抗体的 1/3， 其只包含一个抗原结 合位点。
" F(ab')2片段 "指的是包含两个相连的 Fab片段的二价片段。
"单结构域抗体" 由重链可变区或轻链可变区组成。 由于该抗体片段只由一个 结构域组成， 所以得名。 该片段的大小是一个完整抗体的 1/12。
"抗体的衍生物" 包括例如当通过噬菌体展示技术获得所述抗体的衍生物时， 可使用如 BIAcore系统中使用的表面等离子共振技术来增加与 EGFR或 CD3抗原表 位结合的噬菌体抗体的效率 （Schier, 人抗体杂交瘤 7 ( 1996 ) ， 97-105； Malmborg, 免疫学方法杂志 183 ( 1995 ) ， 7-13 ) 。 还包括， 例如 WO 89/09622中描述的嵌合抗 体的产生的方法， EP-A10239400和 WO90/07861中描述的人源化抗体产生的方法， WO91/10741,WO94/02602和 W096/33735中有描述的产生异种抗体例如小鼠中的人 抗体的方法。
本发明使用的抗体或其片段可单独或联合使用本领域已知的常规技术， 例如氨 基酸缺失、 插入、 取代、 增加、 和 /或重组以及 /或其他修饰方法作进一步修饰。 根 据一种抗体的氨基酸序列在其 DNA序列中引入这种修饰的方法对本领域技术人员 来说是众所周知的； 见例如， Sambrook, 分子克隆： 实验手册， Cold Spring Harbor Laboratory(1989)N.Y.。 所指的修饰优选在核酸水平上进行。
本发明使用的抗体或抗体片段可以是人源化的， 嵌合的或鼠源的。 附图说明
图 1 上半部分为 pH-806/CD3表达载体结构示意图， 下半部分为插入的基因片 段的放大示意图
图 2 pH-7B3/CD3表达载体结构示意图
图 3A 纯化的双功能抗体多肽的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳
( SDS-PAGE) 检测， M为分子量标记 （SDS-聚丙烯酰胺凝胶电泳小分子量标准蛋 白质由上海升正生物技术有限公司提供）， 第一栏为 806/CD3 , 第二栏为 7B3/CD3。
图 3B 纯化的双功能抗体多肽的蛋白质杂交检测第一栏为 806/CD3 , 第二栏为 7B3/CD3。
图 4A 通过荧光激活细胞分选仪（FACS )显示的三种双功能单链抗体 (NGR/CD3 , 7B 3/CD3和 806/CD3 )与 U87 MG肿瘤细胞的特异性结合的测定
图 4B 通过荧光激活细胞分选仪（FACS )显示的三种双功能单链抗体 (NGR/CD3 , 7B3/CD3和 806/CD3)与 U87 MG-EGFRvIII肿瘤细胞的特异性结合的测定
图 4C 通过荧光激活细胞分选仪（FACS )显示的三种双功能单链抗体 (NGR/CD3 , 7B3/CD3和 806/CD3)与 A431肿瘤细胞的特异性结合的测定
图 4D 通过荧光激活细胞分选仪（FACS )显示的三种双功能单链抗体 (NGR/CD3 , 7B3/CD3和 806/CD3)与 U87 MG-de4 EGFR肿瘤细胞的特异性结合的测定
图 4E 通过荧光激活细胞分选仪（FACS )显示的三种双功能单链抗体 (NGR/CD3 , 7B3/CD3和 806/CD3)与 NCI-H1650肿瘤细胞的特异性结合的测定
图 4F 通过荧光激活细胞分选仪（FACS )显示的三种双功能单链抗体 (NGR/CD3 , 7B3/CD3和 806/CD3)与 NCI-H1975肿瘤细胞的特异性结合的测定
图 4G 通过荧光激活细胞分选仪（FACS )显示的三种双功能单链抗体 (NGR/CD3 , 7B 3/CD3和 806/CD3 )与 Jurkat肿瘤细胞的特异性结合的测定
图 4H 通过荧光激活细胞分选仪（FACS )显示的三种双功能单链抗体 (NGR/CD3 , 7B3/CD3和 806/CD3)与 PBMC细胞的特异性结合的测定
图 5A 806/CD3的抗原结合表位分析 （ELISA)
图 5B 7B3/CD3的抗原结合表位分析 （ELISA)
图 6A 系列梯度稀释的三种双功能单链抗体 (NGR/CD3 , 7B3/CD3和 806/CD3 ) 诱导的 T细胞对 U87 MG肿瘤细胞的杀伤率比较。
图 6B 系列梯度稀释的三种双功能单链抗体 (NGR/CD3 , 7B3/CD3和 806/CD3)诱 导的 T细胞对 U87 MG-EGFRvIII肿瘤细胞的杀伤率比较。
图 6C 系列梯度稀释的三种双功能单链抗体 (NGR/CD3 , 7B3/CD3和 806/CD3)诱 导的 T细胞对 A431肿瘤细胞的杀伤率比较。 图 6D 系列梯度稀释的三种双功能单链抗体 (NGR/CD3 , 7B3/CD3和 806/CD3)诱 导的 T细胞对 U87 MG-de4 EGFR肿瘤细胞的杀伤率比较。
图 6E 系列梯度稀释的三种双功能单链抗体 (NGR/CD3 , 7B3/CD3和 806/CD3)诱 导的 T细胞对 NCI-H 1650肿瘤细胞的杀伤率比较。
图 6F 系列梯度稀释的三种双功能单链抗体 (NGR/CD3 , 7B3/CD3和 806/CD3)诱 导的 T细胞对 NCI-H1975肿瘤细胞的杀伤率比较。
图 7 NOD/SCID荷瘤（U87 MG-EGFRvIII)小鼠模型显示的不同浓度双功能抗体 ( 7B3/CD3和 806/CD3)治疗组和对照组的抗肿瘤活性测定
图 8 NOD/SCID荷瘤 （NCI-H1975 ) 小鼠模型显示的不同浓度双功能抗体
( 7 B 3 / C D 3 )治疗组和对照组的抗肿瘤活性测定
图 9A-9C 806/CD3和 ch806对三种不同肿瘤细胞系的杀伤率比较
图 10A-B 基因工程表达的 806/CD3双功能抗体的凝胶过滤层析曲线 具体实施方式
本发明提供针对一系列肿瘤的多功能抗体， 所述一系列肿瘤包括包含扩增的 EGFR基因的肿瘤以及表达变异的 EGFR，如表达缺失第 2-7外显子的 de2-7EGFR的肿 瘤， 包括但不限于肺癌、 结肠癌、 乳腺癌、 胃癌、 脑癌、 膀胱癌、 头颈部肿瘤、 卵 巢癌、 肾癌和前列腺癌。 该多功能抗体包含一个特异性识别包含有扩增的 EGFR基 因或具有突变的 EGFR基因的肿瘤表达的 EGFR的第 287-302位氨基酸序列形成的隐 蔽表位， 如 SEQ ID NO: l所示， 和一个识别人 T细胞表面抗原的第二功能域。
本发明的多功能抗体能够在非常低的浓度，如 100pg/mL - Ing/mL下在体外和体 内诱导针对肿瘤细胞的 T细胞杀伤毒性。 即使在使用较低的效应细胞 (E):靶细胞 比率， 如 10 : 1时， 也能观察到相关肿瘤细胞系的特异性裂解， 而无需任何其他形 式的 T细胞预剌激。 本发明针对的相关肿瘤细胞包括表达突变的 EGFR如 de2-7EGFR、 表达扩增的 EGFR的上述肿瘤细胞可以从商业来源获得， 例如 NCI-1650, NCI-1975, A431 , 得自美国典藏培养物中心 (American Type Culture Collection, ATCC ) ， 又 例如 U87 MG-EGFRvIII, 其是稳定表达 EGFRvIII的 U87 MG细胞系， 其构建方法参 见文献 (Jiang H, J Biol. Chem., 2011,286(7): 5913-20), U87 MG也可以得自 ATCC。
此外， 本发明的多功能抗体几乎不会和 EGFR没有扩增或没有突变的细胞 （如 U87 MG) 结合。 由本发明的多功能抗体制备的抗肿瘤药物具有改善的体内肿瘤靶 向性， 降低对正常组织的杀伤。 本发明的第一功能域特异性识别如 SEQ ID ΝΟ: 1所示的氨基酸序列形成的隐蔽 表位。 特异性识别上述隐蔽表位 （如人野生型 EGFR第 287-302位氨基酸所包含的表 位） 的抗体已有公开， 例如美国专利申请 US2011/0076232A1和 WO/2011/035465所 述 mAb806， 及其衍生抗体， 和中国专利申请 CN101602808A所述的 12H23及其衍生 抗体。 此外， 制备其他特异性针对上述隐蔽表位的抗体可以根据本领域已知的方法 来进行。 本发明的第一功能域能特异性与表达多拷贝 EGFR的肿瘤和表达变异的 EGFR如 de2-7EGFR的肿瘤结合。
本发明的第二功能域包括特异性识别人 T细胞抗原的抗体和抗体片段。 所述 T 细胞表面抗原包括但不限于 CD3， CD16, CD28。优选地，该 T细胞表面抗原是 CD3。 CD3是 T细胞表达的抗原。 它是多分子 T细胞受体复合物 (T cell receptor complex, TCR)的一部分， 含有三条不同链 CD3 e， CD3 δ , CD3 γ。 将 CD3簇集于 Τ细胞上 (例如通过固定的抗 CD3抗体） 可导致 Τ细胞活化， 这类似于 Τ细胞受体的结合， 但 不依赖于其克隆的类型特异性。 实际上， 大多数抗 CD3抗体识别的是 CD3 ε链。 这 种双功能抗体杀伤肿瘤细胞主要是通过剌激免疫系统来完成的， 而且不受主要组织 相容性抗原 (MHC)限制。当双功能抗体中的抗 CD3抗体部分和 Τ细胞表面的 CD3结合 时可以产生杀伤肿瘤细胞的效应。
在本发明的一个实施方案中， 第一功能域包含至少一个具有选自如下序列 SEQ ID NO. 2, SEQ ID NO. 3和 SEQ ID N0.4的抗 EGFR抗体重链可变区的互补决定区 ( CDR) 。 优选地， 第一功能域是顺序包含上述三个 CDR的整个重链可变区。
在本发明的另一个实施方案中， 第一功能域包含至少一个具有选自如下序列 SEQ ID NO. 5， SEQ ID NO. 6和 SEQ ID N0.7的抗 EGFR抗体轻链可变区的互补决 定区 （CDR) 。 优选地， 第一功能域是顺序包含上述三个 CDR的轻链可变区。
更优选地， 第一功能域是包含顺序连接的上述整个重链可变区和整个轻链可变 区的单链抗 EGFR单体。
在本发明的另一个实施方案中， 其中第二功能域为单链抗 CD3抗体。
本发明多功能抗体的两个功能域可以包含两个不同的单链抗体， 因此该抗体又 称作单链双功能抗体。 在一个实施方案中， 所述的双功能抗体多肽具有如 SEQ ID NO. 8所示的氨基酸序列。在另一个实施方案中，所述的双功能抗体多肽具有如 SEQ ID NO. 9所示的氨基酸序列。
在本发明的另一个实施方案中还包含位于所述第一和第二功能域之间或位于 第一或第二功能域内部的不同互补决定区之间的接头。所述多肽接头优选包括几个 亲水性肽键氨基酸， 其长度足够跨越一个含所述结合位点的所述功能域的 C末端和 另一个含所述结合位点的所述功能域的 N末端之间的距离， 所以当本发明多功能抗 体置于水溶液中时能呈现适合于结合的构象。 优选的， 所述多肽接头包括多个甘氨 酸、 丙氨酸和 /或丝氨酸残基。 在本发明的一个特定优选例中， 本发明多肽中的所述 多肽接头的氨基酸序列（GlyGlyGlyGlySer) n，其中 n为 1到 5的整数，优选 n为 1到 3， 更优选 n为 3。
当第一和第二功能域各自含两个和两个以上可变区 （VH， VL ) 时， 优选地， 在 这可变区之间通过上述多肽接头连接。本发明多肽中的连接所述多肽接头的氨基酸 序列 （Gly Gly Gly Gly Ser) n， 其中 n为 1到 5的整数， 优选 n为 1到 3， 更优选 n为 1。
本发明抗体的所述第一和第二功能域可以是相同或不同抗体的一对 VH-VL， VH-VH , 或 VL-VL功能域。 VH和 VL功能域的顺序对本发明不是确定的， 当其顺序 倒转， 通常不会有产生功能损失。 重要的是， VH和 VL功能域的排列使抗原结合位 点可以正确折叠， 从而形成的多功能抗体具有特异性识别和结合多种抗原的功能。
在本 发 明 多 肽 的 一个优选例 中 ， 所述功 能域 的 安排顺序 为 VLEGFR- VHEGFR- HcD3 - VLcD3。
在本发明的另一方面，涉及上述所述的多肽的核苷酸序列。在一个实施方案中， 本方面涉及编码 SEQ ID NO. 8所示的氨基酸序列的核苷酸序列 SEQ ID NO. 10。在另 一个实施方案中， 本方面涉及编码 SEQ ID NO. 9所示的氨基酸序列的核苷酸序列 SEQ ID NO. 11.
在本发明的另一方面， 涉及包含有编码上述多肽的核苷酸序列的载体。 所述载 体可以是真核细胞载体或原核细胞载体， 只要所述载体满足：（a)其编码序列包含复 制起始的序列， 使得该载体能够在宿主细胞中得以复制， （b)其包含有编码筛选标记 的基因序列， 该基因编码的蛋白是该宿主细胞在特定的选择培养基中生存和生长所 必需的。 在宿主细胞如果没有被转染或转化包含该基因的载体的情况下， 宿主细胞 不能在特定选择培养基中生存。典型的筛选标记基因编码的蛋白包括具有对抗生素 或毒素具有耐受性的蛋白，抗生素或毒素包括例如氨苄青霉素、卡那霉素、四环素、 新霉素、 潮霉素、 氨甲蝶呤等； 补偿营养缺陷的相关蛋白， 供应培养基中不存在的 关键营养成分， 例如编码 D-丙氨酸消旋酶基因。 采用抗性筛选的例子包括， 通过转 染包含新霉素抗性基因的外源载体，使宿主细胞获得在含有药物新霉素或 G418的培 养基的情况下， 继续生存生长。 另外一个例子是在哺乳动物细胞例如中国仓鼠卵巢 细胞 （CHO ) 中使用二氢叶酸还原酶 （DHFR) 筛选标记， 哺乳动物细胞宿主细胞 是指 DHFR缺陷型的细胞， 不含二氢叶酸还原酶基因， 不能合成核酸， 必须在含有 HT的培养基里生长。 在利用载体转染宿主细胞时， 可以通过上述培养基条件的选 择筛选得到同时包含目的基因和 DHFR基因的外源载体的阳性克隆。 （c )其编码序 列包含启动子的序列， （d) 表达载体还可能包括其它组成序列， 包括信号肽序列、 转录终止序列、 增强子序列等， 优选地， 本发明的载体为真核细胞载体。 优选地， 本发明的载体为来自用于抗体真核表达的的 pH载体， 其包含了 CMV的启动子、 内 部核糖体进入位点序列 （Internal ribosome entry site, IRES ) 、 DHFR筛选标记等元 件。氨甲喋呤（MTX)是 DHFR的抑制剂， 可阻碍其作用。 当细胞培养基内含有 MTX 时， DHFR被抑制， 通过反馈调节， 使得该基因自我扩增， 连带其上下游基因都会 扩增， 如此目的基因也得到扩增， 即可提高目的蛋白的表达量。
在本发明的另一方面， 涉及包含有所述载体的宿主细胞， 用于表达所需的多功 能抗体多肽。 与使用的载体相适应， 本发明的宿主细胞可以是任意的原核宿主细胞 或真核宿主细胞。 真核宿主细胞， 包括酵母、 昆虫细胞、 植物细胞， 哺乳动物细胞 等可以是优选的，因为真核细胞存在复杂的目的蛋白的翻译后修饰（例如糖基化）， 越来越多的被用于规模化的培养。 常用的宿主细胞系包括猴肾细胞 （COS-7 ATCC CRL 1651 ) 、 人胚胎肾细胞 293及其亚克隆细胞系， 幼仓鼠肾细胞 （BHK， ATCC CCL10 ) ， 中国仓鼠卵巢细胞 （CHO ) 等。 优选地， 本发明的真核宿主细胞是中国 仓鼠卵巢细胞。
在本发明的另一方面，涉及所述的多功能抗体多肽在制备治疗，诊断和 /或预防 肿瘤的药物中的应用。 实施例
实施例 1
抗人 EGFR第 287-302位氨基酸的隐蔽表位的单链抗体序列和抗人 CD3单链抗体 序列的扩增
1. 1 抗人 EGFR第 287-302位氨基酸隐蔽表位的单链抗体 VH和 VL序列的扩增 抗人 EGFR第 287-302位氨基酸所包含的隐蔽表位的单链抗体可以是 1) 806抗体 的 VH和 VL， 其核苷酸序列分别如 US7589180B2中的 SEQ ID NO. 1和 SEQ ID NO.3 所示， 或者 2) 7B3抗体的 VH和 VL， 其核苷酸序列分别如 SEQ ID N0.13和 SEQ ID NO.14所示。
806抗体的 VL和 VH基因分别通过 PCR方法得到， VL基因是利用引物 5Έ806-2和 3'L806; VH基因是利用引物 5Ή806和 3Ή806。
7Β3抗体的 VL或 VH基因分别通过 PCR方法得到， VL基因是利用引物 5'L7B3-2 和 3'L7B3 ; VH基因是利用引物 5Ή7Β3和 3Ή7Β3。
扩增 806抗体的 VL区所用的引物
5'L806-2: gttgctttggtttccaggtgcaagatgtgacatcctgatgaccca (SEQ ID NO. 15)
3'L806: ccgccagagccacctccgcctgaaccgcctccaccacgtttgatttccagcttgg(SEQ ID NO. 16) 扩增 806抗体的 VH区所用的引物
5Ή806: gcggaggtggctctggcggtggcggatcggccgatgtgcagcttcagga (SEQ ID NO. 17)
3Ή806: ggatccaccacctcctgcagagacagtgac (SEQ ID NO. 18) 扩增 7B3抗体的 VL区所用的引物
5'L7B3-2: gttgctttggtttccaggtgcaagatgtgatattcagatgacc(SEQ ID NO. 19)
3 'L7B3： acctccgcctgaaccgcctccacctgaacgtttaatttccac (SEQ ID NO. 21) 扩增 7B3抗体的 VH区所用的引物
5 Ή7Β3： ttcaggcggaggtggctctggcggtggcggatcggatgtgcagctg ( SEQ ID NO. 22)
3Ή7Β3: ggatccaccacctccgctgctcacggtcac (SEQ ID NO. 23)
1.2抗人 CD3单链抗体 VH和 VL序列的扩增
鼠抗人 CD3抗体的 VH和 VL基因的核苷酸序列得自 US71 12324B1中所示的序列 SEQ ID NO. 9 ( 847-1203 )和 SEQ ID NO.9 ( 1258-1575 )通过 PCR方法扩增抗人 CD3 抗体的 VL和 VH结构域的核苷酸序列， 使用的引物如下。
扩增抗人 CD3抗体的 VH区所用的引物
5'HCD3: ggaggtggtggatccgatatcaaactgcagc(SEQ ID NO. 24)
3'HCD3: cacttccaccagaacctccacttccaccttcgactgaggagactgtgag(SEQ ID NO. 25) 扩增抗人 CD3抗体的 VL区所用的引物
5'LCD3: ctggtggaagtggaggttcaggtggagtcgacgacattcagc(SEQ ID NO. 26)
3'LCD3: ctatgcggccgcctaatgatgatggtgatgatgtttcagctcca(SEQ ID NO. 27) 实施例 2 包含编码 806/CD3单链双功能抗体的核苷酸序列的表达载体的构建
上述 P C R扩增得到的 806抗体 VH和 VL区的核苷酸序列与编码接头 1氨基酸 (GlyGlyGlyGlySer)3以及编码接头 2氨基酸 (GlyGlyGlyGlySer)的核苷酸序列通过融 合 PCR ( fusion-PCR)方式扩增得到 VL806-接头 1 -VH806-接头 2; 而上述 PCR扩增得 到的及抗人 CD3抗体 VH和 VL区的核苷酸序列与编码接头 3氨基酸 VE(GGS)4GG的 核酸序列通过融合 PCR ( fusion-PCR) 方式分别扩增得到 VHCD3-接头 3-VLCD3。
然后将上述扩增产物再通过融合 PCR方式扩增得到单链双功能抗体的核苷酸 序列， 其连接顺序如下：
[VL806-接头 1 -VH806-接头 2-VHCD3-接头 3-VLCD3]
然后通过如下的引物对上述接头连接后的序列 （ [VL8。6-接头 1-VH8。6-接头 2-VHCD3-接头 3-VLCD3] )进行第三轮扩增， 以在 N末端添加信号肽序列和引入限制性 内切酶 Nhel的位点， 以及在 C末端添加组氨酸标签并引入限制性内切酶 Notl位点。
5，L806-1 : ctagctagccaccatggtgtccacagctcagttccttgcattcttgttgctttggtttc(SEQ ID NO. 28) 3'LCD3: ctatgcggccgcctaatgatgatggtgatgatgtttcagctcca(SEQ ID NO. 27) 扩增得到的序列 SEQ ID NO： 10用限制性内切酶 Nhel/Notl-HF同时酶切，按照酶 供应商 （New England Biolabs, NEB ) 建议的反应条件在缓冲液 2中进行双酶切。 表 达 pH载体 （参见 WO/2011/035465实施例 7和图 15 ) 也用限制性内切酶 Nhel/Notl-HF 进行同样的酶切。 然后按照酶供应商 （NEB ) 建议的反应条件用 T4 DNA连接酶连 接双酶切后 SEQ ID NO: 10的片段和 pH/DHFR载体片段。由此得到编码 806/CD3单链 双功能抗体多肽的核苷酸序列被克隆到载体中。 所得含有 806/CD3单链双功能抗体 多肽的新载体命名为 pH/806/CD3， 其详细结构如图 1所示。 实施例 3
包含编码双功能抗体 7B3/CD3的核苷酸序列的表达载体的构建
上述 PCR扩增得到的 7B3抗体 VH和 VL区的核苷酸序列与编码接头 1氨基酸 (GlyGlyGlyGlySer)3以及编码接头 2氨基酸 (GlyGlyGlyGlySer)的核苷酸序列通过融 合 PCR ( fusion-PCR) 方式扩增得到 VL7B3 -接头 1 -VH7B3 -接头 2 ; 而上述 PCR扩增得 到的及抗人 CD3抗体 VH和 VL区的核苷酸序列与编码接头 3氨基酸 VE(GGS)4GG的 核酸序列通过融合 PCR ( fusion-PCR) 方式分别扩增得到 VHCD3-接头 3-VLCD3。 然后通过如 SEQ ID NO: 20和 29所示的引物对上述连接好的序列 [VL7B3-接头 1-VH7B3-接头 2]进一步扩增以在 N末端添加引入信号肽序列和 Nhel位点， 和在 C端 引入 BamHI位点。 该进一步扩增的序列 SEQ ID NO: 12通过 Nhel和 BamHI酶切， 按照 酶供应商 （NEB ) 建议的反应条件在缓冲液 2中进行。
5'L7B3-1: ctagctagccaccatggtgtccacagctcagttccttgcattcttgttgctttggtttc(SEQ ID NO. 20) 3Ή7Β3-2: tcttgccagttcagcccctgactgctgcagtttgatatcggatccaccacctccg
(SEQ ID NO. 29) 上述实施例 2中构建的载体 pH-806/CD3通过同样的 Nhel和 BamHI酶切反应。 将 酶切后得到的长片段与 SEQ ID NO: 12连接， 由此编码 7B3/CD3单链双功能抗体多肽 的核苷酸序列 SEQ ID NO: 11被克隆到载体中， 新载体命名为 pH-7B3/CD3， 其详细 结构如图 2所示 实施例 4
单链双功能抗体 806/CD3和 7B3/CD3的表达和纯化
表达载体 PH-806/CD3和 pH-7B3/CD3分别根据 FreeStyle MAX Reagent 转染试 剂 （来自 Invitrogen) 说明书操作步骤转染到中国仓鼠卵巢 （CHO ) 细胞中， 然后根 据 OptiCHO™ 蛋白表达试剂盒 （来自 Invitrogen) 筛选稳定的克隆。 分别转染有上 述表达载体之一的 CHO细胞的稳定克隆在摇瓶中 37°C， 130rpm培养 7天， 所用培养 基为 CD OptiCHO (来自 Gibco ) 。 通过离心获得培养上清， 然后储存于 -20°C。
按照生产商提供的方法步骤， 采用组氨酸亲和层析柱 （His Trap HP column, 来自 GE Healthcare)进行蛋白纯化。 具体而言， 层析柱用缓冲液 A (20mM sodium phosphate pH 7.4, 0.4M NaCl)平衡，然后 PBS透析后的将细胞培养上清 （500 mL上清） 加入到层析柱上 （l mL)， 流速为 3ml/min。 然后用 5倍体积的缓冲液 A和 10倍体积的 含有 50mM咪唑的缓冲液 A清洗柱子， 以去除杂蛋白。结合的目的蛋白用添加 250mM 咪唑的同样缓冲液 A进行洗脱。 所有的纯化步骤都在 4°C下操作。
纯化的 806/CD3和 7B3/CD3蛋白通过还原性 SDS-PAGE进行检测， 如图 3 A所示 这两个单链双功能抗体分子的分子量均在 60kD左右，符合根据氨基酸序列计算得到 的 806/CD3和 7B3/CD3的分子量。
另外通过抗组氨酸抗体对纯化的蛋白进行蛋白杂交（Western blot) ， 其结果如 图 3B表明得到的蛋白都具有组氨酸标记， 其分子量均在 60 kD左右。
经 ELISA检测转染的 CHO细胞上清中 806/CD3和 7B3/CD3的浓度约为 3mg/L。在 280nm波长下检测纯化的蛋白浓度为 0.5mg/L。
经组氨酸亲和层析柱一步纯化法得到的单链双功能抗体进一步采用凝胶过滤 层析法进行单体和聚体的分离。 具体而言， 预装柱 superdex 200 10/300 GL (来自 GE Healthcare ) 用 2倍柱体积的 PBS缓冲液平衡， 样品经上样环上样 500μ1， 流速为 0.4ml/min, 然后用 1倍柱体积的 PBS进行洗脱。 结果如图 10A显示二聚体蛋白在 13ml 出现峰值， 单体蛋白在 15ml出现峰值。 单体蛋白经凝胶过滤层析进行纯度分析具体 步骤同上， 结果如图 10B显示其纯度大于 95%。 实施例 5
双功能抗体的抗原结合特异性及表位分析
5.1 抗原特异性分析
通过荧光激活细胞分选仪 （FACS , 通常又称为流式细胞仪） （FACScalibur, 由 BD公司提供） 分析单链双功能抗体 7B3/CD3和 806/CD3各自与 EGFR的结合能力。
具体方法如下：
1. 取对数生长期的如表 1所列各肿瘤细胞接种到 6cm平皿中， 接种细胞密度 约为 90 %， 37°C孵箱过夜培养。
2. 使用 10mM的 EDTA消化细胞， 200gx5min离心收集细胞。 以 1 χ 106〜
l x l 07/mL的浓度重悬于 1%含小牛血清的磷酸盐缓冲液 （NBS PBS)中， 按 l OOul/管的量加入流式专用管中。
3. 200gx5min离心， 弃上清。
4. 两个实验组分别加入待测抗体 7B3/CD3和 806/CD3 , 同时一个对照组加入 抗体 NGR/CD3作为阴性对照， 另一个对照组为不加抗体的 PB S空白对照。 各抗体的终浓度均为 20μ§/ιη1， 每管加入 100ul。 冰浴， 45分钟。
5. 每管加入 2ml 1%NBS PBS， 以 200gx5min离心， 共二遍。
6. 弃上清， 加入 1 :50稀释的小鼠抗组氨酸标签抗体 （来自上海睿星基因技术 有限公司） ， 每管加入 100ul。 冰浴， 45分钟。
7. 每管加入 2ml 1%NBS PBS， 以 200gx5min离心， 共二遍。
8. 弃上清， 加入 1 :50稀释的 FITC荧光标记的羊抗小鼠抗体（来自上海康成生 物工程有限公司） ， 每管加入 100ul。 冰浴， 45分钟。
9. 每管加入 2ml 1%NBS PBS， 以 200gx5min离心， 共二遍。
10. 弃上清， 重悬于 300ul 1%NBS PBS中， 流式细胞仪检测。
11. 应用流式细胞仪数据分析软件 WinMDI 2.9分析数据。
如图 4B-4C所示， 本发明的绿色所显示的双功能抗体 7B3/CD3的荧光峰和蓝色 所显示的双功能抗体 806/CD 3的荧光峰，与阴性对照（ NGR/CD3 )和空白对照（ PB S ) 相比有显著的差异，表明其均具有和 U87 MG-EGFRvIII以及 A431细胞高效结合的能 力。如图 4D-4F所示， 本发明的两个双功能抗体也能和 U87 MG-de4 EGFR, NCI-1650 及 NCI-1975结合， 但效率不如与 U87 MG-EGFRvIII或 A431的结合。
如图 4A所示， 这两个抗体（7B3/CD3和 806/CD3 )都几乎不能与 U87 MG细胞结 合。 这些结果表明 7B3/CD3和 806/CD3可以特异性地和突变的人 EGFR及过表达的 EGFR的肿瘤细胞结合， 而几乎不与正常表达 EGFR的组织结合。
图 4G显示， 本发明的双功能抗体 （7B3/CD3和 806/CD3 ) 和阴性对照抗体 (NGR/CD3 )以基本相同的水平与表达有 CD3的 Jurkat细胞（人外周血白血病 T细胞） 高效结合。 图 4H显示， 本发明的双功能抗体 （7B3/CD3和 806/CD3 ) 和阴性对照抗 体（NGR/CD3 ) 以相似的水平与人外周血单核细胞（PBMC) 高效结合。 图 4G和 4H 表明本发明的 7B3/CD3和 806/CD3双功能抗体可以特异性地与 T细胞表面的 CD3抗 原结合。
综上所述， 图 4A-4H显示出本发明的双功能抗体（7B3/CD3和 806/CD3 )特异性 地与表达突变的人 EGFR及过表达的 E GFR的肿瘤细胞结合的能力， 同时能够特异性 地与表达 CD3的效应细胞 （T细胞） 结合。
5.2 抗原表位分析
根据已有文献已经表明 806单克隆抗体 （mAb 806 ) 可以与 EGFR的隐蔽表位 CC16多肽 (287CKGYEDSRVMEAGDEC3。2) ( Johns TG, et al" J. Biol. Chem. 2004; 279(29):30375-84. ) 结合。 一般认为单克隆抗体转变成单链抗体应该不会改变其抗 原结合表位特异性， 为了进一步验证本发明的双功能抗体可以与该隐蔽表位结合， 本实验利用含有该表位的两个重组蛋白作为抗原， 进行 ELISA检测。
实验步骤如下：
1 ) 蛋白包被： 三种抗原 rEGFRvIIIex ( EGFRvIII的胞外结构域蛋白， 其制备方 法参见专利 WO/2011/035465 ) ， N12-CC16重组蛋白 （ M13噬菌体 pill蛋白的结构域 N1N2与 CC16的融合多肽，其制备方法参见 Jiang H, et al., J Biol. Chem., 2011 ,286(7): 5913-20 )及 BSA (购自上海生物工程有限公司）对照蛋白， 分别以 50ng/孔 （lng/μΐ, 50μ1/孔） 的量被用于包被 96孔板的各孔， 在 37°C下孵育 2h。
2 ) 封闭： 用 0.1M的磷酸缓冲液 （PBS ) 洗涤 3次， 然后加入 5%PBS 脱脂奶粉 (光明乳业股份有限公司） 在 37°C封闭 2h。
3 ) 将待检测抗体 806/CD3、 783/ 03用5% ?88 脱脂奶粉稀释成 2ng l， 每孔 50μ1， 37°C孵育 lh。
4 ) PBST ( PBS+0.05%Tween20 ) 洗涤 3次后， 加入以 1 : 1000稀释至 50μ1/孔的抗 6xHis-鼠单抗 （购自上海睿星基因技术有限公司） ， 在 37°C孵育 lh。
5 ) PB ST洗涤 3次，加入以 1： 2000稀释的羊抗鼠 IgG-HRP (购自 Santa Cruz公司）， 在 37°C孵育 lh。
6 ) 显色： PBST洗涤 5次， 加 ABTS显色液 100μΙ7孔， 37°C避光显色 10min。 检测： 使用 Bio-Rad Model 680酶标仪， 在波长 405nm下检测吸光度值。
结果：
如图 5A-5B所示： 双功能抗体 806/CD3及 7B3/CD3分别都可以与 Nl 2-CC 16 ( CC16多肽融合表达于 M13噬菌体 pill蛋白的 N1N2结构域的羧基端） 及 rEGFRvIIIex特异性结合。这两个抗体与上述两个抗原的结合强度比其与 BSA的非特 异性结合有非常显著的差异。
由于这两个抗原共有的 EGFR氨基酸序列只有 CC16多肽序列， 因此双特异性抗 体 806/CD3 及 7B3/CD3 结 合 的 表 位 都 是 CC16 多 肽 ， 即 (287CKGYEDSRVMEAGDEC302)。 实施例 6
单链双功能抗体 806/CD3和 7B3/CD3的生物学活性分析 -对各种肿瘤细胞的细胞毒 活性
外周血单核细胞 （PBMC) 用 Ficoll (来自 Biochrom)密度梯度离心方法， 按照标 准步骤从健康人供主的血液中分离。离心后，用浓度为 0.1M的磷酸盐缓冲液（PBS ) 洗涤细胞然后重悬于 RPMI 1640完全培养基 (Gibco)， 将细胞浓度调整到 5 X 105/mL。 PBMC用作细胞毒性实验中的效应细胞。不同的肿瘤细胞作为靶细胞（ target cells) » 用 RPMI 1640完全培养基将靶细胞浓度调整到 5 X 104/mL。同样体积的靶细胞和效应 细胞混合， 使效应细胞： 靶细胞 (E:T)比值为 10:1。
将混合后的细胞悬液以 75 μ L/孔的体积加到 96孔板中。 然后各孔分别添加 25 μ L从 1000ng/mL到 0.1 ng/mL的十倍系列梯度稀释的下列试剂：
1 ) 7B3/CD3单链双功能抗体
2 ) 806/CD3单链双功能抗体
3 ) RPMI 1640完全培养基 （背景对照）
4 ) NGR/CD3单链双功能抗体 （阴性对照， NGR为新生血管靶向肽， 其与 EGFR没有交叉结合位点。 其根据常规方法制备）
在 37 °C， 5% C02的培养箱中孵育 40小时后， 根据生产商的操作说明， 用 CytoTox96®非放射性细胞毒性检测试剂盒 （Non-Radioactive Cytotoxicity Assay kit, 来自 Promega) 检测抗体的细胞毒作用。
CytoTox 96®非放射性细胞毒性检测是基于比色法的检测方法， 可替代 51Cr释放 法。 CytoTox 96®检测定量地测量乳酸脱氢酶（LDH) 。 LDH是一种稳定的胞质酶， 在细胞裂解时会释放出来，其释放方式与 51Cr在放射性分析中的释放方式基本相同。 释放出的 LDH培养基上清中， 可通过 30分钟偶联的酶反应来检测， 在酶反应中 LDH 可使一种四唑盐 （INT ) 转化为红色的甲臜 （formazan) 。 生成的红色产物的量与 裂解的细胞数成正比。
如下表 1所列举的六种与 EGFR有关的肿瘤细胞被用来分别分析本发明的两种 双功能抗体 7B3/CD3和 806/CD3 , 以及作为对照的 EGFR不相关的 NGR/CD3单链双 功能抗体介导的 T细胞肿瘤杀伤能力。
肿瘤细胞的杀伤率 （即， 细胞毒性％) 是根据 CytoTox96® 非放射性细胞毒性 检测 G1780产品使用说明书提供的下列公式计算的： 实验-效应细胞自发-靶细胞自发
细胞毒性0 /0 = X 100 靶细胞最大 -靶细胞自发
其中：
"实验" 指的是加入抗体 /效应细胞 /靶细胞的实验孔所产生的 LDH释放值， "效应细胞自发" 指的是效应细胞自发产生的 LDH释放， "靶细胞自发" 是指细胞不受其他因素处理时产生的 LDH释放，
"靶细胞最大" 是用 0.8%Triton X-100处理后靶细胞完全裂解所产生的 LDH释
"靶细胞最大-靶细胞自发" 代表着细胞受外界处理后完全裂解所产生的 LDH 释放。
表 1
Figure imgf000018_0001
上述表 1的结果表明表达突变的 EGFR和 /或过表达 EGFR的肿瘤细胞如 A431, U87 MG-de4 EGFR等，都会被双功能特异性抗体 7B3/CD3或 806/CD3导向的 T细胞特 异性杀伤。
具体而言， 在用 7B3/CD3处理的上述肿瘤细胞组中， 最小的特异性细胞毒性％ 为 23.3， 最大可达 75.2; 在 806/CD3处理的上述肿瘤细胞组中， 最小的特异性细胞毒 性％为28.7， 最大可达为 97.9。
而上述双功能特异性抗体 7B3/CD3或 806/CD3对表达低水平内源性正常 EGFR 的肿瘤细胞， 如 U87 MG的细胞毒性％非常低， 分别为 1.3和 9.4， 显著低于对上述表 达突变 EGFR和 /或过表达 EGFR的肿瘤细胞的细胞毒性％。
更具体的， 7B3/CD3 , 806/CD3和对照抗体 NGR/CD3在不同浓度下对各肿瘤的 细胞毒性％结果如下列表 2-7所示。 表 2
Figure imgf000019_0001
表 6 ng/ml NCI-H1975
NGR/CD3 7B3/CD3 806/CD3
1000 1 1.57±5.32 75.22±4.51 49.62±0.76
100 9.41±4.88 70.26±5.72 35.87±1.55
10 8.54±4.78 41.67±1.05 15.37±3.51
1 7.15±3.88 6.67±1.22 5.48±4.97
0.1 7.33±3.79 1.10±1.27 4.35±3.53 表 7
ng/ml NCI-H1650
NGR/CD3 7B3/CD3 806/CD3
1000 3.05±0.72 51.97±4.84 69.43±7.97
100 5.90±2.57 43.25±9.84 61.86±3.89
10 3.66±0.63 35.60±6.59 48.10±1.63
1 4.95±1.09 16.38±2.99 20.67±4.27
0.1 3.27±2.49 9.13±1.96 4.26±1.98 据上述表 2-7和图 6A-6F的细胞毒性％数据和所使用的双功能抗体的浓度， 采用 GraphPad Prism 5软件（GraphPad Software inc., San Diego, USA)分析程序计算得到 各双功能抗体针对肿瘤细胞杀伤的 EC5。值 (半最大效应浓度 concentration for 50% of maximal effect)。
例如， 对 U87 MG-EGFRvIII细胞而言， 7B3/CD3单链双功能抗体的 EC5。值为 2.15ng/ml， 而 806/CD3单链双功能抗体的 EC5。值为 0.29ng/ml。
对 NCI-H1975细胞而言， 7B3/CD3单链双功能抗体的 EC5。值为 53.6ng/ml， 而 806/CD3单链双功能抗体的 EC5。值为 1000ng/ml。
上述本发明双功能抗体所显示的针对各种肿瘤细胞系的低 E C 5。值结果表明其 具有显著提高的抗肿瘤生物学活性。 实施例 7
双功能抗体的荷瘤小鼠体内抗肿瘤活性
6 - 10周龄的免疫缺陷的 N 0 D / S C I D小鼠 （由上海斯莱克实验动物有限责任公司 提供） 用于构建人 EGFR相关肿瘤的异种移植模型， 其遗传学特征是缺乏 T细胞， B 细胞， NK细胞以及巨噬细胞功能。
治疗组 （n=6 ) 小鼠右侧皮下接种混合细胞悬液， 该悬液由 1 : 1体积比例混合的 细胞浓度为 1 X 106的 U87 MG-EGFRvIII 或 NCI-H1975肿瘤细胞与细胞浓度为 1 X 106的未剌激的 PBMC制成。
在 U87 MG-EGFRvIII/PBMC接种 1小时后， 小鼠被分别静脉内给药 0.4mg/kg/d 和 0.04mg/kg/d的 7B3/CD3以及 0.04mg/kg 806/CD3 , 该给药重复连续 5天。
在 NCI-H 1975/PBMC接种 1小时后， 小鼠被分别静脉内给药 0.4mg/kg/d和 0.04mg/kg/d的 7B 3/CD3， 该给药重复连续 10天。
对照组包括两个 PBS介质给药的组， 即对照组 1， 只注射肿瘤细胞的组； 和对照 组 2，注射肿瘤细胞和 PBMC的组， 以评估 PBMC效应细胞诱导的非特异性杀伤效果。
在指定日用卡尺测量肿瘤的大小， 肿瘤体积是根据下列公式计算：
长度 X宽度 X宽度
肿瘤体积 =
2 小鼠模型中肿瘤体积的减小被设定为各单链双功能抗体的肿瘤抑制效果的依 据。 下表中对各肿瘤的抑制率的计算是根据如下公式：
治疗组的肿瘤体积
肿瘤的抑制率 = 1- X 100%
对照组 2的肿瘤体积 表 7
Figure imgf000021_0001
如图 7所示， 在 U87 MG-EGFRvIII荷瘤小鼠模型中， 对照组 2 (即只注射人类外 周血淋巴细胞和肿瘤细胞， 而没有双特异抗体的组中） 的小鼠与对照组 1 (即只注 射肿瘤细胞的组） 相比， 没有观察到有显著干预 U87 MG-EGFRVIII肿瘤的生长。
而 806/CD3抗体在浓度 0.04mg/kg/d和 7B3/CD3在浓度 0.4mg/kg/d时都表现出强 烈抑制 U87 MG-EGFRvIII生长的能力， 在细胞接种后的第 23天， 它们的抑制率分别 为 74%和 80% (和对照组 2相比， p<0.05)。 而在较低剂量的 7B3/CD3 (0.04mg/kg/d), 肿 瘤生长抑制率为 35.3% (和对照组 2相比） 。
如图 8显示， 在 NCI-1975荷瘤小鼠模型中， 观察到对照组 2与对照组 1相比， 有 一定的干预 NCI- 1975肿瘤生长的效果， 但效果低于 0.04mg/kg/d剂量 7B3/CD3治疗的 小鼠， 和明显低于 0.4mg/kg/d剂量 7B3/CD3治疗的小鼠。
7B3/CD3治疗的小鼠与对照组 1和 2相比， 表现出剂量依赖性的抗肿瘤生长的效 应。 在接受 0.4mg/kg/d剂量组的 6只小鼠中有 1只在细胞接种后第 30天还没有成瘤， 而 0.04mg/kg/d剂量组的小鼠全部成瘤。 和对照组 2相比， 这两组的肿瘤抑制效果分 别为： 0.4mg/kg/d剂量组， 87% (p<0.05) 和 0.04mg/kg/d剂量组， 35% (p<0.05)。 实施例 8
本发明的单链双功能抗体 806 / C D 3和人源化单克隆抗体 c h 806的生物学活性分析- 对各种肿瘤细胞的细胞毒性
本实验包含两个实验组， SP 806/CD3处理的三种肿瘤细胞和 ch806处理的三种肿 瘤细胞， 涉及的相关实验材料的制备基本同实施例 6所述， 除了（1)人源化单克隆抗 体 ch806， 其制备方法如下： 按照 CN 102405235A公开的序列人工合成编码 ch806抗 体重链和轻链可变区的核酸， 然后在重链编码序列两端添加 Nhel 和 Apal 酶切位 点， 在轻链编码序列的两端添加 EcoRV和 Bsiwl酶切位点， 接着参考如 CN101602808B, 尤其实施例 7中的描述将上述重链可变区和轻链可变区分别装入表 达载体 pH和 pK, 构建得到 pH-ch806和 pK-ch806。 然后根据脂质体转染方法将 pH-ch806和 pK-ch806共转染 CHO-DG44细胞（Invitrogen) ， 经过 MTX筛选， 挑选出 高表达抗体的阳性克隆， 同时进行细胞驯化， 使细胞株逐步适应无血清培养。 对驯 化成功得到的 CHO-ch806细胞进行无血清培养，收集无血清培养上清进行 protein A(Code No.l 7-5280-02,GE Healthcare Life Sciences)亲和纯化， 即可得到纯化 ch806 抗体蛋白。 （2) U87 MG EGFR细胞， 其是过表达 EGFR的 U87 MG细胞株， 其构建方 法参见文献 (Wang H, Neoplasia, 2011 , 13(5): 461-471.)， U87 MG可以得自 ATCC。
将如实施例 6第一段和第二段所述之制备的混合后的肿瘤细胞悬液以 75 μ !7孔 的体积加到 96孔板中。然后各孔分别添加 25 μ L从 20ηΜ到 0.0002ηΜ的十倍系列梯度 稀释的下列试剂： 双功能抗体 806/CD3和人源化抗体 ch806。
对上述抗体进行抗肿瘤细胞毒性实验的方法和步骤同实施例 6中所述。 下面表 8 - 10记载的是双功能抗体 806 / C D 3和人源化抗体 ch 806对
胞毒性的百分比。
表 8
Figure imgf000023_0001
据上述表 8-10和图 9A-9C的细胞毒性％数据和所使用的双功能抗体的浓度， 采 用 GraphPad Prism 5软件（GraphPad Software inc., San Diego, USA)分析程序计算得 到双功能抗体和单克隆抗体针对肿瘤细胞杀伤的 EC50值。 在 U87 MG-EGFRvIII细胞中， 806/CD3单链双功能抗体的 EC50值为 0.136nM， 而 ch806单克隆抗体的 EC50值为 40.79nM。 在 U87 MG-EGFR细胞中， 806/CD3单链 双功能抗体的 EC50值为 23.43nM， 而 ch806单克隆抗体的 EC50值为 6476.08nM。这显 示本发明的单链双功能抗体 806/CD3对肿瘤细胞的细胞毒性相比人源化单克隆抗体 ch806对肿瘤细胞的细胞毒性有非常显著的提高。 表 11 本发明氨基酸序列和核苷酸序列描述:
Figure imgf000024_0001

Claims

权利要求书
1. 一种多功能抗体多肽， 包括：
( a) 第一功能域，其特异性识别 EGFR的第 287-302位氨基酸序列 形成的隐蔽表位， 如 SEQ ID NO: l所示，
( b) 第二功能域， 其特异性识别人 T细胞表面抗原。
2. 权利要求 1所述的多肽， 其中第一功能域包含至少一个具有选自 如下序列 SEQ ID NO. 2, SEQ ID NO. 3和 SEQ ID NO.4的抗 EGFR 抗体重链可变区的互补决定区 （CDR) 。
3. 权利要求 1或 2所述的多肽，其中第一功能域包含至少一个具有选 自如下序列 SEQ ID NO. 5， SEQ ID NO. 6和 SEQ ID NO.7的抗 EGFR抗体轻链可变区的互补决定区 （CDR) 。
4. 权利要求 1所述的多肽， 其中第二功能域为单链抗 CD3抗体。
5. 权利要求 1所述的多肽， 还包含位于所述第一和第二功能域之间 或位于第一或第二功能域内部互补决定区之间的接头。
6. 权利要求 5所述的多肽， 其中所述接头的序列为
( GlyGlyGlyGlySer) n， 其中 n为 1到 5的整数。
7. 权利要求 6所述的多肽， 其中 n=3。
8. 权利要求 1-7之一所述的多肽， 其中第一或第二功能域选自完整 抗体， 单链抗体 (scFV )， Fab片段， Fd片段， Fv片段， F(ab')2片 段和其衍生物。
9. 权利要求 1所述的多肽， 其中第一和 /或第二功能域是人源化的， 嵌合的或鼠源的。
10.权利要求 1所述的多肽，具有如 SEQ ID NO. 8所示的氨基酸序列。 权利要求 1所述的多肽，具有如 SEQ ID NO. 9所示的氨基酸序列。 编码权利要求 1-1 1任一所述的多肽的核苷酸序列。 编码权利要求 10所述的多肽的核苷酸， 其具有如 SEQ ID NO. 10 所示的核苷酸序列。 编码权利要求 11所述的多肽的核苷酸， 其具有如 SEQ ID NO. 11 所示的核苷酸序列。 包含有权利要求 12-14之一所述核苷酸序列的载体。 权利要求 15所述的载体， 其为带有 DHFR营养缺陷性选择标记的 载体。 权利要求 16所述的载体， 其为 pH载体。 包含有权利要求 15或 16所述的载体的真核宿主细胞或原核宿主 细胞。 权利要求 18所述的真核宿主细胞， 其为中国仓鼠卵巢细胞。 权利要求 1-11任一所述的多肽在制备诊断， 治疗和 /或预防肿瘤 的药物中的应用。
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