WO2022037002A1 - 特异性结合糖基化ceacam5的抗体 - Google Patents

特异性结合糖基化ceacam5的抗体 Download PDF

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WO2022037002A1
WO2022037002A1 PCT/CN2021/070825 CN2021070825W WO2022037002A1 WO 2022037002 A1 WO2022037002 A1 WO 2022037002A1 CN 2021070825 W CN2021070825 W CN 2021070825W WO 2022037002 A1 WO2022037002 A1 WO 2022037002A1
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seq
cdr
chain variable
variable region
region comprises
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牟男
于跃
袁纪军
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Shanghai Genbase Biotechnology Co Ltd
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Shanghai Genbase Biotechnology Co Ltd
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Priority to US18/022,113 priority Critical patent/US12037409B2/en
Priority to AU2021329456A priority patent/AU2021329456A1/en
Priority to JP2023512468A priority patent/JP7392200B2/ja
Priority to CN202180051155.6A priority patent/CN116348593A/zh
Priority to EP21857108.1A priority patent/EP4201961A4/en
Priority to KR1020237009445A priority patent/KR20230048150A/ko
Priority to CA3192254A priority patent/CA3192254A1/en
Publication of WO2022037002A1 publication Critical patent/WO2022037002A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to monoclonal antibodies. More specifically, the present application relates to monoclonal antibodies that specifically bind to glycosylated CEACAM5, the preparation and application of humanized antibodies thereof.
  • gastrointestinal-related tumors including colorectal cancer, gastric cancer, and esophageal cancer, are newly diagnosed every year with an annual number of 520,000, 460,000, and 310,000 (WHO, 2018). Therefore, gastrointestinal-related tumors have surpassed lung cancer as the incidence of cancer in my country.
  • the treatment methods for gastrointestinal tumors mainly include surgery, chemotherapy, targeted therapy and immunotherapy.
  • chemotherapy drugs include docetaxel, 5-fluorouracil, mitomycin C, platinum, etc.
  • targeted therapy drugs include VEGFR monoclonal antibody, Her2 monoclonal antibody, etc.
  • immunotherapy is mainly PD1/PDL1 antibody, etc.
  • CEACAM human carcinoembryonic antigen cell adhesion factor
  • tandem IgV domains in the extracellular region is characterized by the tandem IgV domains in the extracellular region to form a highly similar structure and is highly glycosylated (glycosylation accounts for 50% of the molecular weight), and its extracellular region consists of A1-B1 -A2-B2-A3-B3 structure is composed of tandem (A1-3 or B1-3 is a highly homologous structure);
  • a common CEA molecule is CEACAM5 (CD66e), which is coupled to the cell membrane through glycosylphosphatidylinositol (GPI) , and can be released into the blood by enzymatic degradation of GPIs (eg, phospholipase C);
  • GPIs glycosylphosphatidylinositol
  • CEACAM5 molecules are involved in cell adhesion (via CEA family homo- or heterodimers, such as CEACAM6), intracellular signaling, tumor metastasis, and resistance to drug-related; at the same time, CE
  • CEACAM5 is a highly glycosylated protein antigen, so the recombinantly expressed CEACAM5 (eg 293 system) antigen may have different glycosylation from the CEACAM5 protein expressed by tumor cells itself, so there is a need in the art for an antibody that can recognize the natural CEACAM5 antigen.
  • CEACAM5 is expressed in a small amount in normal tissues such as the digestive tract, which is located on the apical surface of the digestive tract; in tumor cells, CEACAM5 is expressed in the apical and basolateral surfaces, and it is difficult for CEACAM5 antibody drugs to reach the tumor site. Therefore, there is a need in the art to improve CEACAM5 antibodies. The problem of local concentration of drugs.
  • One aspect of the invention provides a monoclonal antibody antibody or antigen-binding fragment thereof directed against glycosylated CEACAM5 that specifically binds domains A1-B1, A2-B2, and A2-B2 of glycosylated CEACAM5, and / or A3-B3.
  • the monoclonal antibodies of the invention comprise a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises: CDR-H1 shown by SEQ ID NO:1, CDR-H2 shown by SEQ ID NO:2 and CDR-H3 shown by SEQ ID NO:3; Meanwhile, the light chain variable region comprises: CDR-L1 shown by SEQ ID NO:4, CDR-L2 shown by SEQ ID NO:5 and CDR-L3 shown by SEQ ID NO:6;
  • the heavy chain variable region comprises: CDR-H1 shown by SEQ ID NO:7, CDR-H2 shown by SEQ ID NO:8 and CDR-H3 shown by SEQ ID NO:9; Meanwhile, the light chain variable region comprises: CDR-L1 shown by SEQ ID NO: 10, CDR-L2 shown by SEQ ID NO: 11 and CDR-L3 shown by SEQ ID NO: 12;
  • the heavy chain variable region comprises CDR-H1 as set forth in SEQ ID NO: 13, CDR-H2 as set forth in SEQ ID NO: 14 and CDR-H3 as set forth in SEQ ID NO: 15;
  • the light chain variable region comprises CDR-L1 as set forth in SEQ ID NO:16, CDR-L2 as set forth in SEQ ID NO:17, and CDR-L3 as set forth in SEQ ID NO:18; or
  • the heavy chain variable region comprises CDR-H1 shown in SEQ ID NO: 19, CDR-H2 shown in SEQ ID NO: 20 and CDR-H3 shown in SEQ ID NO: 21;
  • the light chain variable region comprises CDR-L1 shown by SEQ ID NO:22, CDR-L2 shown by SEQ ID NO:23, and CDR-L3 shown by SEQ ID NO:24.
  • the monoclonal antibody of the present invention comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises the polypeptide shown in SEQ ID NO:25, while the light chain variable region comprises the polypeptide shown in SEQ ID NO:26;
  • the heavy chain variable region comprises the polypeptide shown in SEQ ID NO:27, while the light chain variable region comprises the polypeptide shown in SEQ ID NO:28;
  • the heavy chain variable region comprises the polypeptide set forth in SEQ ID NO:29, while the light chain variable region comprises the polypeptide set forth in SEQ ID NO:30; or
  • the heavy chain variable region comprises the polypeptide shown in SEQ ID NO:31, while the light chain variable region comprises the polypeptide shown in SEQ ID NO:32.
  • the invention provides a humanized antibody or antigen-binding fragment thereof that specifically binds glycosylated CEACAM5, wherein the antibody comprises a light chain variable region and a heavy chain variable region, wherein:
  • the light chain variable region comprises:
  • SEQ ID NO:84 selected from the group consisting of SEQ ID NO:84, SEQ ID NO:90, SEQ ID NO:96, SEQ ID NO:102, SEQ ID NO:108, SEQ ID NO:114, SEQ ID NO:120, and SEQ ID NO:126 CDR-L1;
  • SEQ ID NO:85 selected from the group consisting of SEQ ID NO:85, SEQ ID NO:91, SEQ ID NO:97, SEQ ID NO:103, SEQ ID NO:109, SEQ ID NO:115, SEQ ID NO:121 and SEQ ID NO:127 CDR-L2;
  • the heavy chain variable region comprises:
  • SEQ ID NO:88 selected from the group consisting of SEQ ID NO:88, SEQ ID NO:94, SEQ ID NO:100, SEQ ID NO:106, SEQ ID NO:112, SEQ ID NO:118, SEQ ID NO:124 and SEQ ID NO:130 CDR-H2; and
  • SEQ ID NO:89 selected from the group consisting of SEQ ID NO:89, SEQ ID NO:95, SEQ ID NO:101, SEQ ID NO:107, SEQ ID NO:113, SEQ ID NO:119, SEQ ID NO:125 and SEQ ID NO:131 CDR-H3.
  • the light chain variable region of the humanized antibody of the invention comprises at least 90% of the amino acid sequence selected from the group consisting of SEQ ID NOs: 68, 70, 72, 74, 76, 78, 80, 82 identical amino acid sequences
  • the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 69, 71, 73, 75, 77, 79, 81, 83.
  • the humanized antibody of the present invention comprises: a light chain variable region encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 54, 56, 58, 60, 62, 64, 66 , and a heavy chain variable region encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 53, 55, 57, 59, 61, 63, 65, 67.
  • the humanized antibody of the present invention comprises a light chain variable region selected from SEQ ID NO:68, 70, 72, 74, 76, 78, 80, 82 and a light chain variable region selected from SEQ ID NO:69 , 71, 73, 75, 77, 79, 81, 83 heavy chain variable regions.
  • the humanized antibody of the present invention comprises a light chain variable region and a heavy chain variable region, wherein:
  • the light chain variable region comprises: CDR-H1 shown by SEQ ID NO:84, CDR-H2 shown by SEQ ID NO:85 and CDR-H3 shown by SEQ ID NO:86;
  • the heavy chain variable region comprises: CDR-L1 shown by SEQ ID NO:87, CDR-L2 shown by SEQ ID NO:88 and CDR-L3 shown by SEQ ID NO:89;
  • the light chain variable region comprises: CDR-H1 shown by SEQ ID NO:90, CDR-H2 shown by SEQ ID NO:91 and CDR-H3 shown by SEQ ID NO:92; Meanwhile, the heavy chain variable region comprises: CDR-L1 shown by SEQ ID NO:93, CDR-L2 shown by SEQ ID NO:94 and CDR-L3 shown by SEQ ID NO:95;
  • the light chain variable region comprises: CDR-H1 shown by SEQ ID NO:96, CDR-H2 shown by SEQ ID NO:97 and CDR-H3 shown by SEQ ID NO:98; Meanwhile, the heavy chain variable region comprises: CDR-L1 shown by SEQ ID NO:99, CDR-L2 shown by SEQ ID NO:100 and CDR-L3 shown by SEQ ID NO:101;
  • the light chain variable region comprises: CDR-H1 shown by SEQ ID NO:102, CDR-H2 shown by SEQ ID NO:103 and CDR-H3 shown by SEQ ID NO:104; Meanwhile, the heavy chain variable region comprises: CDR-L1 shown by SEQ ID NO: 105, CDR-L2 shown by SEQ ID NO: 106 and CDR-L3 shown by SEQ ID NO: 107;
  • the light chain variable region comprises: CDR-H1 shown by SEQ ID NO:108, CDR-H2 shown by SEQ ID NO:109 and CDR-H3 shown by SEQ ID NO:110;
  • the heavy chain variable region comprises: CDR-L1 shown by SEQ ID NO: 111, CDR-L2 shown by SEQ ID NO: 112 and CDR-L3 shown by SEQ ID NO: 113;
  • the light chain variable region comprises: CDR-H1 shown by SEQ ID NO: 114, CDR-H2 shown by SEQ ID NO: 115 and CDR-H3 shown by SEQ ID NO: 116; Meanwhile, the heavy chain variable region comprises: CDR-L1 shown by SEQ ID NO: 117, CDR-L2 shown by SEQ ID NO: 118 and CDR-L3 shown by SEQ ID NO: 119;
  • the light chain variable region comprises: CDR-H1 shown by SEQ ID NO:120, CDR-H2 shown by SEQ ID NO:121 and CDR-H3 shown by SEQ ID NO:122; Meanwhile the heavy chain variable region comprises: CDR-L1 shown by SEQ ID NO:123, CDR-L2 shown by SEQ ID NO:124 and CDR-L3 shown by SEQ ID NO:125; or
  • the light chain variable region comprises: CDR-H1 shown by SEQ ID NO:126, CDR-H2 shown by SEQ ID NO:127 and CDR-H3 shown by SEQ ID NO:128; Meanwhile the heavy chain variable region comprises: CDR-L1 shown by SEQ ID NO:129, CDR-L2 shown by SEQ ID NO:130 and CDR-L3 shown by SEQ ID NO:131.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof that specifically binds to glycosylated CEACAM5.
  • the present invention relates to an expression vector comprising a nucleic acid molecule encoding an antibody that specifically binds to glycosylated CEACAM5 as disclosed herein.
  • the present invention relates to host cells comprising the expression vectors disclosed herein.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one antibody disclosed herein that specifically binds glycosylated CEACAM5 and a pharmaceutically acceptable carrier.
  • the invention in another aspect, relates to a method for making an antibody that specifically binds glycosylated CEACAM5, comprising expressing in a host cell a nucleic acid sequence encoding an antibody that specifically binds glycosylated CEACAM5 as disclosed herein and extracting from Host cells isolate antibodies that specifically bind to glycosylated CEACAM5.
  • the present invention provides the use of the antibody of the present invention in the manufacture of a medicament for the treatment of gastrointestinal related tumors.
  • the present invention provides a method of treating a gastrointestinal-related tumor comprising administering to a subject in need thereof an antibody of the present invention.
  • Monoclonal antibodies can be prepared as follows. Mice or other suitable host animals are first immunized with the immunogen (adjuvanted if necessary).
  • the immunogen or adjuvant is usually injected subcutaneously at multiple points or intraperitoneally.
  • the immunogen can be preconjugated to certain known proteins, such as serum albumin or soybean trypsin inhibitor, to enhance the immunogenicity of the antigen in the host.
  • the adjuvant may be Freund's adjuvant or MPL-TDM or the like.
  • lymphocytes can also be obtained by in vitro immunization.
  • Lymphocytes of interest are collected and fused with myeloma cells using a suitable fusion agent, such as PEG, to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996).
  • a suitable fusion agent such as PEG
  • the preferred myeloma cells should have the characteristics of high fusion rate, stable antibody secretion ability, and sensitivity to HAT medium.
  • the culture medium of growing hybridoma cells is used to detect the production of monoclonal antibodies against specific antigens.
  • Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the affinity of mAbs can be determined using the Scatchard assay described by Munson et al., Anal. Biochem. 107:220 (1980).
  • the cell line of interest can pass the limited criteria described in (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). Subcloning by dilution.
  • a suitable medium can be DMEM or RPMI-1640 and the like.
  • hybridoma cells can also grow in animals in the form of ascites tumors.
  • immunoglobulin purification methods such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, the monoclonal antibodies secreted by subcloned cells can be purified from cell culture medium, ascites or serum.
  • Monoclonal antibodies can also be obtained by genetic engineering recombinant technology. Using nucleic acid primers that specifically bind to the heavy chain and light chain genes of the monoclonal antibody to perform PCR amplification, the DNA molecules encoding the heavy chain and light chain genes of the monoclonal antibody can be isolated from the hybridoma cells. Insert the obtained DNA molecule into the expression vector, then transfect host cells (such as E.coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulins), and culture under suitable conditions, Recombinantly expressed antibodies of interest can be obtained.
  • host cells such as E.coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulins
  • a tumor cell line with high expression of CEACAM5 (eg, Lovo) is used to immunize mice to obtain an antibody that recognizes the natural CEACAM5 antigen.
  • CEACAM5 knockout cell line was constructed for efficient screening of specific CEACAM5 antibodies.
  • the antibody screened by the present invention simultaneously binds to the three structural domains of CEACAM5 (A1-B1, A2-B2, A3-B3), so it can increase the surface binding amount of CEACAM5 highly expressing tumor cells, thereby increasing its local concentration and improving drug efficacy.
  • Humanized design and screening of mouse-derived antibodies obtained from screening may reduce the HAMA (Human Anti Mouse Antibody) effect in clinical applications, reduce the production of neutralizing antibodies in patients, and increase the blood concentration of drugs to improve drug efficacy.
  • HAMA Human Anti Mouse Antibody
  • a “humanized antibody” is an antibody comprising one or both of a humanized VH domain and a humanized VL domain.
  • One or more immunoglobulin constant regions need not be present, but if present, are derived entirely or substantially from human immunoglobulin constant regions.
  • Humanized antibodies are genetically engineered antibodies in which CDRs from a non-human "donor” antibody are grafted into human "acceptor” antibody sequences (see e.g. Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,205; and Foote, US 6,881,557).
  • the recipient antibody sequence can be, for example, a mature human antibody sequence, a complex of such sequences, a consensus sequence of human antibody sequences, or a germline sequence.
  • the human acceptor sequences can be selected such that the variable region framework has a high degree of sequence identity with the donor sequences to match the typical form between the acceptor CDRs and the donor CDRs and other criteria.
  • a humanized antibody is one in which the CDRs are derived entirely or substantially from the donor antibody and variable region framework sequences and the constant regions, if present, are derived entirely or substantially from human antibody sequences.
  • a humanized heavy chain typically has all three CDRs derived entirely or substantially from the donor antibody heavy and heavy chain variable region framework sequences and substantially heavy chain variable and constant region sequences from a human heavy chain. Chain constant region (if present).
  • a humanized light chain typically has all three CDRs derived entirely or substantially from the light chain and light chain variable region framework sequences of the donor antibody and light substantially derived from the human light chain variable region framework and constant region sequences. Chain constant region (if present).
  • the CDRs in the humanized antibody are substantially derived from the corresponding CDRs in the non-human antibody.
  • variable region framework sequence of an antibody chain or the constant region of an antibody chain is substantially human derived when 99% identical or approximately 100% identical to corresponding residues (variable regions are defined by Kabat numbering and constant regions are defined by EU numbering) Variable region framework sequences or human constant regions.
  • humanized antibodies can also be composed of less than all six CDRs from mouse antibodies (eg, at least 3 1, 4 or 5) CDR composition (eg, Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320:415-428, 2002; Iwahashi et al., Mol. Immunol. 36 : 1079-1091, 1999; Tamura et al., Journal of Immunology, 164: 1432-1441, 2000).
  • CDR composition eg, Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320:415-428, 2002; Iwahashi et al., Mol. Immunol. 36 : 1079-1091, 1999; Tamura et al., Journal of Immunology, 164: 1432-1441, 2000).
  • a CDR in a humanized antibody is "substantially" when at least 60%, at least 85%, at least 90%, at least 95%, or 100% identical between the individual CDRs that correspond to residues (as defined by Kabat or IMGT) Corresponding CDRs from "non-human antibodies.
  • the CDRs are substantially derived from humanized VH or VL domains of non-human immunoglobulins
  • the CDRs of the humanized VH or VL domains relative to the corresponding non-human VH or VL CDRs have across all three CDRs No more than six (eg, no more than five, no more than four, no more than three, no more than two, or no more than one) amino acid substitutions (preferably conservative substitutions).
  • variable region framework sequence of an antibody VH or VL domain or an immunoglobulin constant region The sequences, if present, are "substantially derived from" human VH or VL framework sequences or human constant regions, respectively. Thus, all parts (except the CDRs) of a humanized antibody are usually derived, in whole or in substantial part, from the corresponding parts of native human immunoglobulin sequences.
  • antibody refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having a light chain and a heavy chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus.
  • the assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917 ; Definition by Chothia et al. (1989) Nature 342:878-883.
  • antibody is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3, or IgG4 subtype
  • IgAl IgA2, IgD, IgE, or IgM antibodies.
  • antibody when the term “antibody” is referred to, it includes not only whole antibodies but also antigen-binding fragments of antibodies.
  • antigen-binding fragment of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to, and/or compete with, the same antigen to which the full-length antibody binds Specific binding to an antigen, which is also referred to as an "antigen binding moiety". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is hereby incorporated by reference in its entirety for all purposes.
  • antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining regions (CDRs) Fragments, single chain antibodies (eg, scFvs), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of the antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • CDRs complementarity determining regions
  • Antigen-binding fragments of an antibody can be obtained from a given antibody using conventional techniques known to those of skill in the art (eg, recombinant DNA techniques or enzymatic or chemical fragmentation methods), and used in the same Antibodies are screened specifically for antigen-binding fragments in the same manner as antibodies.
  • the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, that is, excluding natural mutations that may arise spontaneously , a group of identical antibody molecules.
  • Monoclonal antibodies are highly specific for a single epitope on an antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which generally comprise at least two or more different antibodies that generally recognize different epitopes on an antigen.
  • Monoclonal antibodies are typically obtained using the hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA technology (eg, see U.S.P 4,816,567).
  • monoclonal antibodies can be prepared as follows. Mice or other suitable host animals are first immunized with the immunogen (adjuvanted if necessary).
  • the immunogen or adjuvant is usually injected subcutaneously at multiple points or intraperitoneally.
  • the immunogen can be preconjugated to certain known proteins, such as serum albumin or soybean trypsin inhibitor, to enhance the immunogenicity of the antigen in the host.
  • the adjuvant may be Freund's adjuvant or MPL-TDM or the like.
  • lymphocytes can also be obtained by in vitro immunization.
  • Lymphocytes of interest are collected and fused with myeloma cells using a suitable fusion agent, such as PEG, to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996).
  • a suitable fusion agent such as PEG
  • the hybridoma cells prepared above can be inoculated into a suitable culture medium for growth, and the culture medium preferably contains one or more substances capable of inhibiting the growth of unfused, parental myeloma cells.
  • hypoxanthine guanine phosphotransferase HGPRT or HPRT
  • HAT medium hypoxanthine guanine phosphotransferase
  • the preferred myeloma cells should have the characteristics of high fusion rate, stable antibody secretion ability, and sensitivity to HAT medium.
  • murine myeloma cells are preferred, such as MOP-21 or MC-11 mouse tumor-derived strains (THE Salk Institute Cell Distribution Center, San Diego, Calif. USA), and SP-2/0 or X63-Ag8 -653 cell line (American Type Culture Collection, Rockville, Md. USA).
  • Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the affinity of mAbs can be determined using the Scatchard assay described by Munson et al., Anal. Biochem. 107:220 (1980).
  • the cell line of interest can pass the limited criteria described in (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). Subcloning by dilution.
  • a suitable medium can be DMEM or RPMI-1640 and the like.
  • hybridoma cells can also grow in animals in the form of ascites tumors.
  • immunoglobulin purification methods such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, the monoclonal antibodies secreted by subcloned cells can be purified from cell culture medium, ascites or serum.
  • Monoclonal antibodies can also be obtained by genetic engineering recombinant technology. Using nucleic acid primers that specifically bind to the heavy chain and light chain genes of the monoclonal antibody to perform PCR amplification, the DNA molecules encoding the heavy chain and light chain genes of the monoclonal antibody can be isolated from the hybridoma cells. Insert the obtained DNA molecule into the expression vector, then transfect host cells (such as E.coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulins), and culture under suitable conditions, Recombinantly expressed antibodies of interest can be obtained.
  • host cells such as E.coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulins
  • chimeric antibody refers to an antibody in which a portion of the light or/and heavy chain is derived from an antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or a different species or belong to the same or a different antibody class or subclass), but in any case it remains Binding activity to target antigens (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
  • human antibody refers to a humanized antibody in which all or part of the CDR regions of a human immunoglobulin (acceptor antibody) are replaced by the CDR regions of a non-human antibody (donor antibody)
  • donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of corresponding non-human antibodies, or by amino acid residues of other antibodies, to further improve or optimize the performance of the antibody.
  • epitope refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody.
  • Epitopes are also known in the art as “antigenic determinants”.
  • Epitopes or antigenic determinants typically consist of chemically active surface groups of molecules such as amino acids or carbohydrate or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be "linear” “or conformational”. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol.
  • epitopic peptide refers to a peptide segment on an antigen that can be used as an epitope.
  • individual epitope peptides are capable of being specifically recognized/bound by antibodies directed against the epitope.
  • carrier protein refers to a protein that can act as a carrier for epitope peptides, ie, which can insert epitope peptides at specific positions (eg, inside the protein, N-terminal or C-terminal) , so that the epitope peptide can be presented so that the epitope peptide can be recognized by the antibody or immune system.
  • carrier proteins are well known to those skilled in the art and include, for example, the HPV L1 protein (epitope peptides can be inserted between amino acids 130-131 or between amino acids 426-427 of the protein, see Slupetzky , K. et al.
  • HBV core particles allow the insertion and surface exposure of the entire potentially protective region of Puumala hantavirus nucleocapsid protein [J]. Biol Chem, 1999, 380: 325-333) , woodchuck hepatitis virus core protein (amino acids 79-81 of the protein can be replaced with epitope peptides, see Sabine Gertrud Beterams and Michael Nassal, J. Virol. 1998, 72(6):4997), CRM197 protein (epitope peptides can be attached to the N-terminus or C-terminus of the protein or fragments thereof).
  • a linker eg, a flexible or rigid linker
  • Antibodies can be screened competitively for binding to the same epitope using routine techniques known to those of skill in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete for binding to an antigen (eg, influenza virus hemagglutinin protein). A high-throughput method for obtaining antibodies that bind the same epitope based on their cross-competition is described in International Patent Application WO 03/48731. Thus, antibodies and antigen-binding fragments thereof (ie, antigen-binding portions) that compete with the monoclonal antibodies of the invention for binding to the same epitope on the influenza virus hemagglutinin protein can be obtained using conventional techniques known to those skilled in the art.
  • an antigen eg, influenza virus hemagglutinin protein
  • an antibody that specifically binds to an antigen refers to an antibody that is less than about 10-5 M, such as less than about 10-6 M, 10-7 M, Binds the antigen with an affinity (K D ) of 10-8 M, 10-9 M, or 10-10 M or less.
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen.
  • hybridoma and “hybridoma cell line” are used interchangeably, and when referring to the terms “hybridoma” and “hybridoma cell line”, it also includes subclones of hybridomas and progeny cells. For example, when referring to the hybridoma cell line 2F4, it also refers to subclones and progeny cells of the hybridoma cell line 2F4.
  • Figure 1 shows the results of CEACAM5 gene knockout by CRISPR method using a vector (CEACAM5 KO1-3) in Lovo 6-1, a cell highly expressing CEACAM5.
  • Figure 2 shows the results of CEACAM5 gene knockout using the CEACAM5 KO3 vector in Lovo 6-1, a cell highly expressing CEACAM5.
  • Figure 3 shows the binding results of murine monoclonal antibodies to CEACAM5 recombinant protein.
  • Figure 4 shows binding of murine monoclonal antibodies to the extracellular domain of CEACAM5.
  • Figure 5 shows the binding of murine monoclonal antibodies to CEACAM5 highly expressing LS174T cells and KATO3 cells.
  • Figure 6 shows the binding of humanized antibodies to recombinant CEACAM5 antigen.
  • Figure 7 shows the binding of humanized antibodies to KATO3 cells highly expressing CEACAM5.
  • Figure 8 shows the competitive binding of humanized antibody and murine m2F4 antibody to CEACAM5.
  • a CEACAM5-expressing tumor cell line was used to immunize mice to prepare monoclonal antibodies.
  • the Lovo cell line ATCC CCL-229 with high expression of CEACAM5 was cultured in RPMI1640 medium containing 10% FBS. After the Lovo cells were trypsinized with TrypLE, they were resuspended in DPBS solution. Each SJL mouse was immunized subcutaneously at multiple points. 10 7 Lovo cells were immunized once a week for a total of 5 times. After the mice tested for serum titer were sacrificed, the spleen was taken, ground and sieved, and SP20 myeloma cells were fused according to the standard fusion process to obtain hybridoma cells.
  • Lovo cells were recombinantly expressed with the anti-CEACAM5 antibody hMN14 (Immunomedics, Phase2 drug, the sequence is as follows
  • clone 6-1 has a higher purity than other clones, reaching 98.6%, and its CEACAM5 expression MFI is significantly higher than other clones, so clone 6-1 was selected for CEACAM5 knockout.
  • the Lovo 6-1 clone was knocked out of the CEACAM5 gene using the CRISPR method, and the Lovo CEACAM5 KO cell line (hereinafter referred to as the Lovo CEA KO cell line) was screened.
  • the vector carrying CRISPR and sgRNA was packaged into a lentiviral vector (CEACAM5 KO1-3) and transduced into Lovo 6-1 cells. After transduction, the expression of CEACAM5 (MN14 antibody) was detected by FACS, and the binding of MN14 was detected by mouse Fc-APC secondary antibody . The results are shown in Figure 1.
  • CEACAM5 KO1-3 could knock out CEACAM5, and the CEACAM5 KO3 vector had a higher knockout efficiency, showing a clearer CEACAM5 negative population.
  • the sequences of the three sgRNAs targeting CEACAM5 are as follows:
  • CEACAM5 knockout population appeared in its cell population, accounting for 100%-29.3%, indicating that CEACAM5 was successfully knocked out.
  • Lovo cells and Lovo CEA KO cells were inoculated in a 96-well plate at 10 4 per well for overnight culture, and 1-10 ⁇ l of hybridoma supernatant was added to Lovo/Lovo CEA KO cell culture plate respectively, incubated for 1 hour, and the supernatant was discarded. After 1 hour of incubation, discard the supernatant, add DPBS solution containing 2% BSA, read and analyze the fluorescent signal and the area of FITC staining in Celigo.
  • VH and VL heavy chain and light chain variable regions
  • the heavy and light chain sequences were linked to pcDNA3.4 vector and transiently expressed in 293 system and purified by protein A/G.
  • the obtained chimeric recombinant antibody was subjected to buffer exchange into PBS solution by ultrafiltration. The sequencing results are shown in the table below.
  • CEACAM5 antigen (Sinobiological, 11077-H08H) was diluted to 1 ⁇ g/ml with DPBS solution, and 100 ⁇ l per well was added to a 96-well plate, and coated overnight at 2-8 °C; the coating solution was discarded, and PBS solution was used.
  • the CEACAM5 molecule was split according to its extracellular domain (A1-B1-A2-B2-A3-B3), and A1-B1-His Tag, A2-B2-His Tag, A3-B3-His Tag were constructed for expression
  • the vector was expressed in the 293 system and purified using a Ni column. The sequence is shown in the following table:
  • CEACAM5 fragment was diluted to 1 ⁇ g/ml with DPBS solution and added to 96-well plate according to 100 ⁇ l per well, and coated overnight at 2-8 °C; the coating solution was discarded, washed twice with PBS solution, and added containing 2 %BSA in PBS solution, blocked for 2 hours at room temperature; discard the blocking solution, add the antibody diluted in concentration gradient, and incubate at 37°C for 1 hour; discard the antibody solution and wash with PBS solution (PBST solution) containing 0.05% Tween20 4 times; add anti-human IgG Fc-HRP secondary antibody and incubate at 37°C for 30 minutes; wash 4 times with PBST solution, add TMB chromogenic substrate, and after 5-10 minutes of color development, an equal volume of 1M H2SO4 terminates the reaction ; Read the absorbance at 450nm on a microplate reader. The results are shown in Figure 4.
  • the epitopes of the above four antibodies M7, M17, M18 and M19 that bind to the CEACAM5 molecule are as follows:
  • LS174T cells and KATO3 cells (high expression of CEACAM5; ATCC, CL-188) were cultured in RPMI1640 medium containing 10% FBS, cells were trypsinized with TrypLE, centrifuged and resuspended in DPBS containing 2% BSA (FACS buffer).
  • the M19 antibody (m2F4) was compared with the IMGT database, and the humanized Framework sequence with the highest VH/VL homology was selected, CDR graft was performed, and computational chemical simulation was performed to maintain its binding to the antigen.
  • the humanized antibody was designed as shown in the following table. Show.
  • VH and VL regions of the above antibodies were linked to the human IgG1 Fc region and kappa constant region, and the heavy and light chain sequences of the antibody were inserted into the pcDNA3.4 vector, transiently expressed in 293 cells, and purified by protein A or G.
  • CEACAM5 antigen (Sinobiological, 11077-H08H) was diluted to 1 ⁇ g/ml with DPBS solution, and 100 ⁇ l per well was added to a 96-well plate, and coated overnight at 2-8 °C; the coating solution was discarded, and PBS solution was used.
  • KATO3 cells highly expressing CEACAM5 were cultured in RPMI1640 medium containing 10% FBS, the cells were trypsinized with TrypLE, centrifuged and resuspended in DPBS solution containing 2% BSA (FACS buffer, 4°C) according to 5 ⁇ 10 5 /100 ⁇ l/well was added to a U-bottom 96-well plate, and the antibody diluted in concentration gradient was added, incubated at 4°C for 1 hour, and the supernatant was discarded after centrifugation; 100 ⁇ l of anti-human IgG Fc-APC II was added to each well. Antibody and incubated at 4°C for 1 hour, washed once with FACS buffer, resuspended in 200 ⁇ l FACS buffer, and read the fluorescence signal value on BD C6plus. The results are shown in Figure 7.
  • KATO3 cells highly expressing CEACAM5 were cultured in RPMI1640 medium containing 10% FBS, the cells were trypsinized with TrypLE, centrifuged and resuspended in DPBS solution containing 2% BSA (FACS buffer, 4°C) according to 5 ⁇ 10 5 /100 ⁇ l/well was added to a U-bottom 96-well plate, and the mouse monoclonal antibody M19 antibody (m2F4) was added to 1 ⁇ g/ml and the humanized antibody (50 ⁇ g/ml, 3-fold dilution) was diluted in a concentration gradient.
  • DPBS solution containing 2% BSA FACS buffer, 4°C

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Abstract

本申请提供了特异性结合糖基化CEACAM5的抗体，其人源化抗体的制备及应用。

Description

特异性结合糖基化CEACAM5的抗体 技术领域
本发明涉及单克隆抗体。更具体地，本申请涉及与糖基化CEACAM5特异性结合的单克隆抗体，其人源化抗体的制备及应用。
背景技术
在中国，胃肠道相关肿瘤包含结直肠癌、胃癌、食管癌等每年新发数量为52万、46万和31万(WHO，2018)，因此胃肠道相关肿瘤已超过肺癌成为我国癌症发病率第一的癌种。目前，针对胃肠道肿瘤的治疗方法，主要包括手术治疗、化疗、靶向治疗和免疫治疗。常用的化疗药物包括多西他赛、5-氟尿嘧啶、丝裂霉素C、铂类等；靶向治疗药物包括VEGFR单抗、Her2单抗等；免疫治疗主要为PD1/PDL1抗体等。
人癌胚抗原细胞粘附因子(CEACAM)家族在上世纪60年代被发现，CEA家族在多种胃肠道及肺癌肿瘤中高度表达，例如结直肠癌、胰腺癌、肺癌、胃癌、肝细胞癌、乳腺癌等。CEACAM家族由CEACAM亚群和PSG家族构成，其特点为胞外区有IgV结构域串联形成高度相似的结构并高度糖基化(糖基化占50％分子量)，其胞外区由A1-B1-A2-B2-A3-B3结构串联组成(A1-3或B1-3为高度同源结构)；常见CEA分子为CEACAM5(CD66e)，通过糖基磷脂酰肌醇(GPI)偶联至细胞膜上，并可通过酶降解GPI(例如磷脂酶C)释放至血液中；CEACAM5分子参与细胞粘附(通过CEA家族同源或异源二聚体，例如CEACAM6)、胞内信号传递、肿瘤转移和耐药发生相关；同时CEACAM5与胃肠道大肠杆菌的粘附相关。
CEACAM5是高度糖基化的蛋白抗原，因此重组表达的CEACAM5(例如293系统)抗原可能与肿瘤细胞自身表达的CEACAM5蛋白的糖基化不同，因此本领域需要一种能够识别天然CEACAM5抗原的抗体。另外，CEACAM5少量表达于正常组织例如消化道，其位于消化道的apical面；在肿瘤细胞中CEACAM5表达于apical和basolateral面，CEACAM5抗体药物较难到达肿瘤部位，因此，本领域需要解决提高CEACAM5抗体药物局部浓度的问题。
发明内容
本发明的一个方面提供了针对糖基化CEACAM5的单克隆抗体抗体或其抗原结合片段，所述抗体或其抗原结合片段特异性结合糖基化CEACAM5的结构域A1-B1，A2-B2，和/或A3-B3。
在具体实施方案中，本发明的单克隆抗体包含重链可变区及轻链可变区，其中：
a.所述重链可变区包含：由SEQ ID NO:1所示的CDR-H1、由SEQ ID NO:2所示的CDR-H2和由SEQ ID NO:3所示的CDR-H3；同时所述轻链可变区包含：由SEQ ID NO:4所示的CDR-L1、由SEQ ID NO:5所示的CDR-L2和由SEQ ID NO:6所示的CDR-L3；
b.所述重链可变区包含：由SEQ ID NO:7所示的CDR-H1、由SEQ ID NO:8所示的CDR-H2和由SEQ ID NO:9所示的CDR-H3；同时所述轻链可变区包含：由SEQ ID NO:10所示的CDR-L1、由SEQ ID NO:11所示的CDR-L2和由SEQ ID NO:12所示的CDR-L3；
c.所述重链可变区包含由SEQ ID NO:13所示的CDR-H1、由SEQ ID NO:14所示的CDR-H2和由SEQ ID NO:15所示的CDR-H3；同时所述轻链可变区包含由SEQ ID NO:16所示的CDR-L1、由SEQ ID NO:17所示的CDR-L2和由SEQ ID NO:18所示的CDR-L3；或
d.所述重链可变区包含由SEQ ID NO:19所示的CDR-H1、由SEQ ID NO:20所示的CDR-H2和由SEQ ID NO:21所示的CDR-H3；同时所述轻链可变区包含由SEQ ID NO:22所示的CDR-L1、由SEQ ID NO:23所示的CDR-L2和由SEQ ID NO:24所示的CDR-L3。
在一个具体实施方式中，本发明的单克隆抗体包含重链可变区及轻链可变区，其中：
a)所述重链可变区包含由SEQ ID NO:25所示的多肽，同时所述轻链可变区包含由SEQ ID NO:26所示的多肽；
b)所述重链可变区包含由SEQ ID NO:27所示的多肽，同时所述轻链可变区包含由SEQ ID NO:28所示的多肽；
c)所述重链可变区包含由SEQ ID NO:29所示的多肽，同时所述轻链可变区包含由SEQ ID NO:30所示的多肽；或
d)所述重链可变区包含由SEQ ID NO:31所示的多肽，同时所述轻链可变区包含由SEQ ID NO:32所示的多肽。
在一个方面，本发明提供了特异性结合糖基化CEACAM5的人源化抗体或其抗原结合片段，其中所述抗体包含轻链可变区及重链可变区，其中：
所述轻链可变区包含：
选自SEQ ID NO:84、SEQ ID NO:90、SEQ ID NO:96、SEQ ID NO:102、SEQ ID NO:108、SEQ ID NO:114、SEQ ID NO:120和SEQ ID NO:126的CDR-L1；
选自SEQ ID NO:85、SEQ ID NO:91、SEQ ID NO:97、SEQ ID NO:103、SEQ ID NO:109、SEQ ID NO:115、SEQ ID NO:121和SEQ ID NO:127的CDR-L2；和
选自SEQ ID NO:86、SEQ ID NO:92、SEQ ID NO:98、SEQ ID NO:104、SEQ ID NO:110、SEQ ID NO:116、SEQ ID NO:122和SEQ ID NO:128的CDR-L3，
并且所述重链可变区包含：
选自SEQ ID NO:87、SEQ ID NO:93、SEQ ID NO:99、SEQ ID NO:105、SEQ ID NO:111、SEQ ID NO:117、SEQ ID NO:123和SEQ ID NO:129的CDR-H1；
选自SEQ ID NO:88、SEQ ID NO:94、SEQ ID NO:100、SEQ ID NO:106、SEQ ID NO:112、SEQ ID NO:118、SEQ ID NO:124和SEQ ID NO:130的CDR-H2；和
选自SEQ ID NO:89、SEQ ID NO:95、SEQ ID NO:101、SEQ ID NO:107、SEQ ID NO:113、SEQ ID NO:119、SEQ ID NO:125和SEQ ID NO:131的CDR-H3。
在一个具体实施方案中，本发明的人源化抗体的轻链可变区包含与选自SEQ ID NO:68、70、72、74、76、78、80、82的氨基酸序列具有至少90％同一性的氨基酸序列，并且重链可变区包含与选自SEQ ID NO:69、71、73、75、77、79、81、83的氨基酸序列具有至少90％同一性的氨基酸序列。
在优选的技术方案中，本发明的人源化抗体包含：由选自SEQ ID NO:52、54、56、58、60、62、64、66的核苷酸序列编码的轻链可变区，以及由选自SEQ ID NO:53、55、57、59、61、63、65、67的核苷酸序列编码的重链可变区。
在优选的技术方案中，本发明的人源化抗体包含选自SEQ ID NO:68、70、72、74、76、78、80、82的轻链可变区和选自SEQ ID NO:69、71、73、75、77、79、81、83的重链可变区。
在优选的技术方案中，本发明的人源化抗体包含轻链可变区及重链可变区，其中：
a.所述轻链可变区包含：由SEQ ID NO:84所示的CDR-H1、由SEQ ID NO:85所示的CDR-H2和由SEQ ID NO:86所示的CDR-H3；同时所述重链可变区包含：由SEQ ID NO:87所示的CDR-L1、由SEQ ID NO:88所示的CDR-L2和由SEQ ID NO:89所示的CDR-L3；
b.所述轻链可变区包含：由SEQ ID NO:90所示的CDR-H1、由SEQ ID NO:91所示的CDR-H2和由SEQ ID NO:92所示的CDR-H3；同时所述重链可变区包含：由SEQ ID NO:93所示的CDR-L1、由SEQ ID NO:94所示的CDR-L2和由SEQ ID NO:95所示的CDR-L3；
c.所述轻链可变区包含：由SEQ ID NO:96所示的CDR-H1、由SEQ ID NO:97所示的CDR-H2和由SEQ ID NO:98所示的CDR-H3；同时所述重链可变区包含：由SEQ ID NO:99所示的CDR-L1、由SEQ ID NO:100所示的CDR-L2和由SEQ ID NO:101所示的CDR-L3；
d.所述轻链可变区包含：由SEQ ID NO:102所示的CDR-H1、由SEQ ID NO:103所示的CDR-H2和由SEQ ID NO:104所示的CDR-H3；同时所述重链可变区包含：由SEQ ID NO:105所示的CDR-L1、由SEQ ID NO:106所示的CDR-L2和由SEQ ID NO:107所示的CDR-L3；
e.所述轻链可变区包含：由SEQ ID NO:108所示的CDR-H1、由SEQ ID NO:109所示的CDR-H2和由SEQ ID NO:110所示的CDR-H3；同时所述重链可变区包含：由SEQ ID NO:111所示的CDR-L1、由SEQ ID NO:112所示的CDR-L2和由SEQ ID NO:113所示的CDR-L3；
f.所述轻链可变区包含：由SEQ ID NO:114所示的CDR-H1、由SEQ ID NO:115所示的CDR-H2和由SEQ ID NO:116所示的CDR-H3；同时所述重链可变区包含：由SEQ ID NO:117所示的CDR-L1、由SEQ ID NO:118所示的CDR-L2和由SEQ ID NO:119所示的CDR-L3；
g.所述轻链可变区包含：由SEQ ID NO:120所示的CDR-H1、由SEQ ID NO:121所示的CDR-H2和由SEQ ID NO:122所示的CDR-H3；同时所述重链可变区包含：由SEQ ID NO:123所示的CDR-L1、由SEQ ID NO:124所示的CDR-L2和由SEQ ID NO:125所示的CDR-L3；或
h.所述轻链可变区包含：由SEQ ID NO:126所示的CDR-H1、由SEQ ID NO:127 所示的CDR-H2和由SEQ ID NO:128所示的CDR-H3；同时所述重链可变区包含：由SEQ ID NO:129所示的CDR-L1、由SEQ ID NO:130所示的CDR-L2和由SEQ ID NO:131所示的CDR-L3。
在另一个方面，本发明提供了一种分离的核酸分子，其包含编码特异性结合糖基化CEACAM5的抗体或其抗原结合片段的核苷酸序列。
在另一个方面，本发明涉及包含编码如本文所公开的特异性结合糖基化CEACAM5的抗体的核酸分子的表达载体。
在另一个方面，本发明涉及包含本文公开的表达载体的宿主细胞。
在另一个方面，本发明涉及包含至少一种本文公开的特异性结合糖基化CEACAM5的抗体和药学上可接受的载体的药物组合物。
在另一个方面，本发明涉及用于制备特异性结合糖基化CEACAM5的抗体的方法，其包括在宿主细胞中表达编码如本文所公开的特异性结合糖基化CEACAM5的抗体的核酸序列并从宿主细胞分离特异性结合糖基化CEACAM5的抗体。
在另一个方面，本发明提供了本发明的抗体在制备用于治疗胃肠道相关肿瘤的药物中的用途。
在另一个方面，本发明提供了一种治疗胃肠道相关肿瘤的方法，包括向有此需要的受试者施用本发明的抗体。
单克隆抗体的制备及筛选
可以如下来制备单克隆抗体。首先用免疫原(必要时候添加佐剂)免疫注射小鼠或其它合适的宿主动物。免疫原或佐剂的注射方式通常为皮下多点注射或腹腔注射。可将免疫原预先偶联到某些已知蛋白，如血清白蛋白或大豆胰酶抑制剂上，以增强抗原在宿主内的免疫原性。佐剂可以是弗氏佐剂或MPL-TDM等。动物在接受免疫后，体内将产生分泌特异性结合免疫原的抗体的淋巴细胞。另外，淋巴细胞也可以利用体外免疫获得。收集目的淋巴细胞，并用合适的融合剂，如PEG，使其与骨髓瘤细胞融合以获得杂交瘤细胞(Goding,Monoclonal Antibodies:Principles and Practice,pp.59-103,Academic Press,1996)。优选的骨髓瘤细胞应该具有融合率高，抗体分泌能力稳定，对HAT培养液敏感等特征。生长杂交瘤细胞的培养液用于检测针对特异抗原的单抗的产生。测定杂交瘤细胞产生的单抗的结合特异性的方法包括例如，免疫沉淀或体外结合 试验，如放射免疫试验(RIA)、酶联免疫吸附试验(ELISA)。例如，可利用Munson等在Anal.Biochem.107:220(1980)描述的Scatchard分析法来测定单抗的亲和力。当确定了杂交瘤产生的抗体的特异性、亲和力和反应性之后，目的细胞株可以通过(Goding,Monoclonal Antibodies:Principles and Practice,pp.59-103,Academic Press,1996)所描述的标准的有限稀释法进行亚克隆化。合适的培养液可以是DMEM或RPMI-1640等。另外，杂交瘤细胞还可以腹水瘤的形式在动物体内生长。利用传统的免疫球蛋白纯化方法，如蛋白A琼脂糖凝胶、羟基磷灰石层析、凝胶电泳、透析或亲和层析等，可以将亚克隆细胞分泌的单抗从细胞培养液、腹水或血清中分离出来。
还可以通过基因工程重组技术获得单克隆抗体。利用特异性结合单抗重链和轻链基因的核酸引物进行PCR扩增，可以从杂交瘤细胞中分离得到编码单抗重链和轻链基因的DNA分子。将所得的DNA分子插入表达载体内，然后转染宿主细胞(如E.coli细胞、COS细胞、CHO细胞、或其它不产生免疫球蛋白的骨髓瘤细胞)，并在合适的条件下进行培养，可以获得重组表达的目标抗体。
本发明采用CEACAM5高表达的肿瘤细胞系(例如Lovo)进行小鼠免疫，以获取识别天然CEACAM5抗原的抗体。
通过CRISPR技术利用sgRNA引导Cas9对目标基因组进行切割，高效敲除基因表达，构建了CEACAM5敲除细胞系，用于高效筛选特异性CEACAM5抗体。本发明筛选得到的抗体同时结合CEACAM5三个结构域(A1-B1，A2-B2，A3-B3)，因此可提高CEACAM5高表达肿瘤细胞的表面结合量，从而提高其局部浓度，提高药效。
人源化抗体
对筛选得到鼠源抗体进行人源化设计和筛选可能降低临床应用中HAMA(Human Anti Mouse Antibody)效应，降低患者体内中和抗体产生，提高药物的血药浓度从而提高药效。
“人源化抗体”是包含人源化VH结构域和人源化VL结构域中的一者或两者的抗体。一个或多个免疫球蛋白恒定区无需存在，但如果存在的话，那么其完全或大体上来自人类免疫球蛋白恒定区。
人源化抗体是基因工程改造的抗体，其中来自非人类“供体”抗体的CDR接枝至人类“接受体”抗体序列中(参见例如Queen，US 5,530,101和5,585,089；Winter，US 5,225,539；Carter，US 6,407,213；Adair，US 5,859,205；以及Foote，US 6,881,557)。 接受体抗体序列可为例如成熟人类抗体序列、此类序列的复合物、人类抗体序列的共有序列或生殖区序列。可选择人类接受体序列以使可变区构架与供体序列具有高程度序列同一性，以匹配接受体CDR与供体CDR之间的典型形式和其它准则。因此，人源化抗体是CDR完全或大体上来自供体抗体和可变区构架序列并且恒定区(如果存在的话)完全或大体上来自人类抗体序列的抗体。类似地，人源化重链通常具有完全或大体上来自供体抗体重链和重链可变区构架序列的所有三个CDR以及大体上来自人类重链可变区构架和恒定区序列的重链恒定区(如果存在的话)。类似地，人源化轻链通常具有完全或大体上来自供体抗体轻链和轻链可变区构架序列的所有三个CDR以及大体上来自人类轻链可变区构架和恒定区序列的轻链恒定区(如果存在的话)。当各别CDR之间对应的残基(如依Kabat编号定义)的至少约80％、约81％、约82％、约83％、约84％、约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％相同时或其中对应的残基(如依Kabat编号定义)的约100％相同，人源化抗体中的CDR大体上来自非人类抗体中的对应的CDR。当对应的残基(可变区依Kabat编号定义并且恒定区依EU编号定义)的至少约80％、约81％、约82％、约83％、约84％、约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％相同或对应的残基(可变区依Kabat编号定义并且恒定区依EU编号定义)的约100％相同时，抗体链的可变区构架序列或抗体链的恒定区分别大体上来自人类可变区构架序列或人类恒定区。
虽然人源化抗体常常合并有来自小鼠抗体的所有六个CDR(优选如依Kabat或IMGT定义)，但人源化抗体还可由来自小鼠抗体的少于所有六个CDR(例如，至少3个、4个或5个)CDR组成(例如，Pascalis等,J.Immunol.169:3076,2002；Vajdos等,Journal of Molecular Biology,320:415-428,2002；Iwahashi等,Mol.Immunol.36:1079-1091,1999；Tamura等,Journal of Immunology,164:1432-1441,2000)。
当个别CDR之间对应的残基(如依Kabat或IMGT)定义)的至少60％、至少85％、至少90％、至少95％或100％相同时，人源化抗体中的CDR“大体上来自”非人类抗体中的对应的CDR。在CDR大体上来自非人类免疫球蛋白的人源化VH或VL结构域的特定变化中，人源化VH或VL结构域的CDR相对于对应的非人类VH或VL CDR 跨越所有三个CDR具有不多于六个(例如，不多于五个、不多于四个、不多于三个、不多于两个或不多于一个)氨基酸取代(优选保守性取代)。当对应的残基(可变区依Kabat编号定义并且恒定区依EU编号定义)的至少约80％、约81％、约82％、约83％、约84％、约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％相同或对应的残基(可变区依Kabat编号定义并且恒定区依EU编号定义)的约100％相同时，抗体VH或VL结构域的可变区构架序列或免疫球蛋白恒定区的序列(如果存在的话)分别“大体上来自”人类VH或VL构架序列或人类恒定区。因此，人源化抗体的所有部分(CDR除外)通常完全或大体上来自天然人类免疫球蛋白序列的对应部分。
一般性术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“抗体”是指，通常由两对多肽链(每对具有一条轻链和一条重链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991))，或Chothia& Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。在一些情况下，抗原结合片段包括Fab、Fab'、F(ab')2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即，除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(Nature,256:495,1975)，但也可采用重组DNA技术获得(如参见U.S.P 4,816,567)。
例如，可以如下来制备单克隆抗体。首先用免疫原(必要时候添加佐剂)免疫注射小鼠或其它合适的宿主动物。免疫原或佐剂的注射方式通常为皮下多点注射或腹腔注射。可将免疫原预先偶联到某些已知蛋白，如血清白蛋白或大豆胰酶抑制剂上，以增强抗原在宿主内的免疫原性。佐剂可以是弗氏佐剂或MPL-TDM等。动物在接受免疫后，体内将产生分泌特异性结合免疫原的抗体的淋巴细胞。另外，淋巴细胞也可以利用体外免疫获得。收集目的淋巴细胞，并用合适的融合剂，如PEG，使其与骨髓瘤细胞融 合以获得杂交瘤细胞(Goding,Monoclonal Antibodies:Principles and Practice,pp.59-103,Academic Press,1996)。上述制备的杂交瘤细胞可以接种到合适的培养液中生长，培养液中优选含有一种或多种能够抑制未融合的、母体骨髓瘤细胞生长的物质。例如，对于缺乏次黄嘌呤鸟嘌呤磷酸转移酶(HGPRT或HPRT)的母体骨髓瘤细胞，在培养液中添加次黄嘌呤、氨基喋呤和胸腺嘧啶(HAT培养基)等物质将可以抑制HGPRT-缺陷细胞的生长。优选的骨髓瘤细胞应该具有融合率高，抗体分泌能力稳定，对HAT培养液敏感等特征。其中，骨髓瘤细胞首选鼠源骨髓瘤，如MOP-21或MC-11小鼠肿瘤衍生株(THE Salk Institute Cell Distribution Center,San Diego,Calif.USA)，和SP-2/0或X63-Ag8-653细胞株(American Type Culture Collection,Rockville,Md.USA)。另外也有研究报道，利用人骨髓瘤和人鼠异源骨髓瘤细胞株制备人单抗(Kozbor,J.Immunol.,133:3001(1984)；Brodeur et al.,Monoclonal Antibody Production Techniques and Applications,pp.51-63,Marcel Dekker,Inc.,New York,1987)。生长杂交瘤细胞的培养液用于检测针对特异抗原的单抗的产生。测定杂交瘤细胞产生的单抗的结合特异性的方法包括例如，免疫沉淀或体外结合试验，如放射免疫试验(RIA)、酶联免疫吸附试验(ELISA)。例如，可利用Munson等在Anal.Biochem.107:220(1980)描述的Scatchard分析法来测定单抗的亲和力。当确定了杂交瘤产生的抗体的特异性、亲和力和反应性之后，目的细胞株可以通过(Goding,Monoclonal Antibodies:Principles and Practice,pp.59-103,Academic Press,1996)所描述的标准的有限稀释法进行亚克隆化。合适的培养液可以是DMEM或RPMI-1640等。另外，杂交瘤细胞还可以腹水瘤的形式在动物体内生长。利用传统的免疫球蛋白纯化方法，如蛋白A琼脂糖凝胶、羟基磷灰石层析、凝胶电泳、透析或亲和层析等，可以将亚克隆细胞分泌的单抗从细胞培养液、腹水或血清中分离出来。
还可以通过基因工程重组技术获得单克隆抗体。利用特异性结合单抗重链和轻链基因的核酸引物进行PCR扩增，可以从杂交瘤细胞中分离得到编码单抗重链和轻链基因的DNA分子。将所得的DNA分子插入表达载体内，然后转染宿主细胞(如E.coli细胞、COS细胞、CHO细胞、或其它不产生免疫球蛋白的骨髓瘤细胞)，并在合适的条件下进行培养，可以获得重组表达的目标抗体。
如本文中所使用的，术语“嵌合抗体”是指这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和 重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性(U.S.P 4,816,567 to Cabilly et al.；Morrison et al.,Proc.Natl.Acad.Sci.USA,81:6851 6855(1984))。
如本文中所使用的，术语“人抗体”是指人源化抗体，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.,Nature,321:522525(1986)；Reichmann et al.,Nature,332:323 329(1988)；Presta,Curr.Op.Struct.Biol.,2:593 596(1992)；和Clark,Immunol.Today 21:397 402(2000)。
如本文中所使用的，术语“表位”是指，抗原上被免疫球蛋白或抗体特异性结合的部位。“表位”在本领域内也称为“抗原决定簇”。表位或抗原决定簇通常由分子的化学活性表面基团例如氨基酸或碳水化合物或糖侧链组成，并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸，其可以是“线性的”或“构象的”。参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
如本文中所使用的，术语“表位肽”是指，抗原上能够用作表位的肽段。在一些情况下，单独的表位肽即能够被针对所述表位的抗体特异性识别/结合。在另一些情况下，可能需要将表位肽与载体蛋白融合，以便表位肽能够被特异性抗体识别。如本文中所使用的，术语“载体蛋白”是指这样的蛋白，其可以充当表位肽的载体，即，其可以在特定位置处(例如蛋白内部，N端或C端)插入表位肽，以便该表位肽能够呈现出来，从而该表位肽能够被抗体或免疫系统识别。此类载体蛋白是本领域技术人员熟知的，包括例如，HPV L1蛋白(可以将表位肽插入在所述蛋白的第130-131位氨基酸之间或在第426-427位氨基酸之间，参见Slupetzky,K.等Chimeric papillomavirus-like particles expressing a foreign epitope on capsid surface loops[J].J Gen Virol,2001,82: 2799-2804；Varsani,A.等Chimeric human papillomavirus type 16(HPV-16)L1 particles presenting the common neutralizing epitope for the L2 minor capsid protein of HPV-6 and HPV-16[J].J Virol,2003,77:8386-8393)，HBV核心抗原(可以用表位肽替换所述蛋白的第79-81位氨基酸，参见Koletzki,D.,et al.HBV core particles allow the insertion and surface exposure of the entire potentially protective region of Puumala hantavirus nucleocapsid protein[J].Biol Chem,1999,380:325-333)，土拨鼠肝炎病毒核心蛋白(可以用表位肽替换所述蛋白的第79-81位氨基酸，参见Sabine
Figure PCTCN2021070825-appb-000001
Gertrud Beterams and Michael Nassal，J.Virol.1998,72(6):4997)，CRM197蛋白(可以将表位肽连接至该蛋白或其片段的N末端或C末端)。任选地，可以在表位肽与载体蛋白之间使用连接体(例如柔性或刚性连接体)，以促进二者各自的折叠。
可使用本领域技术人员已知的常规技术，就与相同表位的结合竞争性筛选抗体。例如，可进行竞争和交叉竞争研究，以获得彼此竞争或交叉竞争与抗原(例如，流感病毒血凝素蛋白)的结合的抗体。基于它们的交叉竞争来获得结合相同表位的抗体的高通量方法描述于国际专利申请WO 03/48731中。因此，可使用本领域技术人员已知的常规技术，获得与本发明的单克隆抗体竞争结合流感病毒血凝素蛋白上的相同表位的抗体及其抗原结合片段(即，抗原结合部分)。
如本文中使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的亲和力(K D)结合该抗原。
如本文中所使用的，术语“K D”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。可以使用多种方法测定，例如使用表面等离子体共振术(SPR)在BIACORE仪中测定。
如本文中所使用的，术语“杂交瘤”和“杂交瘤细胞株”可互换使用，并且当提及术语“杂交瘤”和“杂交瘤细胞株”时，其还包括杂交瘤的亚克隆和后代细胞。例如，当提及杂交瘤细胞株2F4时，其还指杂交瘤细胞株2F4的亚克隆和后代细胞。
附图说明
图1显示了通过CRISPR方法使用载体(CEACAM5 KO1-3)对高表达CEACAM5的细胞Lovo 6-1进行CEACAM5基因敲除的结果。
图2显示了使用CEACAM5 KO3号载体对高表达CEACAM5的细胞Lovo 6-1进行CEACAM5基因敲除的结果。
图3显示了鼠单克隆抗体对CEACAM5重组蛋白的结合结果。
图4显示了鼠单克隆抗体对CEACAM5胞外结构域的结合。
图5显示了鼠单克隆抗体对高表达CEACAM5的LS174T细胞和KATO3细胞的结合。
图6显示了人源化抗体对重组CEACAM5抗原的结合。
图7显示了人源化抗体对高表达CEACAM5的KATO3细胞的结合。
图8显示了人源化抗体与鼠m2F4抗体对CEACAM5的竞争结合。
序列信息
本申请涉及的部分序列信息描述于下表中，其余描述于实施例中。
Figure PCTCN2021070825-appb-000002
Figure PCTCN2021070825-appb-000003
Figure PCTCN2021070825-appb-000004
Figure PCTCN2021070825-appb-000005
Figure PCTCN2021070825-appb-000006
Figure PCTCN2021070825-appb-000007
具体实施方式
下面将结合实施例对本申请的实施方案进行详细描述，但是本领域技术人员将理解，下列实施例仅用于说明本申请，而不是对本申请的范围的限定。根据优选实施方案的下列详细描述，本申请的各种目的和有利方面对于本领域技术人员来说将变得显然。
实施例1.单克隆抗体的制备
本实施例利用表达CEACAM5的肿瘤细胞系免疫小鼠制备单克隆抗体。
1.SJL小鼠免疫/杂交瘤融合
高表达CEACAM5的Lovo细胞系ATCC CCL-229培养于含10％FBS RPMI1640培养基中，将Lovo细胞用TrypLE胰酶消化后，重悬于DPBS溶液中，每只SJL小鼠皮下多点免疫，每次免疫10 7Lovo细胞，每周免疫一次，共5次。将经过血清滴度检测的小鼠处死后，取脾脏研磨过筛，按标准融合流程融合SP20骨髓瘤细胞，获得杂交瘤细胞。
2.Lovo CEACAM5 KO细胞系的构建及筛选
2.1 Lovo CEACAM5高表达单克隆细胞系筛选
将Lovo细胞用抗CEACAM5抗体hMN14(Immunomedics，Phase2药物，经重组表达，序列如下
>hMN14 VH
Figure PCTCN2021070825-appb-000008
Figure PCTCN2021070825-appb-000009
>hMN14 VL
DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC标记后，加入抗鼠Fc-PE荧光标记二抗，用BD FACS Aria分选至96孔板，并培养单克隆后，用MN14检测单克隆CEACAM5表达，结果如下：
克隆 CEACAM5阳性比例％ CEACAM5阳性MFI
Lovo+二抗 0.3％ NA
1-1 90.5％ 1105790
1-2 95.6％ 783976
2-1 95.6％ 1009535
2-2 96.4％ 999722
3-1 97.8％ 915297
3-2 95.9％ 974630
4-1 93.7％ 904109
4-2 82.7％ 1142303
5-1 76.6％ 513840
5-2 96.9％ 1053108
6-1 98.6％ 1470927
6-2 88.1％ 751552
7-1 93.9％ 759412
7-2 96.9％ 990410
8-1 97.9％ 1188614
8-2 96.8％ 1099974
以上结果表明:6-1号克隆相比其他克隆纯度更高，达到98.6％，且其CEACAM5表达MFI显著高于其他克隆，因此挑选6-1号克隆进行CEACAM5敲除。
2.2 Lovo CEACAM5 KO细胞系筛选
将Lovo 6-1克隆用CRISPR方法进行CEACAM5基因敲除，并筛选Lovo  CEACAM5 KO细胞系(以下简称Lovo CEA KO细胞系)。将携带CRISPR及sgRNA载体包装为慢病毒载体(CEACAM5 KO1-3)，并转导Lovo 6-1细胞，转导后通过FACS检测CEACAM5表达(MN14抗体)，通过鼠Fc-APC二抗检测MN14结合。结果如图1所示。结果表明，CEACAM5 KO1-3均可对CEACAM5敲除，其中CEACAM5 KO3号载体敲除效率更高，表现为CEACAM5阴性群体更加清晰。靶向CEACAM5三条sgRNA的序列如下：
No sgRNA
CEACAM5 KO1 GATCTGACTTTATGACGTGT
CEACAM5 KO2 GATGACTGAATCACTGCGCC
CEACAM5 KO3 CAGGGGATGCACCATCTGTG
如图2所示，Lovo 6-1克隆经CRISPR方法敲除CEACAM5后，其细胞群体中出现CEACAM5敲除群体，占比为100％-29.3％，表明成功进行了CEACAM5敲除。
3.杂交瘤克隆及筛选
将Lovo细胞及Lovo CEA KO细胞按每孔10 4接种于96孔板中过夜培养，将杂交瘤上清1-10μl分别加入Lovo/Lovo CEA KO细胞培养板中，孵育1小时，将上清弃掉，加入抗鼠Fc-FITC荧光二抗，孵育1小时后，弃掉上清，加入含有2％BSA的DPBS溶液，于Celigo中读取和分析荧光信号及FITC染色区域面积。
筛选得到M19(2F4)、M7(11B6)、M17(6A8)、M18(7G1)四个克隆。
克隆 Lovo结合百分比 Lovo CEA KO结合百分比
M7 54.6％ 0.5％
M17 34.7％ 1.1％
M18 89.5％ 0.7％
M19 78.4％ 0.3％
4.杂交瘤测序/重组表达载体构建
筛选得到M19(2F4)、M7(11B6)、M17(6A8)、M18(7G1)四个克隆。将挑取的杂交瘤克隆按照标准的杂交瘤测序方法进行杂交瘤测序得到所挑取克隆的重链 及轻链可变区(VH和VL)。将VH和VL经全基因合成的方式合成并连接human IgG1及kappa链恒定区，将重链及轻链序列连接至pcDNA3.4载体中并于293系统中瞬时表达并进行protein A/G纯化。所得到的嵌合重组抗体经超滤进行缓冲液置换为PBS溶液。测序结果如下表所示。
Figure PCTCN2021070825-appb-000010
Figure PCTCN2021070825-appb-000011
Figure PCTCN2021070825-appb-000012
Figure PCTCN2021070825-appb-000013
Figure PCTCN2021070825-appb-000014
实施例2.抗原结合ELISA实验
将重组CEACAM5抗原(Sinobiological，11077-H08H)用DPBS溶液稀释至1μg/ml，并按照每孔100μl加入96孔板中，于2-8℃包被过夜；将包被液弃掉，并用PBS溶液清洗2次，加入含2％BSA的PBS溶液，室温封闭2小时；将封闭液弃掉，加入浓度梯度稀释的抗体，于37℃孵育1小时；将抗体溶液弃掉，并用含有0.05％Tween20的PBS溶液(PBST溶液)清洗4次；加入抗人IgG Fc-HRP二抗并于37℃ 孵育30分钟；用PBST溶液清洗4次，加入TMB显色底物，显色5-10分钟后，等体积1M H 2SO4终止反应；于酶标仪读取450nm光吸收。以上四个抗体M7、M17、M18、M19对CEACAM5重组蛋白的结合结果如图3和下表所示。结果表明：以上4个抗体均可结合CEACAM5-His重组蛋白。
抗体 EC50μg/ml
M7 0.008
M17 0.013
M18 0.016
M19 0.055
实施例3.抗体Epitope结合实验
将CEACAM5分子，按照其胞外结构域(A1-B1-A2-B2-A3-B3)进行拆分，分别构建A1-B1-His Tag、A2-B2-His Tag、A3-B3-His Tag表达载体，并于293系统表达后，利用Ni柱进行纯化，序列如下表所示：
Figure PCTCN2021070825-appb-000015
Figure PCTCN2021070825-appb-000016
Figure PCTCN2021070825-appb-000017
Figure PCTCN2021070825-appb-000018
Figure PCTCN2021070825-appb-000019
将上述CEACAM5片段，以DPBS溶液稀释至1μg/ml并按照每孔100μl加入96孔板中，于2-8℃包被过夜；将包被液弃掉，并用PBS溶液清洗2次，加入含2％BSA的PBS溶液，室温封闭2小时；将封闭液弃掉，加入浓度梯度稀释的抗体，于37℃孵育1小时；将抗体溶液弃掉，并用含有0.05％Tween20的PBS溶液(PBST溶液)清洗4次；加入抗人IgG Fc-HRP二抗并于37℃孵育30分钟；用PBST溶液清洗4次，加入TMB显色底物，显色5-10分钟后，等体积1M H 2SO4终止反应；于酶标仪读取450nm光吸收。结果如图4所示。
上述四种抗体M7、M17、M18、M19结合CEACAM5分子的表位如下：
抗体 CEACAM5结构域
M7 A1-B1
M17 A1-B1
M18 A1-B1
M19 A1-B1，A2-B2，A3-B3
以上结果表明，M19抗体可以识别并结合所有三个CEACAM5结构域，其中对A2-B2结构域结合EC50最小(EC50＝0.003μg/ml)，远小于A1-B1(EC50＝0.95μg/ml)和A3-B3(EC50＝3.23μg/ml)结构域结合；这说明，M19抗体主要结合位置位于CEACAM5分子的A2-B2结构域，但同时也可以结合A1-B1和A3-B3结构域，可能结合B1-A2和B2-A3结构域。
实施例4.抗原结合FACS实验
将LS174T细胞和KATO3细胞(CEACAM5高表达；ATCC,CL-188)培养于含10％FBS RPMI1640培养基，将细胞用TrypLE胰酶消化后，离心重悬于含2％BSA的DPBS溶液(FACS缓冲液，4℃)中，按照5×10 5/100μl/孔加入U型底96孔板中， 并加入浓度梯度稀释的抗体，于4℃孵育1小时，离心后弃掉上清；每孔加入100μl含有抗人IgG Fc-APC二抗并于4℃孵育1小时后，用FACS缓冲液清洗1次，重悬于200μl FACS缓冲液中，于BD CantoII读取荧光信号值。结果如图5所示。结果表明：以上抗体均结合LS174T细胞和KATO3细胞。
抗体 LS174T细胞结合EC50μg/ml KATO3细胞结合EC50μg/ml
M7 1.9 2.6
M17 1.5 2.7
M18 11.9 13.9
M19 6.5 10.8
实施例5.人源化抗体设计与表达
将M19抗体(m2F4)与IMGT数据库比对，选取与其VH/VL同源度最高的人源Framework序列，进行CDR graft，并进行计算化学模拟维持其与抗原结合，人源化抗体设计如下表所示。
Figure PCTCN2021070825-appb-000020
Figure PCTCN2021070825-appb-000021
Figure PCTCN2021070825-appb-000022
Figure PCTCN2021070825-appb-000023
Figure PCTCN2021070825-appb-000024
Figure PCTCN2021070825-appb-000025
Figure PCTCN2021070825-appb-000026
Figure PCTCN2021070825-appb-000027
将以上抗体VH与VL区域链接human IgG1Fc区域及kappa恒定区，并将抗体的重链和轻链序列插入pcDNA3.4载体，瞬时表达于293细胞，通过protein A或G进行抗体纯化。
实施例6.人源化抗体ELISA结合实验
将重组CEACAM5抗原(Sinobiological，11077-H08H)用DPBS溶液稀释至1μg/ml，并按照每孔100μl加入96孔板中，于2-8℃包被过夜；将包被液弃掉，并用PBS溶液清洗2次，加入含2％BSA的PBS溶液，室温封闭2小时；将封闭液弃掉，加入浓度梯度稀释的人源化抗体，于37℃孵育1小时；将抗体溶液弃掉，并用含有0.05％Tween20的PBS溶液(PBST溶液)清洗4次；加入抗人IgG Fc-HRP二抗并于37℃孵育30分钟；用PBST溶液清洗4次，加入TMB显色底物，显色5-10分钟后，等体积1M H 2SO4终止反应；于酶标仪读取450nm光吸收。以上人源化抗体对CEACAM5重组蛋白的结合结果如图6所示。结果表明：除hAb-005抗体，其他人源化抗体均可结合CEACAM5-His抗原。
人源化抗体 EC50μg/ml
M2F4 3.53
hAb-003 3.69
hAb-005 No binding
hAb-006 3.11
hAb-009 2.87
hAb-010 3.56
hAb-013 6.10
hAb-016 4.32
hAb-017 7.05
实施例7.人源化抗体细胞结合实验
将KATO3细胞(CEACAM5高表达)培养于含10％FBS RPMI1640培养基，将细胞用TrypLE胰酶消化后，离心重悬于含2％BSA的DPBS溶液(FACS缓冲液，4℃)中，按照5×10 5/100μl/孔加入U型底96孔板中，并加入浓度梯度稀释的抗体，于4℃孵育1小时，离心后弃掉上清；每孔加入100μl含有抗人IgG Fc-APC二抗并于4℃孵育1小时后，用FACS缓冲液清洗1次，重悬于200μl FACS缓冲液中，于BD C6plus读取荧光信号值。结果如图7所示。
人源化抗体结合KATO3细胞EC50和Emax如下表所示
抗体 EC50μg/ml Emax
m2F4-hIgG1 5.3 100％
hAb-003 2.7 85.1％
hAb-005 3250 9.1％
hAb-006 5.7 118.2％
hAb-009 1.8 108.6％
hAb-010 1.3 60.6％
hAb-013 12.7 49.3％
hAb-016 3.2 48.4％
hAb-017 2.2 57.7％
以上结果表明，hAb-005丢失了与CEACAM5蛋白和KATO3细胞系结合；其中hAb-009人源化抗体对KATO3细胞结合能力最强，且保持了最大结合。
实施例8.抗体结合阻断实验
将KATO3细胞(CEACAM5高表达)培养于含10％FBS RPMI1640培养基，将细胞用TrypLE胰酶消化后，离心重悬于含2％BSA的DPBS溶液(FACS缓冲液，4℃)中，按照5×10 5/100μl/孔加入U型底96孔板中，加入鼠单抗M19抗体(m2F4)至1μg/ml和浓度梯度稀释的人源化抗体(50μg/ml，3倍稀释)，于4℃孵育1小时，离心后弃掉上清；每孔加入100μl含有抗鼠IgG Fc-APC二抗并于4℃孵育1小时后，用FACS缓冲液清洗1次，重悬于200μl FACS缓冲液中，于BD C6plus读取荧光信号值。结果如图8所示。
人源化抗体对鼠单抗M19抗体(m2F4)结合阻断结果如下表所示
抗体 IC50μg/ml Emax Inhibition％
m2F4-hIgG1 6.6 43.7％
hAb-003 3.9 51.3％
hAb-005 ND ND
hAb-006 6.9 47.2％
hAb-009 2.7 62.7％
hAb-010 2.3 75.0％
hAb-013 5.2 89.0％
hAb-016 6.4 73.2％
hAb-017 4.5 73.9％
以上结果表明，hAb-003、hAb-006、hAb-009、hAb-010、hAb-013、hAb-016、hAb-017抗体与m2F4抗体能够产生竞争作用，表明其结合CEACAM5分子上的表位一致。

Claims (13)

  1. 一种特异性结合糖基化CEACAM5的人源化抗体或其抗原结合片段，其中所述抗体包含轻链可变区及重链可变区，其中：
    所述轻链可变区包含：
    选自SEQ ID NO:84、SEQ ID NO:90、SEQ ID NO:96、SEQ ID NO:102、SEQ ID NO:108、SEQ ID NO:114、SEQ ID NO:120和SEQ ID NO:126的CDR-L1；
    选自SEQ ID NO:85、SEQ ID NO:91、SEQ ID NO:97、SEQ ID NO:103、SEQ ID NO:109、SEQ ID NO:115、SEQ ID NO:121和SEQ ID NO:127的CDR-L2；和
    选自SEQ ID NO:86、SEQ ID NO:92、SEQ ID NO:98、SEQ ID NO:104、SEQ ID NO:110、SEQ ID NO:116、SEQ ID NO:122和SEQ ID NO:128的CDR-L3，
    并且所述重链可变区包含：
    选自SEQ ID NO:87、SEQ ID NO:93、SEQ ID NO:99、SEQ ID NO:105、SEQ ID NO:111、SEQ ID NO:117、SEQ ID NO:123和SEQ ID NO:129的CDR-H1；
    选自SEQ ID NO:88、SEQ ID NO:94、SEQ ID NO:100、SEQ ID NO:106、SEQ ID NO:112、SEQ ID NO:118、SEQ ID NO:124和SEQ ID NO:130的CDR-H2；和
    选自SEQ ID NO:89、SEQ ID NO:95、SEQ ID NO:101、SEQ ID NO:107、SEQ ID NO:113、SEQ ID NO:119、SEQ ID NO:125和SEQ ID NO:131的CDR-H3。
  2. 根据权利要求1所述的人源化抗体或其抗原结合片段，其中所述轻链可变区包含与选自SEQ ID NO:68、70、72、74、76、78、80、82的氨基酸序列具有至少90％同一性的氨基酸序列，并且所述重链可变区包含与选自SEQ ID NO:69、71、73、75、77、79、81、83的氨基酸序列具有至少90％同一性的氨基酸序列；
    优选地，所述抗体包含：由选自SEQ ID NO:52、54、56、58、60、62、64、66的核苷酸序列编码的轻链可变区，以及由选自SEQ ID NO:53、55、57、59、61、63、65、67的核苷酸序列编码的重链可变区；
    优选地，所述抗体包含选自SEQ ID NO:68、70、72、74、76、78、80、82的轻链可变区和选自SEQ ID NO:69、71、73、75、77、79、81、83的重链可变区；
    优选地，所述抗体包含轻链可变区及重链可变区，其中：
    a.所述轻链可变区包含：由SEQ ID NO:84所示的CDR-H1、由SEQ ID NO:85所示的CDR-H2和由SEQ ID NO:86所示的CDR-H3；同时所述重链可变区包含：由SEQ ID NO:87所示的CDR-L1、由SEQ ID NO:88所示的CDR-L2和由SEQ ID NO:89所示的CDR-L3；
    b.所述轻链可变区包含：由SEQ ID NO:90所示的CDR-H1、由SEQ ID NO:91所示的CDR-H2和由SEQ ID NO:92所示的CDR-H3；同时所述重链可变区包含：由SEQ ID NO:93所示的CDR-L1、由SEQ ID NO:94所示的CDR-L2和由SEQ ID NO:95所示的CDR-L3；
    c.所述轻链可变区包含：由SEQ ID NO:96所示的CDR-H1、由SEQ ID NO:97所示的CDR-H2和由SEQ ID NO:98所示的CDR-H3；同时所述重链可变区包含：由SEQ ID NO:99所示的CDR-L1、由SEQ ID NO:100所示的CDR-L2和由SEQ ID NO:101所示的CDR-L3；
    d.所述轻链可变区包含：由SEQ ID NO:102所示的CDR-H1、由SEQ ID NO:103所示的CDR-H2和由SEQ ID NO:104所示的CDR-H3；同时所述重链可变区包含：由SEQ ID NO:105所示的CDR-L1、由SEQ ID NO:106所示的CDR-L2和由SEQ ID NO:107所示的CDR-L3；
    e.所述轻链可变区包含：由SEQ ID NO:108所示的CDR-H1、由SEQ ID NO:109所示的CDR-H2和由SEQ ID NO:110所示的CDR-H3；同时所述重链可变区包含：由SEQ ID NO:111所示的CDR-L1、由SEQ ID NO:112所示的CDR-L2和由SEQ ID NO:113所示的CDR-L3；
    f.所述轻链可变区包含：由SEQ ID NO:114所示的CDR-H1、由SEQ ID NO:115所示的CDR-H2和由SEQ ID NO:116所示的CDR-H3；同时所述重链可变区包含：由SEQ ID NO:117所示的CDR-L1、由SEQ ID NO:118所示的CDR-L2和由SEQ ID NO:119所示的CDR-L3；
    g.所述轻链可变区包含：由SEQ ID NO:120所示的CDR-H1、由SEQ ID NO:121所示的CDR-H2和由SEQ ID NO:122所示的CDR-H3；同时所述重链可变区包含：由SEQ ID NO:123所示的CDR-L1、由SEQ ID NO:124所示的CDR-L2和由SEQ ID NO:125所示的CDR-L3；或
    h.所述轻链可变区包含：由SEQ ID NO:126所示的CDR-H1、由SEQ ID NO:127 所示的CDR-H2和由SEQ ID NO:128所示的CDR-H3；同时所述重链可变区包含：由SEQ ID NO:129所示的CDR-L1、由SEQ ID NO:130所示的CDR-L2和由SEQ ID NO:131所示的CDR-L3。
  3. 一种特异性结合糖基化CEACAM5的单克隆抗体或其抗原结合片段，其中所述抗体包含重链可变区及轻链可变区，其中：
    a.所述重链可变区包含：由SEQ ID NO:1所示的CDR-H1、由SEQ ID NO:2所示的CDR-H2和由SEQ ID NO:3所示的CDR-H3；同时所述轻链可变区包含：由SEQ ID NO:4所示的CDR-L1、由SEQ ID NO:5所示的CDR-L2和由SEQ ID NO:6所示的CDR-L3；
    b.所述重链可变区包含：由SEQ ID NO:7所示的CDR-H1、由SEQ ID NO:8所示的CDR-H2和由SEQ ID NO:9所示的CDR-H3；同时所述轻链可变区包含：由SEQ ID NO:10所示的CDR-L1、由SEQ ID NO:11所示的CDR-L2和由SEQ ID NO:12所示的CDR-L3；
    c.所述重链可变区包含由SEQ ID NO:13所示的CDR-H1、由SEQ ID NO:14所示的CDR-H2和由SEQ ID NO:15所示的CDR-H3；同时所述轻链可变区包含由SEQ ID NO:16所示的CDR-L1、由SEQ ID NO:17所示的CDR-L2和由SEQ ID NO:18所示的CDR-L3；或
    d.所述重链可变区包含由SEQ ID NO:19所示的CDR-H1、由SEQ ID NO:20所示的CDR-H2和由SEQ ID NO:21所示的CDR-H3；同时所述轻链可变区包含由SEQ ID NO:22所示的CDR-L1、由SEQ ID NO:23所示的CDR-L2和由SEQ ID NO:24所示的CDR-L3。
  4. 根据权利要求3所述的单克隆抗体或其抗原结合片段，其中：
    a)所述重链可变区包含由SEQ ID NO:25所示的多肽，同时所述轻链可变区包含由SEQ ID NO:26所示的多肽；
    b)所述重链可变区包含由SEQ ID NO:27所示的多肽，同时所述轻链可变区包含由SEQ ID NO:28所示的多肽；
    c)所述重链可变区包含由SEQ ID NO:29所示的多肽，同时所述轻链可变区包含 由SEQ ID NO:30所示的多肽；或
    d)所述重链可变区包含由SEQ ID NO:31所示的多肽，同时所述轻链可变区包含由SEQ ID NO:32所示的多肽。
  5. 根据权利要求3所述的单克隆抗体或其抗原结合片段，其中：
    a)所述重链可变区包含由SEQ ID NO:38编码的多肽，同时所述轻链可变区包含由SEQ ID NO:39所示的多肽；
    b)所述重链可变区包含由SEQ ID NO:40编码的多肽，同时所述轻链可变区包含由SEQ ID NO:41所示的多肽；
    c)所述重链可变区包含由SEQ ID NO:42编码的多肽，同时所述轻链可变区包含由SEQ ID NO:43所示的多肽；或
    d)所述重链可变区包含由SEQ ID NO:44编码的多肽，同时所述轻链可变区包含由SEQ ID NO:45所示的多肽。
  6. 一种分离的核酸分子，其包含编码权利要求1-5任一项的抗体或其抗原结合片段的核苷酸序列。
  7. 一种载体，其包含权利要求6所述的核酸分子。
  8. 一种宿主细胞，其包含权利要求7所述的载体。
  9. 一种药物组合物，其包含权利要求1-5任一项的抗体或其抗原结合片段和药学上可接受的载体。
  10. 一种制备特异性结合糖基化CEACAM5的抗体的方法，其包括在宿主细胞中表达权利要求6的核酸分子并从宿主细胞分离特异性结合糖基化CEACAM5的抗体。
  11. 权利要求1-5任一项的抗体在制备用于治疗胃肠道相关肿瘤的药物中的用途。
  12. 一种治疗胃肠道相关肿瘤的方法，包括向有此需要的受试者施用权利要求1-5任一项的抗体。
  13. 权利要求1-5任一项的抗体，用于治疗胃肠道相关的肿瘤。
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