WO2023178645A1 - 靶向cd3的抗体及其应用 - Google Patents

靶向cd3的抗体及其应用 Download PDF

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WO2023178645A1
WO2023178645A1 PCT/CN2022/083004 CN2022083004W WO2023178645A1 WO 2023178645 A1 WO2023178645 A1 WO 2023178645A1 CN 2022083004 W CN2022083004 W CN 2022083004W WO 2023178645 A1 WO2023178645 A1 WO 2023178645A1
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seq
heavy chain
variable region
light chain
following sequence
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PCT/CN2022/083004
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English (en)
French (fr)
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丁茜
张雪琨
彭菲
李健
冯蒙
张琳琳
李贞�
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嘉和生物药业有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • 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

Definitions

  • the present application relates to the field of biomedicine, and in particular to an antibody targeting CD3 or an antigen-binding fragment thereof, as well as preparation methods and applications thereof.
  • CD3 is a protein complex composed of four different chains (CD3 ⁇ chain, CD3 ⁇ chain, CD3 ⁇ chain and CD3 ⁇ chain), which contains three dimers, namely CD3 ⁇ / ⁇ (or CD3e/g), CD3 ⁇ / ⁇ (or CD3e/d) and CD3 ⁇ / ⁇ (or CD3z/z), their intracellular regions contain immunoreceptor tyrosine-based activation motifs (Immunoreceptor tyrosine-based activation) necessary to initiate signal transmission. motif, ITAM).
  • ITAM immunoreceptor tyrosine-based activation
  • TCR is a heterodimer of covalently linked ⁇ and ⁇ chains (“TCR ⁇ "). It mainly recognizes antigens presented by MHC molecules, but its intracellular domain is very short. It needs to form a TCR/CD3 complex with CD3 to transmit the stimulating signal. into cells (Dong, D., Zheng, L., Lin, J., et al. Nature 573, 546–552 (2019)).
  • CD3-targeting antibodies used interchangeably in this disclosure with "CD3 antibody”, “anti-CD3 antibody”, “antibody that specifically binds to CD3"
  • CD3 antibodies include OKT3.
  • T cell function usually returns to normal within a week after treatment with OKT3, but OKT3 also has considerable side effects at the beginning, including OKT3 influenza-like syndrome, cytokine storm, etc.
  • OKT3 does not bind to CD3 derived from cynomolgus, which brings inconvenience to the selection of primate cynomolgus monkeys in preclinical safety evaluation studies.
  • the present disclosure provides new anti-CD3 antibodies that can bind to human and cynomolgus monkey CD3 with low side effects, providing new potential treatments for diseases including cancer and autoimmune diseases.
  • the antibodies of the present disclosure or antigen-binding fragments thereof have excellent biological activity (eg, high affinity). Its ability to bind to human CD3e/g (hCD3e/g), human CD3e/d (hCD3e/d) and cynomolgus monkey CD3e/g (cCD3e/g) enables primates of choice for preclinical safety assessment studies.
  • the disease model of cynomolgus monkey (cynomolgus, cyno) is used for pharmacological and toxicological experiments, which brings great convenience to preclinical pharmacological and toxicological research.
  • the antibodies or antigen-binding fragments thereof of the present disclosure can effectively activate the NFAT downstream signaling pathway of human T cell line Jurkat cells, which is beneficial to subsequent biological activities.
  • the present disclosure relates to antibodies or antigen-binding fragments thereof that specifically bind to CD3 and uses thereof.
  • the present disclosure relates to an antibody or antigen-binding fragment thereof that specifically binds to CD3, the heavy chain of which comprises CDR1, CDR2 and CDR3, wherein:
  • the heavy chain CDR1 contains at least 80% identity with NYYMH (SEQ ID NO:1), DNYIH (SEQ ID NO:2), DYTVN (SEQ ID NO:3) or DYTIH (SEQ ID NO:4) sequence;
  • the heavy chain CDR2 contains a sequence that is at least 80% identical to WTYPGNNNIKYNEKFKG (SEQ ID NO: 18), WIYPGSVNIKYNEKFKD (SEQ ID NO: 6), or YINPFNSYTKYNQKFKD (SEQ ID NO: 22); and
  • the heavy chain CDR3 contains at least 80% identity with DGYGYYFFDY (SEQ ID NO:8), DISRYYFDY (SEQ ID NO:9), SVSTY (SEQ ID NO:10) or SVSIY (SEQ ID NO:11) sequence.
  • the antibody or antigen-binding fragment thereof comprises:
  • Heavy chain CDR1 which includes a sequence selected from: NYYMH (SEQ ID NO: 1), DNYIH (SEQ ID NO: 2), DYTVN (SEQ ID NO: 3), DYTIH (SEQ ID NO: 4) Or a sequence with one amino acid addition, substitution and/or deletion compared to any of SEQ ID NO: 1-4;
  • Heavy chain CDR2 comprising a sequence selected from: WTYPGX 1 A sequence of amino acid additions, substitutions and/or deletions, or YINPFX 4 X 5 YTKYNQKFKD (SEQ ID NO: 7 ); wherein X 1 , X 2 and X 5 is S or D;
  • Heavy chain CDR3 comprising a sequence selected from the group consisting of: DGYGYYFFDY (SEQ ID NO:8), DISRYYFDY (SEQ ID NO:9), SVSTY (SEQ ID NO:10), SVSIY (SEQ ID NO:11) Or a sequence similar to any one of SEQ ID NO:8-11 with a total of 2 amino acid additions, substitutions and/or deletions;
  • a light chain CDR1 comprising a sequence selected from: KSSQSLLNX 6 RTRKNYLA (SEQ ID NO: 12) or KSSQSLLDX 7 DX 8 KTYLN (SEQ ID NO: 13); wherein X 6 is N, Q or S, X 7 is S, A or G, X 8 is G or A;
  • a light chain CDR2 comprising a sequence selected from: WASTRES (SEQ ID NO: 14) or LVSKLNS (SEQ ID NO: 15); and
  • a light chain CDR3 comprising a sequence selected from: KQSX 9 X 10 LRT (SEQ ID NO: 16) or WQGTHFPRT (SEQ ID NO: 17 ); wherein or i.
  • the antibody or antigen-binding fragment thereof comprises:
  • Heavy chain CDR1 which contains or consists of the following sequence: NYYMH (SEQ ID NO: 1); heavy chain CDR2, which contains or consists of the following sequence: WTYPGX 1 X 2 X 3 IKYNEKFKG (SEQ ID NO: 5), wherein each of X 1 , X 2 and or consisting of: KSSQSLLNX 6 RTRKNYLA (SEQ ID NO: 12) , wherein , which includes or consists of the following sequence: KQSYTLRT (SEQ ID NO: 31); or
  • Heavy chain CDR1 which contains or consists of the following sequence: DNYIH (SEQ ID NO: 2); heavy chain CDR2, which contains or consists of the following sequence: WIYPGSVNIKYNEKFKD (SEQ ID NO: 6); heavy chain CDR3 , which contains or consists of the following sequence: DISRYYFDY (SEQ ID NO:9); light chain CDR1, which contains or consists of the following sequence: KSSQSLLNSRTRKNYLA (SEQ ID NO:26); light chain CDR2, which contains the following sequence or Consisting of: WASTRES (SEQ ID NO:14); and light chain CDR3, which contains or consists of the following sequence: KQSFILRT (SEQ ID NO:32); or
  • Heavy chain CDR1 which includes or consists of the following sequence: DYTVN (SEQ ID NO: 3); heavy chain CDR2, which includes or consists of the following sequence: YINPFNSYTKYNQKFKD (SEQ ID NO: 22); heavy chain CDR3 , which contains or consists of the following sequence: SVSTY (SEQ ID NO: 10); light chain CDR1, which contains or consists of the following sequence: KSSQSLLDX 7 DX 8 KTYLN (SEQ ID NO: 13), wherein X 7 is S , 17); or
  • Heavy chain CDR1 which includes or consists of the following sequence: DYTIH (SEQ ID NO: 4); heavy chain CDR2, which includes or consists of the following sequence: YINPFSDYTKYNQKFKD (SEQ ID NO: 23); heavy chain CDR3 , which contains or consists of the following sequence: SVSIY (SEQ ID NO: 11); light chain CDR1, which contains or consists of the following sequence: KSSQSLLDX 7 DX 8 KTYLN (SEQ ID NO: 13), wherein X 7 is G or A , 17).
  • the heavy chain CDR2 comprises a sequence selected from: WTYPGNNNIKYNEKFKG (SEQ ID NO: 18), WTYPGQNNIKYNEKFKG (SEQ ID NO: 19), WTYPGNQNIKYNEKFKG (SEQ ID NO: 20), WTYPGQNQIKYNEKFKG (SEQ ID NO: 20) :21), WIYPGSVNIKYNEKFKD(SEQ ID NO:6), YINPFNSYTKYNQKFKD(SEQ ID NO:22) and YINPFSDYTKYNQKFKD(SEQ ID NO:23).
  • the light chain CDR1 comprises a sequence selected from: KSSQSLLNNRTKNYLA (SEQ ID NO: 24), KSSQSLLNQRTRKNYLA (SEQ ID NO: 25), KSSQSLLNSRTRKNYLA (SEQ ID NO: 26), KSSQSLLDSDGKTYLN (SEQ ID NO :27), KSSQSLLDSDAKTYLN (SEQ ID NO:28), KSSQSLLDGDGKTYLN (SEQ ID NO:29) and KSSQSLLDADGKTYLN (SEQ ID NO:30).
  • the light chain CDR3 comprises a sequence selected from: KQSYTLRT (SEQ ID NO:31), KQSFILRT (SEQ ID NO:32), or WQGTHFPRT (SEQ ID NO:17).
  • the antibody or antigen-binding fragment thereof comprises:
  • Heavy chain CDR1 which contains or consists of the following sequence: NYYMH (SEQ ID NO: 1); heavy chain CDR2, which contains or consists of the following sequence: WTYPGNNNIKYNEKFKG (SEQ ID NO: 18); heavy chain CDR3 , which contains or consists of the following sequence: DGYGYYFFDY (SEQ ID NO:8); light chain CDR1, which contains or consists of the following sequence: KSSQSLLNNRTRKNYLA (SEQ ID NO:24); light chain CDR2, which contains the following sequence or Consisting of: WASTRES (SEQ ID NO:14); and light chain CDR3, which contains or consists of the following sequence: KQSYTLRT (SEQ ID NO:31); or
  • Heavy chain CDR1 which contains or consists of the following sequence: NYYMH (SEQ ID NO:1); heavy chain CDR2, which contains or consists of the following sequence: WTYPGNNNIKYNEKFKG (SEQ ID NO:18); heavy chain CDR3 , which contains or consists of the following sequence: DGYGYYFFDY (SEQ ID NO:8); light chain CDR1, which contains or consists of the following sequence: KSSQSLLNQRTRKNYLA (SEQ ID NO:25); light chain CDR2, which contains the following sequence or Consisting of: WASTRES (SEQ ID NO:14); and light chain CDR3, which contains or consists of the following sequence: KQSYTLRT (SEQ ID NO:31); or
  • Heavy chain CDR1 which contains or consists of the following sequence: NYYMH (SEQ ID NO: 1); heavy chain CDR2, which contains or consists of the following sequence: WTYPGQNNIKYNEKFKG (SEQ ID NO: 19); heavy chain CDR3 , which contains or consists of the following sequence: DGYGYYFFDY (SEQ ID NO:8); light chain CDR1, which contains or consists of the following sequence: KSSQSLLNNRTRKNYLA (SEQ ID NO:24); light chain CDR2, which contains the following sequence or Consisting of: WASTRES (SEQ ID NO:14); and light chain CDR3, which contains or consists of the following sequence: KQSYTLRT (SEQ ID NO:31); or
  • Heavy chain CDR1 which contains or consists of the following sequence: NYYMH (SEQ ID NO: 1); heavy chain CDR2, which contains or consists of the following sequence: WTYPGQNNIKYNEKFKG (SEQ ID NO: 19); heavy chain CDR3 , which contains or consists of the following sequence: DGYGYYFFDY (SEQ ID NO:8); light chain CDR1, which contains or consists of the following sequence: KSSQSLLNQRTRKNYLA (SEQ ID NO:25); light chain CDR2, which contains the following sequence or Consisting of: WASTRES (SEQ ID NO:14); and light chain CDR3, which contains or consists of the following sequence: KQSYTLRT (SEQ ID NO:31); or
  • Heavy chain CDR1 which contains or consists of the following sequence: NYYMH (SEQ ID NO: 1); heavy chain CDR2, which contains or consists of the following sequence: WTYPGNQNIKYNEKFKG (SEQ ID NO: 20); heavy chain CDR3 , which contains or consists of the following sequence: DGYGYYFFDY (SEQ ID NO:8); light chain CDR1, which contains or consists of the following sequence: KSSQSLLNNRTRKNYLA (SEQ ID NO:24); light chain CDR2, which contains the following sequence or Consisting of: WASTRES (SEQ ID NO:14); and light chain CDR3, which contains or consists of the following sequence: KQSYTLRT (SEQ ID NO:31); or
  • Heavy chain CDR1 which contains or consists of the following sequence: NYYMH (SEQ ID NO: 1); heavy chain CDR2, which contains or consists of the following sequence: WTYPGNQNIKYNEKFKG (SEQ ID NO: 20); heavy chain CDR3 , which contains or consists of the following sequence: DGYGYYFFDY (SEQ ID NO:8); light chain CDR1, which contains or consists of the following sequence: KSSQSLLNQRTRKNYLA (SEQ ID NO:25); light chain CDR2, which contains the following sequence or Consisting of: WASTRES (SEQ ID NO:14); and light chain CDR3, which contains or consists of the following sequence: KQSYTLRT (SEQ ID NO:31); or
  • Heavy chain CDR1 which contains or consists of the following sequence: NYYMH (SEQ ID NO: 1); heavy chain CDR2, which contains or consists of the following sequence: WTYPGQNQIKYNEKFKG (SEQ ID NO: 21); heavy chain CDR3 , which contains or consists of the following sequence: DGYGYYFFDY (SEQ ID NO:8); light chain CDR1, which contains or consists of the following sequence: KSSQSLLNNRTRKNYLA (SEQ ID NO:24); light chain CDR2, which contains the following sequence or Consisting of: WASTRES (SEQ ID NO:14); and light chain CDR3, which contains or consists of the following sequence: KQSYTLRT (SEQ ID NO:31); or
  • Heavy chain CDR1 which contains or consists of the following sequence: NYYMH (SEQ ID NO: 1); heavy chain CDR2, which contains or consists of the following sequence: WTYPGQNQIKYNEKFKG (SEQ ID NO: 21); heavy chain CDR3 , which contains or consists of the following sequence: DGYGYYFFDY (SEQ ID NO:8); light chain CDR1, which contains or consists of the following sequence: KSSQSLLNQRTRKNYLA (SEQ ID NO:25); light chain CDR2, which contains the following sequence or Consisting of: WASTRES (SEQ ID NO:14); and light chain CDR3, which contains or consists of the following sequence: KQSYTLRT (SEQ ID NO:31); or
  • Heavy chain CDR1 which contains or consists of the following sequence: DYTVN (SEQ ID NO: 3); heavy chain CDR2, which contains or consists of the following sequence: YINPFNSYTKYNQKFKD (SEQ ID NO: 22); heavy chain CDR3 , which contains or consists of the following sequence: SVSTY (SEQ ID NO: 10); light chain CDR1, which contains or consists of the following sequence: KSSQSLLDSDGKTYLN (SEQ ID NO: 27); light chain CDR2, which contains the following sequence or Consisting of: LVSKLNS (SEQ ID NO:15); and light chain CDR3, which contains or consists of the following sequence: WQGTHFPRT (SEQ ID NO:17); or
  • Heavy chain CDR1 which contains or consists of the following sequence: DYTVN (SEQ ID NO:3); heavy chain CDR2, which contains or consists of the following sequence: YINPFNSYTKYNQKFKD (SEQ ID NO:22); heavy chain CDR3 , which contains or consists of the following sequence: SVSTY (SEQ ID NO: 10); light chain CDR1, which contains or consists of the following sequence: KSSQSLLDSDAKTYLN (SEQ ID NO: 28); light chain CDR2, which contains the following sequence or Consisting of: LVSKLNS (SEQ ID NO:15); and light chain CDR3, which contains or consists of the following sequence: WQGTHFPRT (SEQ ID NO:17); or
  • Heavy chain CDR1 which contains or consists of the following sequence: DYTIH (SEQ ID NO:4); heavy chain CDR2, which contains or consists of the following sequence: YINPFSDYTKYNQKFKD (SEQ ID NO:23); heavy chain CDR3 , which contains or consists of the following sequence: SVSIY (SEQ ID NO: 11); light chain CDR1, which contains or consists of the following sequence: KSSQSLLDADGKTYLN (SEQ ID NO: 30); light chain CDR2, which contains the following sequence or Consisting of: LVSKLNS (SEQ ID NO:15); and light chain CDR3, which contains or consists of the following sequence: WQGTHFPRT (SEQ ID NO:17); or
  • Heavy chain CDR1 which contains or consists of the following sequence: DYTIH (SEQ ID NO:4); heavy chain CDR2, which contains or consists of the following sequence: YINPFSDYTKYNQKFKD (SEQ ID NO:23); heavy chain CDR3 , which contains or consists of the following sequence: SVSIY (SEQ ID NO: 11); light chain CDR1, which contains or consists of the following sequence: KSSQSLLDGDGKTYLN (SEQ ID NO: 29); light chain CDR2, which contains the following sequence or Consisting of: LVSKLNS (SEQ ID NO:15); and light chain CDR3, which contains or consists of the following sequence: WQGTHFPRT (SEQ ID NO:17).
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the following sequence:
  • the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the following sequence:
  • the antibody or antigen-binding fragment thereof comprises the following group of heavy chain variable regions and light chain variable regions:
  • a heavy chain variable region selected from the amino acid sequence of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 or SEQ ID NO:36; and a heavy chain variable region selected from the group consisting of SEQ ID NO:58, SEQ ID NO :59, the light chain variable region of the amino acid sequence of SEQ ID NO:60 or SEQ ID NO:61; or
  • a heavy chain variable region selected from the amino acid sequence of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 or SEQ ID NO:44; and a heavy chain variable region selected from the group consisting of SEQ ID NO:66, SEQ ID NO :67, the light chain variable region of the amino acid sequence of SEQ ID NO:68 or SEQ ID NO:69; or
  • a heavy chain variable region selected from the amino acid sequence of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 or SEQ ID NO: 48; and a heavy chain variable region selected from the group consisting of SEQ ID NO: 70, SEQ ID NO :71, the light chain variable region of the amino acid sequence of SEQ ID NO:72 or SEQ ID NO:73; or
  • a heavy chain variable region selected from the amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 or SEQ ID NO: 53; and selected from SEQ ID NO :74, the light chain variable region of the amino acid sequence of SEQ ID NO:75 or SEQ ID NO:76; or
  • a heavy chain variable region selected from the amino acid sequence of SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 or SEQ ID NO:57; and a heavy chain variable region selected from the group consisting of SEQ ID NO:77, SEQ ID NO :78, the light chain variable region of the amino acid sequence of SEQ ID NO:79 or SEQ ID NO:80.
  • the antibody or antigen-binding fragment thereof comprises the set of a heavy chain variable region and a light chain variable region of any one of the following (1) to (18):
  • the antibody or antigen-binding fragment thereof is a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a single chain antibody, a diabody, a triabody, a quadribody, Chain antibodies, Fab fragments, F(ab') 2 fragments, scFv fragments, Fv fragments, Fab' fragments, domain antibodies.
  • the antibody or antigen-binding fragment thereof is an IgA antibody, IgD antibody, IgE antibody, IgM antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody, or IgG4 antibody.
  • the antibody or antigen-binding fragment thereof binds human CD3 and/or cynomolgus CD3.
  • the antibody or antigen-binding fragment thereof binds to CD3e/d and/or CD3e/g.
  • the antibody or antigen-binding fragment thereof (i) significantly activates human total T cells, upregulates the expression of CD25 and/or induces the release of IFN- ⁇ ; and/or (ii) activates human total T cells at a concentration of less than 4 ⁇ 10 - K D value of 8 M binds to human CD3.
  • the present disclosure relates to an antibody or antigen-binding fragment thereof that competes with or cross-blocks binding to CD3, particularly CD3e/d or CD3e/g, with an antibody or antigen-binding fragment as described in the first aspect.
  • the present disclosure relates to an isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof as described in the first or second aspect.
  • the present disclosure relates to a nucleic acid vector comprising an isolated nucleic acid as described in the third aspect.
  • the present disclosure relates to a host cell comprising an isolated nucleic acid as described in the third aspect or a nucleic acid vector as described in the fourth aspect.
  • the present disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in the first or second aspect, an isolated nucleic acid as described in the third aspect, or an isolated nucleic acid as described in the fourth aspect.
  • the nucleic acid carrier, and a pharmaceutically acceptable carrier are provided.
  • the present disclosure relates to a method of producing an antibody or an antigen-binding fragment thereof as described in the first or second aspect, comprising subjecting a host cell as described in the fifth aspect to allowing it to express said antibody or its antigen-binding fragments.
  • the present disclosure relates to an antibody or an antigen-binding fragment thereof as described in any one of the first or second aspects, an isolated nucleic acid as described in the third aspect, a nucleic acid vector as described in the fourth aspect Or the use of the pharmaceutical composition as described in the sixth aspect in the preparation of drugs for suppressing immune responses or activating T cells.
  • the present disclosure relates to an antibody or an antigen-binding fragment thereof as described in any one of the first or second aspects, an isolated nucleic acid as described in the third aspect, a nucleic acid vector as described in the fourth aspect Or the use of the pharmaceutical composition as described in the sixth aspect in the preparation of medicaments for treating cancer or autoimmune diseases.
  • the present disclosure relates to an antibody or an antigen-binding fragment thereof as described in any one of the first or second aspects, an isolated nucleic acid as described in the third aspect, a nucleic acid vector as described in the fourth aspect Or the pharmaceutical composition as described in the sixth aspect, which is used to suppress immune responses or activate T cells, or to treat cancer or autoimmune diseases.
  • the present disclosure relates to a method of suppressing an immune response or activating T cells, or treating cancer or an autoimmune disease, comprising administering to an individual in need thereof an inhibitory effective amount, an activating effective amount, or a therapeutically effective amount as described above.
  • the antibody or antigen-binding fragment thereof as described in any one of the first aspect or the second aspect, the isolated nucleic acid as described in the third aspect, the nucleic acid vector as described in the fourth aspect, or the pharmaceutical combination as described in the sixth aspect things.
  • the aforementioned cancer is selected from: melanoma (e.g., metastatic malignant melanoma), renal cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, nasopharyngeal cancer, oral cancer, liver cancer, Bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin lymphoma tumour, non-Hodgkin lymphoma, esophageal cancer, small bowel cancer, colorectal cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethra cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leuk
  • the aforementioned autoimmune disease is selected from: rheumatoid arthritis, multiple sclerosis, Sjogren's syndrome, insulin-dependent diabetes, autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma , polymyositis, dermatomyositis, psoriasis, Wegener's granulomatosis, Crohn's disease, ulcerative colitis, lupus such as systemic lupus erythematosus, atherosclerosis, chronic obstructive pulmonary disease, cirrhosis , renal transplant fibrosis, renal transplant nephropathy, pulmonary fibrosis, and combinations thereof.
  • Figure 1 shows a graph showing the results of serum titers against immunogens in mouse serum detected by ELISA.
  • Figure 2 shows the results of binding of CD3 antibodies to human CD3e/g detected by ELISA.
  • Figure 3 shows the results of CD3 antibody binding to cynomolgus monkey CD3e/g detected by ELISA.
  • Figure 4 shows the results of CD3 antibody binding to human CD3e/d detected by ELISA.
  • Figures 5(a)-(e) show the results of binding of CD3 antibodies to Jurkat cells detected by FACS.
  • Figures 6(a)-(h) show the effect of CD3 antibodies on CD25 expression detected by FACS.
  • FIGS 7(a)-(f) show the effect of CD3 antibodies on IFN- ⁇ secretion detected by FACS. Detailed description of the invention
  • a range is used as a shorthand form to describe each value and all values within the range. Any value within the range, especially an integer value, can be selected as the end point of the range.
  • the range "at least 80% identity” is used to describe all values within the range, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%, and includes all subranges, such as at least 85%, at least 90%, at least 95% etc.
  • Antibody refers to monomers as well as multimers. Whole antibodies (including multimers) or antibody fragments carrying an antigen-binding fragment of the antibody may be used.
  • the antibody may be a human antibody, humanized antibody, chimeric antibody, monoclonal antibody, polyclonal antibody or recombinant antibody.
  • Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • a “Fab fragment” is a monovalent fragment having VL, VH , CL and CH domains; a “F( ab ') 2 fragment” is a bivalent fragment having two Fab fragments connected by a disulfide bond in the hinge region fragment.
  • Single chain antibodies are antibodies in which the V L and V H regions are linked by a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain, where the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site (see, eg, Bird et al., Science 242:423-26 (1988) and Huston et al., 1988, Proc Natl Acad Sci USA 85:5879-83 (1988)).
  • linker e.g., a synthetic sequence of amino acid residues
  • tribodies and tetrabodies (or “tetrabodies”) contain three and four polypeptide chains, which may be the same or different, respectively, and form three and four polypeptide chains, respectively.
  • Antibodies with the same or different antigen binding sites are also included in the same or different antigen binding sites.
  • CDRs complementarity determining regions
  • FRs framework regions
  • One or more CDRs can be covalently or non-covalently incorporated into a molecule to turn the molecule into an antigen-binding protein such as an antibody.
  • the antigen-binding protein may incorporate the CDRs as part of a larger polypeptide chain, may have the CDRs covalently linked to another polypeptide chain, or may incorporate the CDRs non-covalently.
  • CDRs allow an antigen-binding protein to specifically bind to a specific antigen of interest.
  • the CDRs of an antibody can be defined by a variety of methods in the art. Methods and techniques for identifying CDRs within the variable region within the antibody molecule and within the amino acid sequence of the variable region of the antibody are also well known in the art, and can be used to identify the CDRs within the variable region of the antibody molecule. CDRs within the amino acid sequences of the variable regions of the specific antibodies disclosed. In this application, the amino acid sequences of the listed CDRs are all shown in accordance with Kabat definition rules (the claims in this application are also sequences shown in accordance with Kabat definition rules).
  • CDR complementarity determining region
  • Antibody or antigen-binding fragment thereof may also include an antibody heavy chain constant region and/or an antibody light chain constant region.
  • the antibody heavy chain constant region is a mouse antibody heavy chain constant region or a human antibody heavy chain constant region;
  • the antibody light chain constant region is a mouse light chain antibody constant region or a human antibody light chain constant region.
  • chain constant region More preferably, the antibody heavy chain constant region is a human antibody heavy chain constant region, such as the heavy chain constant region of a human IgG1, IgG2a, IgG2b, IgG2c, IgG3 or IgG4 antibody;
  • the antibody light chain constant region is a human Source antibody light chain kappa or lambda chain constant region.
  • humanized antibody includes antibodies having variable and constant regions of human germline immunoglobulin sequences in which CDR sequences derived from another germline have been grafted onto human backbone sequences.
  • nucleic acid includes single-stranded and double-stranded nucleotide polymers.
  • the nucleotides that make up a polynucleotide may be ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide.
  • the modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide bond modifications such as phosphorothioate, phosphorodithioate, Selenophosphates, diselenophosphates, thioaniline phosphates and phosphoramide esters.
  • vector means any molecule or entity (eg, nucleic acid, plasmid, phage, or virus) used to transfer protein-encoding information into a host cell.
  • the term "host cell” means a cell that has been transformed or is capable of being transformed with a nucleic acid sequence and thereby expresses a gene of interest.
  • the term includes the progeny of the parent cell, regardless of whether the progeny is identical in morphology or genetic makeup to the original parent cell, as long as the gene of interest is present.
  • the host cell can be a prokaryote (eg, Escherichia coli), or it can be a eukaryote (eg, a single-celled eukaryote (eg, yeast or other fungi), a plant cell (eg, tobacco or tomato plant cells), an animal cells (eg, human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells)) or hybridomas.
  • a prokaryote eg, Escherichia coli
  • a eukaryote eg, a single-celled eukaryote (eg, yeast or other fungi)
  • a plant cell eg, tobacco or tomato plant cells
  • an animal cells eg, human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells
  • identity refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. Generally speaking, identity refers to the number or percentage of identical positions shared by two amino acid or nucleic acid sequences, taking into account the number of gaps and the length of each gap that need to be introduced to achieve optimal alignment of the two sequences. . When an amino acid sequence is described as being at least 85%, or at least 90%, or at least 95% identical to another amino acid sequence, the amino acid sequence may differ by conservative substitutions (including where all substitutions are conservative substitutions).
  • a "conservative substitution” is considered in the art to be the substitution of one amino acid for another amino acid that has similar properties.
  • Exemplary conservative substitutions are well known in the art (see, e.g., WO 97/09433, page 10, published March 13, 1997; Lehninger, Biochemistry, 2nd ed.; Worth Publishers, Inc. NY: NY (1975), pp. 71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), p. 8).
  • Naturally occurring amino acids can be divided into the following groups based on common side chain properties:
  • Conservative substitutions may involve exchanging a member of one of these class groups with another member of the same class group.
  • Conservative amino acid substitutions may include unnatural amino acid residues that are typically incorporated by chemical peptide synthesis without resorting to synthesis in biological systems. These include peptide mimetics and other reversed or inverted forms of amino acid moieties.
  • Non-conservative substitutions may include the exchange of a member of one of the above class groups with a member from another class group. Such substituted residues can be introduced into regions of the antibody that are homologous to the human antibody or into non-homologous regions of the molecule.
  • an “effective amount” is generally one sufficient to reduce the severity and/or frequency of symptoms, eliminate the symptoms and/or underlying causes, prevent the occurrence of symptoms and/or their underlying causes, and/or ameliorate or treat the disease (e.g., cancer or autoimmune disease). ) caused by or related to the amount of damage.
  • an effective amount is a therapeutically effective amount or a prophylactically effective amount.
  • a “therapeutically effective amount” is one sufficient to correct in any manner a disease state (e.g., cancer or autoimmune disease) or symptoms, a specific state or symptom associated with said disease state, or to prevent, hinder, retard, or reverse a disease state or to be associated with a disease The amount of progression of any other adverse symptoms.
  • the therapeutically effective amount will depend, for example, on the context and goals of the treatment.
  • dosage levels for treatment will be based, in part, on the molecule being delivered, the indication for which the antibody or antigen-binding protein thereof is being used, the route of administration, and the size (body weight, body surface, or organ size) of the patient. and/or changes in condition (age and general health).
  • the clinician can adjust the dosage and adjust the route of administration to achieve optimal therapeutic effects.
  • subject refers to warm-blooded animals, such as mammals.
  • the term includes, but is not limited to, domestic animals, rodents (eg, rats and mice), primates, and humans. Preferably the term refers to humans.
  • EC50 refers to the half-maximum effect concentration, ie, the concentration that causes 50% of the maximum effect.
  • CD3 antibody refers to an antibody that is capable of specifically binding to human or other mammalian CD3.
  • the antibody can react with less than 1 ⁇ 10 -4 , less than 1 ⁇ 10 -5 , less than 1 ⁇ 10 -6 , less than 1 ⁇ 10 -7 , less than 1 ⁇ 10 -8 , less than 1 ⁇ 10 -9 , less than 1 ⁇ 10 -10 or less than 1 ⁇ 10 -11 M K D value specifically binds to CD3 antigen.
  • CD3e/g antibody refers to an antibody capable of specifically binding to the human or other mammalian CD3e/g subunit.
  • the CD3e/g antigen has the sequence set forth in SEQ ID NO:83 and/or SEQ ID NO:84.
  • the antibody can react with less than 1 ⁇ 10 -4 , less than 1 ⁇ 10 -5 , less than 1 ⁇ 10 -6 , less than 1 ⁇ 10 -7 , less than 1 ⁇ 10 -8 , less than 1 ⁇ 10 -9 , less than 1 ⁇ 10 -10 or less than 1 ⁇ 10 -11 M K D value specifically binds to CD3e/g antigen.
  • CD3e/d antibody refers to an antibody capable of specifically binding to the human or other mammalian CD3e/d subunit.
  • the antibody can react with less than 1 ⁇ 10 -4 , less than 1 ⁇ 10 -5 , less than 1 ⁇ 10 -6 , less than 1 ⁇ 10 -7 , less than 1 ⁇ 10 -8 , less than 1 ⁇ 10 -9 , less than 1 ⁇ 10 -10 or less than 1 ⁇ 10 -11 M K D value specifically binds to CD3e/d antigen.
  • the antibody can react with less than 1 ⁇ 10 -6 , less than 1 ⁇ 10 -7 , less than 1 ⁇ 10 -8 , less than 1 ⁇ 10 -9 , less than 1 ⁇ 10 -10 or less than 1 A K D value of ⁇ 10 -11 M specifically binds to CD3e/g antigen and/or CD3e/d antigen.
  • K D is the equilibrium dissociation constant, a commonly used parameter to characterize antibody affinity.
  • the larger the K D the more dissociation, which represents the relationship between the antibody and the antigen.
  • the weaker the affinity between the antibodies and the smaller the KD the less dissociation, which means the stronger the affinity between the antibody and the antigen.
  • the antibodies described above bind to human CD3 with a KD value of less than 4 ⁇ 10 ⁇ 8 M.
  • the expression of CD25 may mean at least about 50%, at least about 60%, at least about 70 %, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the cells are activated; or the expression of CD25 is upregulated by at least about 50%, at least about 60%, at least about 70%, at least about 80 %, at least about 85%, at least about 90%, or at least about 95%; or the amount of induced IFN- ⁇ release is at least about 5000pg/mL, about 6000pg/mL, about 7000pg/mL, about 8000pg/mL, about 9000pg/ mL, about 10000pg/mL, about 11000pg/mL, about 12000pg/mL, about 13000pg/mL, about 14000pg/mL, about 15000pg/mL
  • "significantly” may refer to activating total T cells, upregulating the expression of CD25, and/or inducing the release of IFN- ⁇ to be at least about 1.5 times, at least about 2 times that of the negative control, that is, the isotype control or the blank control. , at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times or more. In some preferred embodiments, "significantly” may refer to activating total T cells, upregulating the expression of CD25, and/or inducing the release of IFN- ⁇ to be at least about 3 times that of the negative control, that is, the isotype control or the blank control.
  • isotype control refers to an antibody that maintains similar properties to the test antibody but lacks a specific target. In flow cytometry experiments, isotype controls serve as negative controls, which can distinguish specific antibody signals from non-specific background staining, and eliminate interfering factors to ensure the accuracy of experimental results.
  • the present disclosure relates to antibodies or antigen-binding fragments thereof that specifically bind to CD3 (eg, CD3e, especially CD3e/d and/or CD3e/g).
  • CD3 eg, CD3e, especially CD3e/d and/or CD3e/g.
  • the CD3 is human CD3 and/or cynomolgus CD3.
  • the antibodies of the present disclosure, or antigen-binding fragments thereof generally comprise one or more CDRs (eg, 1, 2, 3, 4, 5, or 6 CDRs) as described herein.
  • the heavy chain CDR1 comprises at least 80% identity to any one of SEQ ID NOs: 1-4 (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99 % or 100% identity) or a sequence similar thereto by a total of 3 (eg 1, 2 and 3) amino acid additions, substitutions and/or deletions.
  • the heavy chain CDR2 comprises at least 80% identity to any one of SEQ ID NOs: 5-7 and 18-23 (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity) or a sequence similar thereto by a total of 3 (eg 1, 2 and 3) amino acid additions, substitutions and/or deletions.
  • the heavy chain CDR3 comprises at least 80% identity to any one of SEQ ID NOs: 8-11 (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99 % or 100% identity) or a sequence similar thereto by a total of 3 (eg 1, 2 and 3) amino acid additions, substitutions and/or deletions.
  • the light chain CDR1 comprises at least 80% identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity) or a sequence similar thereto by a total of 3 (eg 1, 2 and 3) amino acid additions, substitutions and/or deletions.
  • the light chain CDR2s comprise at least 80% identical to any one of SEQ ID NOs: 14-15 (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99 % or 100% identity) or a sequence similar thereto by a total of 3 (eg 1, 2 and 3) amino acid additions, substitutions and/or deletions.
  • the light chain CDR3 comprises at least 80% identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity) or a sequence similar thereto by a total of 3 (eg 1, 2 and 3) amino acid additions, substitutions and/or deletions.
  • the substitutions are conservative substitutions.
  • the antibodies of the present disclosure, or antigen-binding fragments thereof comprise heavy chain variable regions and/or light chain variable regions.
  • the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 33-57 or has at least 85% identity (e.g., at least 90%, 95%, 96) to any of the foregoing sequences. %, 97%, 98%, 99% or 100% identity).
  • the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 58-82 or is at least 85% identical (e.g., at least 90%, 95%, 96) to any of the foregoing sequences. %, 97%, 98%, 99% or 100% identity).
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO: 33 and a light chain variable region as set forth in SEQ ID NO: 58; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 33 and a light chain variable region as shown in SEQ ID NO: 59; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 33 and a light chain variable region as shown in SEQ ID NO: 60; in some preferred embodiments, the The antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 33 and a light chain variable region as shown in SEQ ID NO: 61.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO: 34 and a light chain variable region as set forth in SEQ ID NO: 58; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 34 and a light chain variable region as shown in SEQ ID NO: 59; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:34 and a light chain variable region as shown in SEQ ID NO:60; in some preferred embodiments, the The antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 34 and a light chain variable region as shown in SEQ ID NO: 61.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO: 35 and a light chain variable region as set forth in SEQ ID NO: 58; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 35 and a light chain variable region as shown in SEQ ID NO: 59; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 35 and a light chain variable region as shown in SEQ ID NO: 60; in some preferred embodiments, the The antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 35 and a light chain variable region as shown in SEQ ID NO: 61.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO: 36 and a light chain variable region as set forth in SEQ ID NO: 58; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:36 and a light chain variable region as shown in SEQ ID NO:59; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 36 and a light chain variable region as shown in SEQ ID NO: 60; in some preferred embodiments, the The antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 36 and a light chain variable region as shown in SEQ ID NO: 61.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 37 and a light chain variable region as shown in SEQ ID NO: 62; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 37 and a light chain variable region as shown in SEQ ID NO: 63; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 37 and a light chain variable region as shown in SEQ ID NO: 64; in some preferred embodiments, the The antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 37 and a light chain variable region as shown in SEQ ID NO: 65.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO: 38 and a light chain variable region as set forth in SEQ ID NO: 62; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 38 and a light chain variable region as shown in SEQ ID NO: 63; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:38 and a light chain variable region as shown in SEQ ID NO:64; in some preferred embodiments, the The antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 38 and a light chain variable region as shown in SEQ ID NO: 65.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:39 and a light chain variable region as shown in SEQ ID NO:62; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 39 and a light chain variable region as shown in SEQ ID NO: 63; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:39 and a light chain variable region as shown in SEQ ID NO:64; in some preferred embodiments, the The antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 39 and a light chain variable region as shown in SEQ ID NO: 65.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:40 and a light chain variable region as shown in SEQ ID NO:62; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:40 and a light chain variable region as shown in SEQ ID NO:63; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:40 and a light chain variable region as shown in SEQ ID NO:64; in some preferred embodiments, the The antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:40 and a light chain variable region as shown in SEQ ID NO:65.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:41 and a light chain variable region as set forth in SEQ ID NO:66; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:41 and a light chain variable region as shown in SEQ ID NO:67; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:41 and a light chain variable region as shown in SEQ ID NO:68; in some preferred embodiments, the The antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:41 and a light chain variable region as shown in SEQ ID NO:69.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:42 and a light chain variable region as set forth in SEQ ID NO:66; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:42 and a light chain variable region as shown in SEQ ID NO:67; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:42 and a light chain variable region as shown in SEQ ID NO:68; in some preferred embodiments, the The antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 42 and a light chain variable region as shown in SEQ ID NO: 69.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO: 43 and a light chain variable region as set forth in SEQ ID NO: 66; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 43 and a light chain variable region as shown in SEQ ID NO: 67; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:43 and a light chain variable region as shown in SEQ ID NO:68; in some preferred embodiments, the The antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 43 and a light chain variable region as shown in SEQ ID NO: 69.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:44 and a light chain variable region as set forth in SEQ ID NO:66; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:44 and a light chain variable region as shown in SEQ ID NO:67; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:44 and a light chain variable region as shown in SEQ ID NO:68; in some preferred embodiments, the The antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 44 and a light chain variable region as shown in SEQ ID NO: 69.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:45 and a light chain variable region as shown in SEQ ID NO:70; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:45 and a light chain variable region as shown in SEQ ID NO:71; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:45 and a light chain variable region as shown in SEQ ID NO:72; in some preferred embodiments, the The antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 45 and a light chain variable region as shown in SEQ ID NO: 73.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:46 and a light chain variable region as shown in SEQ ID NO:70; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:46 and a light chain variable region as shown in SEQ ID NO:71; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:46 and a light chain variable region as shown in SEQ ID NO:72; in some preferred embodiments, the The antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 46 and a light chain variable region as shown in SEQ ID NO: 73.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:47 and a light chain variable region as shown in SEQ ID NO:70; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:47 and a light chain variable region as shown in SEQ ID NO:71; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:47 and a light chain variable region as shown in SEQ ID NO:72; in some preferred embodiments, the The antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:47 and a light chain variable region as shown in SEQ ID NO:73.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:48 and a light chain variable region as shown in SEQ ID NO:70; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:48 and a light chain variable region as shown in SEQ ID NO:71; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:48 and a light chain variable region as shown in SEQ ID NO:72; in some preferred embodiments, the The antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 48 and a light chain variable region as shown in SEQ ID NO: 73.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:49 and a light chain variable region as shown in SEQ ID NO:74; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:49 and a light chain variable region as shown in SEQ ID NO:75; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:49 and a light chain variable region as shown in SEQ ID NO:76.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 50 and a light chain variable region as shown in SEQ ID NO: 74; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 50 and a light chain variable region as shown in SEQ ID NO: 75; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:50 and a light chain variable region as shown in SEQ ID NO:76.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 51 and a light chain variable region as shown in SEQ ID NO: 74; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 51 and a light chain variable region as shown in SEQ ID NO: 75; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:51 and a light chain variable region as shown in SEQ ID NO:76.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO: 52 and a light chain variable region as set forth in SEQ ID NO: 74; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 52 and a light chain variable region as shown in SEQ ID NO: 75; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:52 and a light chain variable region as shown in SEQ ID NO:76.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO: 53 and a light chain variable region as set forth in SEQ ID NO: 74; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 53 and a light chain variable region as shown in SEQ ID NO: 75; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:53 and a light chain variable region as shown in SEQ ID NO:76.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO: 54 and a light chain variable region as set forth in SEQ ID NO: 77; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 54 and a light chain variable region as shown in SEQ ID NO: 78; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 54 and a light chain variable region as shown in SEQ ID NO: 79; in some preferred embodiments, the The antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:54 and a light chain variable region as shown in SEQ ID NO:80.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO: 55 and a light chain variable region as set forth in SEQ ID NO: 77; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 55 and a light chain variable region as shown in SEQ ID NO: 78; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:55 and a light chain variable region as shown in SEQ ID NO:79; in some preferred embodiments, the The antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:55 and a light chain variable region as shown in SEQ ID NO:80.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO: 56 and a light chain variable region as set forth in SEQ ID NO: 77; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 56 and a light chain variable region as shown in SEQ ID NO: 78; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:56 and a light chain variable region as shown in SEQ ID NO:79; in some preferred embodiments, the The antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:56 and a light chain variable region as shown in SEQ ID NO:80.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO: 57 and a light chain variable region as set forth in SEQ ID NO: 77; in some preferred In embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 57 and a light chain variable region as shown in SEQ ID NO: 78; in some preferred embodiments In, the antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO:57 and a light chain variable region as shown in SEQ ID NO:79; in some preferred embodiments, the The antibody or antigen-binding fragment thereof has a heavy chain variable region as shown in SEQ ID NO: 57 and a light chain variable region as shown in SEQ ID NO: 80.
  • the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:85 and a light chain variable region as set forth in SEQ ID NO:82. In some preferred embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region as set forth in SEQ ID NO:89 and a light chain variable region as set forth in SEQ ID NO:81.
  • the antibodies of the present disclosure, or antigen-binding fragments thereof (i) significantly activate human total T cells, upregulate the expression of CD25 and/or induce the release of IFN- ⁇ ; and/or (ii) react at less than 4 ⁇ 10 -8 (e.g. less than 1 ⁇ 10 -8 , less than 1 ⁇ 10 -9 , less than 1 ⁇ 10 -10 or less than 1 ⁇ 10 -11 ) M K D values are similar to those of human CD3 (e.g. CD3e, especially CD3e/d and /or CD3e/g) binding.
  • antibodies that compete with or cross-block the binding of the antibodies disclosed herein to CD3, or are themselves cross-blocked from binding to CD3 by the antibodies disclosed herein may also be used in the present invention.
  • these competing antibodies, cross-blocking antibodies, or cross-blocked antibodies have epitopes that intersect and/or overlap with epitopes of the antibodies disclosed herein, or antigen-binding fragments thereof.
  • these competing antibodies, cross-blocking antibodies, or cross-blocked antibodies have the same epitope as the antibodies disclosed herein, or antigen-binding fragments thereof.
  • Competing antibodies, cross-blocking antibodies, and cross-blocked antibodies can be identified using any suitable method known in the art, including competition ELISA or Assays in which binding of a competing or cross-blocking antibody to human CD3 prevents binding of an antibody disclosed herein, or vice versa.
  • the competing antibody or cross-blocked antibody is competed or cross-blocked to greater than 80%, greater than 85%, greater than 90%, or greater than 95%. In some embodiments, the competing antibody, cross-blocking antibody, or cross-blocked antibody is chimeric, fully human, or humanized.
  • the antibodies or antigen-binding fragments thereof disclosed herein can also be used to construct a chimeric antigen receptor or a genetically modified cell, the chimeric antigen receptor comprising the antibodies or antigen-binding fragments thereof of the present disclosure as described above.
  • the genetically modified cells are preferably eukaryotic cells, more preferably isolated human cells, and even more preferably immune cells such as T cells, or NK cells such as NK92 cell lines.
  • chimeric antigen receptor refers to a structure that contains an extracellular domain (extracellular binding domain) capable of binding antigen, a hinge domain, a transmembrane domain (transmembrane region), and a structure that transmits cytoplasmic signals to Domain polypeptide (i.e., intracellular signaling domain).
  • the hinge domain can be thought of as the portion that provides flexibility to the extracellular antigen-binding region.
  • Intracellular signaling domains refer to proteins that transmit information into cells to regulate cell activity by producing second messengers through a defined signaling pathway, or proteins that function as effectors by corresponding to such messengers, producing proteins that can promote CAR Signaling of immune effector functions of cells (e.g., CART cells).
  • the intracellular signaling domain includes a signaling domain and may also include a costimulatory intracellular domain derived from a costimulatory molecule.
  • immunode refers to a cell that can elicit an immune response
  • immunodet cells include, for example, white blood cells (leukocytes) derived from hematopoietic stem cells (HSCs) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells, and bone marrow-derived cells (neutrophils) , eosinophils, basophils, monocytes, macrophages, dendritic cells).
  • HSCs hematopoietic stem cells
  • T cells lymphocytes
  • B cells natural killer cells
  • neutrils bone marrow-derived cells
  • eosinophils basophils
  • monocytes monocytes
  • macrophages dendritic cells
  • dendritic cells dendritic cells
  • an immune cell may be derived from blood, Such as autologous T cells, allogeneic T cells, autologous NK cells, allogeneic NK cells, or they can also be derived from cell lines, such as using EBV virus infection to prepare NK cell lines, NK cells induced and differentiated from embryonic stem cells and iPSCs, and NK92 cells Department etc.
  • the antibodies or antigen-binding fragments thereof disclosed herein can also be used to construct an antibody-drug conjugate, which contains a cytotoxic agent, and the antibodies or antigen-binding fragments thereof of the present disclosure as described above.
  • the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent, and antibody-drug conjugates comprising the antibodies or antigen-binding fragments thereof can covalently or non-covalently bind the therapeutic agent.
  • the therapeutic agent is a cytotoxic agent or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenox Poside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, maytansinoid alkaloids (such as DM-1 and DM-4), diketone, serine, Mitomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin and cyclophosphamide and similar things).
  • cytotoxic agent or cytostatic agent e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenox Poside
  • compositions comprising an antibody of the present disclosure, or an antigen-binding fragment thereof.
  • These pharmaceutical compositions comprise a therapeutically effective amount of an antibody or antigen-binding fragment thereof and one or more additional components, such as a physiologically acceptable carrier, excipient or diluent.
  • additional components may include buffers, carbohydrates, polyols, amino acids, chelating agents, stabilizers, and/or preservatives, and the like.
  • compositions may include as active ingredients a therapeutically effective amount of an antibody or antigen-binding fragment thereof and one or more other therapeutic agents.
  • the above-mentioned antibodies or antigen-binding fragments thereof, the above-mentioned antibody-drug conjugates, etc., and/or additional therapeutic or diagnostic agents may each be administered as a single agent within any time frame suitable for performing the intended treatment or diagnosis. Accordingly, these single agents may be administered substantially simultaneously (ie, as a single formulation or within minutes or hours) or in sequence and consecutively.
  • the additional therapeutic agent may comprise one or more inhibitors selected from: B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c -Met inhibitor, anaplastic lymphoma kinase (ALK) inhibitor, phosphatidylinositol 3-kinase (PI3K) inhibitor, Akt inhibitor, mTOR inhibitor, PI3K/mTOR dual inhibitor, Bruton tyrosine kinase (BTK ) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and/or isocitrate dehydrogenase 2 (IDH2) inhibitors.
  • the other therapeutic agent is an indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor (eg, epacastat).
  • additional therapeutic agents may include one or more inhibitors selected from the group consisting of: HER3 inhibitors, LSD 1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, inhibitors of activated hedgehog signaling pathway , and drugs that selectively degrade estrogen receptors.
  • the additional therapeutic agent may comprise one or more therapeutic agents selected from: Trabectedin, Nabupaclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib , everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alibita, ceritinib, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient , pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitor, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, phthalamine piperidine, datamine Carbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomib, amrubicin, carfilzomib, prala
  • the additional therapeutic agent may comprise one or more therapeutic agents selected from: adjuvants, TLR agonists, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonist, IL-13 antagonist, IL-17 antagonist, HVEM antagonist, ICOS agonist, therapeutic agents targeting CX3CL1, therapeutic agents targeting CXCL9, therapeutic agents targeting CXCL10, therapeutic agents targeting CCL5 therapeutic agents, LFA-1 agonists, ICAM1 agonists and selectin agonists.
  • TNF tumor necrosis factor
  • compositions can be administered according to known methods.
  • the route of administration is, for example, oral, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intrahilar or intralesional route injection; by sustained release system or by implanted device Apply.
  • the compositions may be administered by bolus injection or continuously by infusion or by an implanted device.
  • the antibodies of the present disclosure can be prepared by methods known in the art for antibody preparation (eg, by chemical synthesis or by recombinant expression techniques).
  • Therapeutic antibodies such as monoclonal antibodies can be developed by a variety of technologies and approaches, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc.
  • the preparation of monoclonal antibodies through hybridoma technology is still the mainstream method of preparing monoclonal antibodies for treatment.
  • variable region genes can be grafted into human antibody constant region genes to form human Mouse chimeric antibodies (U.S. Patent No. 4,816,567 to Cabilly et al.) to greatly reduce immunogenicity when used in humans. Furthermore, the CDR domain of the mouse antibody variable region can be grafted onto the human antibody structure, thereby reducing the mouse antibody component to less than 5%, greatly increasing the safety of the antibody for use in the human body.
  • the antibodies obtained through this approach are called humanized antibodies and are currently the main products in the antibody drug market (U.S. Patent No. 5,225,539 by Winter and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762 and 6,180,370 by Queen et al.).
  • the preparation method of the nucleic acid is a conventional preparation method in the art, and may include, for example, the following steps: obtaining the nucleic acid molecule encoding the above antibody through gene cloning technology, or obtaining the nucleic acid molecule encoding the above antibody through artificial full sequence synthesis.
  • the vector for producing the antibody can be generated by recombinant DNA technology.
  • the vector can be obtained by conventional methods in the art, for example, it is constructed by connecting the nucleic acid molecule described in this application to a suitable expression vector.
  • the expression vector is a variety of conventional vectors in the art, as long as it can accommodate the aforementioned nucleic acid molecules.
  • the vector includes a eukaryotic cell expression vector and/or a prokaryotic cell expression vector.
  • the expression vector is transferred to the host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the antibodies of the present disclosure.
  • the host cells are various conventional host cells in the art, as long as the recombinant expression vector can be stably replicated and the nucleic acid carried can be effectively expressed.
  • the host cell can be a prokaryotic cell (e.g., E. coli cell) and/or a eukaryotic cell (e.g., HEK293 cell or CHO cell).
  • the antibodies or antigen-binding fragments thereof, isolated nucleic acids, nucleic acid vectors, host cells, antibody drug conjugates, and pharmaceutical compositions of the present disclosure can be used to treat a variety of conditions, including, for example, various forms of cancer, infections, autoimmunity or inflammatory conditions and/or fibrotic conditions.
  • the antibodies or antigen-binding fragments thereof, isolated nucleic acids, nucleic acid vectors, host cells, antibody drug conjugates and pharmaceutical compositions of the present disclosure can also be used to inhibit immune responses or activate T cells.
  • the antibodies or antigen-binding fragments thereof, isolated nucleic acids, nucleic acid vectors, host cells, antibody drug conjugates, and pharmaceutical compositions of the present disclosure are provided for use in the preparation of inhibitors of immune responses or activation of T cells. Use in medicines or kits.
  • the antibodies or antigen-binding fragments thereof, isolated nucleic acids, nucleic acid vectors, host cells, antibody drug conjugates, and pharmaceutical compositions of the present disclosure are provided for use in the preparation of medicaments for preventing, treating, or ameliorating diseases. or use in the kit.
  • the disease is cancer or an autoimmune disease.
  • the cancer includes, but is not limited to: melanoma, kidney cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, nasopharyngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer , head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin lymphoma, non-Hodgkin lymphoma , Esophageal cancer, small intestine cancer, colorectal cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethra cancer, penile cancer, chronic or acute leukemia, bladder cancer, renal pelvis cancer, central nervous system tumors, Glioma, spinal cord axial tumor, brainstem glioma, pituitary adenoma, Kaposi's
  • the autoimmune diseases include, but are not limited to: rheumatoid arthritis, multiple sclerosis, Sjogren's syndrome, insulin-dependent diabetes, autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma diseases, polymyositis, dermatomyositis, psoriasis, Wegener's granulomatosis, Crohn's disease, ulcerative colitis, lupus such as systemic lupus erythematosus, atherosclerosis, chronic obstructive pulmonary disease, liver Sclerosis, renal allograft fibrosis, renal allograft nephropathy, or pulmonary fibrosis.
  • CD3e/g is a soluble ligand.
  • CD3e/g recombinant protein the product of CD3e/g protein and human constant region, as an immunogen.
  • the specific sequence of CD3e/g protein is shown in Table 1.
  • the above immunogens were used to immunize 6-8 week old SJL mice (purchased from Shanghai Slack Company), and the mice were raised under SPF conditions.
  • the immunogen was emulsified with Freund's complete adjuvant and then injected into the tail vein with 0.25 ml, that is, 50 ⁇ g of the immunogen was injected into each mouse.
  • the immunogen was emulsified with Freund's incomplete adjuvant and then injected into the tail vein with 0.25 ml, that is, 50 ⁇ g of the immunogen was injected into each mouse.
  • the interval between the initial immunization and the first booster immunization is 2 weeks, and the interval between each subsequent booster immunization is 3 weeks.
  • each selected mouse was intraperitoneally injected with 100 ⁇ g of CD3e/g recombinant protein for the last immunization.
  • the mice were sacrificed 3 days later, and spleen cells were collected.
  • NH 4 OH was added to a final concentration of 1% (w/w) to lyse the red blood cells doped in the splenocytes to obtain a splenocyte suspension.
  • the cells were centrifuged and washed 3 times with DMEM basic medium at 1000 rpm, and then mixed with mouse myeloma cells SP2/0 (purchased from ATCC #CRL-1581) at a ratio of 5:1 to the number of viable cells, and a high-efficiency electrofusion method was used ( See METHODS IN ENZYMOLOGY, VOL. 220) for cell fusion.
  • the fused cells were diluted into DMEM medium containing 20% fetal calf serum and 1 ⁇ HAT, and the percentages are mass percentages. Then add 1 ⁇ 10 5 /200 ⁇ l per well into a 96-well cell culture plate, and place it in a 5% CO 2 , 37°C incubator. The percentage is a volume percentage.
  • the cell fusion plate supernatant was screened using ELISA plates coated with antigen CD3e/g, and the positive clones with an OD value greater than 2 in the ELISA results were amplified into 24-well plates and cultured in 10% (w/w) HT fetal bovine Serum was expanded in DMEM (purchased from Invitrogen, catalog number 11995-065) at 37°C, 5% (v/v) CO2 . After 3 days of culture, take the expanded culture medium in the 24-well plate and centrifuge, collect the supernatant, conduct antibody subtype analysis on the supernatant, and determine the antigen-binding activity with ELISA.
  • DMEM purchased from Invitrogen, catalog number 11995-065
  • hybridoma cells with OD 450nm >2 in the ELISA test were selected as qualified positive clones.
  • the qualified hybridoma cells were selected for subcloning in a 96-well plate using the limiting dilution method.
  • ELISA was used for preliminary screening 10 days after subcloning, and a single positive clone was selected and expanded to a 24-well plate for further culture. After 3 days, ELISA was used to determine the positive antigen binding and the CD3e/g receptor ligand binding experiment was used to evaluate the biological activity (the evaluation standard was OD 450nm value > 2 in the ELISA experiment).
  • clones were selected and cultured in DMEM medium (purchased from Invitrogen) containing 10% (w/w) FBS at 37°C and 5% (v/v) CO2 .
  • DMEM medium purchased from Invitrogen
  • the clones obtained are expanded and cultured, and then frozen in liquid nitrogen to obtain the hybridoma cells of the present disclosure, which can be used for subsequent antibody production and purification.
  • the hybridoma cells obtained in Example 1 were inoculated into T-75 cell culture flasks and acclimated and passaged for 3 generations using production medium (Hybridoma serum free medium, purchased from Invitrogen Company).
  • production medium Hybridoma serum free medium, purchased from Invitrogen Company.
  • inoculate the cell culture spinner flask Add 500 ml of production medium to each 2-liter culture roller bottle, and inoculate the cells at a density of 1.0 ⁇ 10 5 /ml. Place the culture spinner bottle on a spinner in a 37°C incubator with a rotation speed of 3 rpm. After 14 days of continuous rotating culture, the cell culture fluid was collected, filtered to remove cells, and filtered with a 0.45-micron filter until the culture supernatant was clear.
  • the clarified culture supernatant can be purified immediately or frozen at -30°C.
  • Antibodies in the clarified hybridoma cell culture supernatant were purified using a 2 mL protein A column (purchased from GE Healthcare).
  • the protein A column was first equilibrated with equilibrium buffer (PBS phosphate buffer, pH 7.2), and then the clarified culture supernatant was loaded onto the protein A column, and the flow rate was controlled at 3 mL/min. After loading the sample, wash the Protein A column with equilibration buffer. The volume of the equilibration buffer is 4 times the bed volume of the Protein A column.
  • eluent 0.1M glycine hydrochloride buffer, pH 2.5
  • a UV detector to monitor the elution (A 280 UV absorption peak).
  • Collect the eluted antibodies add 10% 1.0M Tris-HCl buffer to neutralize the pH, the percentage is the volume percentage, and then immediately dialyze with PBS phosphate buffer overnight, change the medium once the next day, and continue dialysis for 3 hours.
  • Collect the dialyzed CD3e/g antibody sterile filter it with a 0.22 micron filter, and store it aseptically to obtain the purified CD3e/g antibody.
  • Enzyme-linked immunosorbent assay was used to detect antigen-antibody binding sites.
  • the purified CD3e/g antibody obtained in Example 2 was subjected to a binding reaction with CD3e/g (see Table 1 for the sequence) and CD3e/d protein (purchased from Sinobiological, CAT: CT038-H2508H-B).
  • the blocking solution was discarded, and 100 ⁇ l of the purified CD3e/g antibody obtained in Example 2 was added to each well. After incubation at 37°C for 2 hours, the plate was washed three times with plate washing solution [PBS+0.01% (v/v) Tween20]. HRP (horseradish peroxidase)-labeled secondary antibody (purchased from Sigma) was added, and after incubation at 37°C for 2 hours, the plate was washed three times with washing solution [PBS+0.01% (v/v) Tween20]. Add 100 ⁇ l of TMB substrate to each well, and after incubation at room temperature for 30 minutes, add 100 ⁇ l of stop solution (1.0N HCl) to each well.
  • stop solution 1.0N HCl
  • FIG. 1 Use an ELISA plate reader (SpectraMax 384plus, purchased from Molecular Device) to read the A 450nm value.
  • Figure 2 and Table 3 show the ELISA detection of the binding activity of CD3e/g antibodies to hCD3e/g.
  • Figure 3 and Table 4 show the ELISA detection.
  • HIT3a was purchased from BD pharmigen, the catalog number is 555336; SP34 was purchased from BD pharmigen, the catalog number is 551916.
  • OKT3 was purchased from Biolegend.
  • the antibody of the present disclosure can bind to human CD3e/g (hCD3e/g), and the binding affinity of the antibody of the present disclosure to human CD3e/g is higher than that of OKT3. good.
  • the antibodies of the present disclosure can bind to cyno CD3e/g, while OKT3 does not have binding affinity or significant binding to cyno CD3e/g.
  • the antibody of the present disclosure can also bind to human CD3e/d (hCD3e/d), and the binding affinity of the antibody of the present disclosure to human CD3e/d is better than that of SP34.
  • the heavy and light chain variable region (VH and VL) sequences of murine anti-human CD3 antibodies were used to search the corresponding human antibody heavy and light chain variable region sequences in the IMGT database.
  • MOE software by comparing the heavy chain and light chain variable regions of mouse antibodies with the retrieved human antibody heavy and light chain variable region gene sequences, we can select heavy and light chains with high homology to mouse antibodies.
  • the variable region germline gene is used as a template, and the CDRs of the mouse antibody are grafted into the corresponding human template respectively to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the key amino acids in the backbone sequence are back mutated to the corresponding amino acids of the mouse antibody.
  • the amino acid residues in the CDR region are determined and annotated by the Kabat numbering system.
  • the light and heavy chain variable regions of the above-mentioned murine antibody are connected to the light and heavy chain constant regions of the human antibody to form a chimeric antibody.
  • the chimeric antibody corresponding to the mAb001 antibody is named CAb001, and the analogy is applied to other antibodies.
  • the humanized light chain template of the mouse antibody mAb001 is IGKV1-39 IGKV1-39*01 X59315 V-KAPPA, and the humanized heavy chain template is IGHV1-8 IGHV1-8*01 M99637 VH.
  • human Humanized antibody hAb001 the humanized variable region sequence is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, underlined is the CDR sequence, no underlined is the FR sequence,
  • Q3V means numbering according to the natural sequence of the amino acid sequence, mutating Q at position 3 back to V.
  • the grafted representative mouse antibody CDRs were implanted with human germline FR region sequences.
  • This table represents the sequences obtained by various mutation combinations. As indicated by hAb001-6, the humanized antibody hAb001-6 has four reverse mutations of the light chain mAb001VL_hum2 and the heavy chain mAb001VH_hum2. Other analogies.
  • the humanized light chain template of the mouse antibody mAb001 is IGKV4-1 IGKV4-1*01 Z0023 V-KAPPA, and the humanized heavy chain template is IGHV1-3 IGHV1-3*01 X62109 VH.
  • human Humanized antibody hAb001 the humanized variable region sequence is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, underlined is the CDR sequence, no underlined is the FR sequence,
  • S69T means numbering according to the natural sequence of the amino acid sequence, mutating S at position 69 back to T.
  • the grafted representative mouse antibody CDRs were implanted with human germline FR region sequences.
  • This table represents the sequences obtained by various mutation combinations. As indicated by hAb001-22, the humanized antibody hAb001-22 has three reverse mutations of light chain mAb001VL_hum6 and heavy chain mAb001VH_hum6. Other analogies.
  • the humanized light chain template of the mouse antibody mAb007 is IGKV1-27 IGKV1-27*01 X63398 V-KAPPA F, and the humanized heavy chain template is IGHV1-8 IGHV1-8*01 M99637 VH F.
  • the humanized antibody hAb007 was obtained.
  • the sequence of the humanized variable region is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, underlined is the CDR sequence, no underlined is the FR sequence,
  • A49S means numbering according to the natural sequence of the amino acid sequence, mutating A at position 49 back to S.
  • the grafted representative mouse antibody CDRs were implanted with human germline FR region sequences.
  • This table represents the sequences obtained by various mutation combinations. As indicated by hAb007-5, the humanized antibody hAb007-5 has four reverse mutations of the light chain mAb007VL_hum1 and the heavy chain mAb007VH_hum2. Other analogies.
  • the humanized light chain template of the mouse antibody mAb007 is IGKV4-1 IGKV4-1*01 Z0023 V-KAPPA, and the humanized heavy chain template is IGHV1-3 IGHV1-3*01 X62109 VH.
  • human Humanized antibody hAb007 the humanized variable region sequence is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, underlined is the CDR sequence, no underlined is the FR sequence,
  • S69T means numbering according to the natural sequence of the amino acid sequence, mutating S at position 69 back to T.
  • the grafted representative mouse antibody CDRs were implanted with human germline FR region sequences.
  • This table represents the sequences obtained by various mutation combinations. As indicated by hAb007-22, the humanized antibody hAb007-22 has three reverse mutations of light chain mAb007VL_hum6 and heavy chain mAb007VH_hum6. Other analogies.
  • the humanized light chain template of mouse antibody mAb008 is IIGKV2-30 IGKV2-30*01 X63403 V-KAPPA F, and the humanized heavy chain template is IGHV1-8 IGHV1-8*01 M99637 VH F.
  • the humanized antibody hAb008 was obtained.
  • the sequence of the humanized variable region is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, underlined is the CDR sequence, no underlined is the FR sequence,
  • F41L means numbering according to the natural sequence of the amino acid sequence, mutating F at position 41 back to L.
  • the grafted representative mouse antibody CDRs were implanted with human germline FR region sequences.
  • This table represents the sequences obtained by various mutation combinations. As indicated by hAb008-5, the humanized antibody hAb008-5 has three reverse mutations of light chain mAb008VL_hum2 and heavy chain mAb007VH_hum2. Other analogies.
  • the humanized light chain template of the mouse antibody mAb008 is IIGKV2-30 IGKV2-30*01 X63403 V-KAPPA F, and the humanized heavy chain template is IGHV1-3 IGHV1-3*01 X62109 VH, which is obtained after humanization Humanized antibody hAb008, the humanized variable region sequence is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, underlined is the CDR sequence, no underlined is the FR sequence,
  • Y54Q means numbering according to the natural sequence of the amino acid sequence, mutating Y at position 54 back to Q.
  • the grafted representative mouse antibody CDRs were implanted with human germline FR region sequences.
  • This table represents the sequences obtained by various mutation combinations. As indicated by hAb008-22, the humanized antibody hAb008-22 has six reverse mutations of light chain mAb008VL_hum6 and heavy chain mAb007VH_hum7. Other analogies.
  • Point mutations were performed on antibody mAb008 hotspots.
  • the light chain mAb008 antibody has one mutable site.
  • the 34-position G of the light chain is mutated back to A, resulting in a hotspot mutation chimeric antibody CAb008DA.
  • Point mutations were performed on antibody mAb009 hotspots.
  • the light chain mAb009 antibody has two mutable sites.
  • the 32-position G of the light chain is mutated back to A, resulting in a hotspot mutation chimeric antibody CAb009DADG.
  • Jurkat cells were expanded and cultured in a T-175 cell culture flask to 3 ⁇ 10 6 /ml, centrifuged to absorb the culture medium, washed twice with PBS buffer (purchased from Invitrogen), and after cell counting, the cells were washed with PBS buffer. Dilute to 2 ⁇ 10 6 cells per ml, add 1% BSA blocking solution, the percentage is mass percentage, incubate on ice for 30 minutes, and then centrifuge and wash twice with PBS buffer. The collected cells were suspended in FACS buffer (PBS+1% BSA, the percentage is mass percentage) to 2 ⁇ 10 6 cells/mL, and 100 ⁇ l per well was added to a 96-well FACS reaction plate, and the Example was added 4.
  • PBS buffer purchased from Invitrogen
  • Total human T cells were extracted using a human total T cell isolation kit (EasySep TM Direct Human T Cell Isolation Kit, purchased from Stemcell, catalog number 19661). Wash 3 times with complete medium. The T cells and diluted CD3 antibodies were mixed evenly in a 96-well plate and cultured at 37°C for 3 days. Collect the culture supernatant after 3 days, and use an ELISA kit (IFN gamma Human Uncoated ELISA Kit, purchased from Invitrogen Company, catalog number 88-7316-88) to detect the human IFN- ⁇ level of the culture supernatant.
  • EasySep TM Direct Human T Cell Isolation Kit purchased from Stemcell, catalog number 19661.
  • the cultured cells were washed once with PBS, then labeled anti-human CD25 flow cytometry antibody (PE-Cy7 Mouse Anti-Human CD25, purchased from BD Pharmingen, catalog number 557741) was added for staining for 30 minutes in the dark, and then washed twice with PBS.
  • the expression of CD25 in human T cells was analyzed using FACS (BD FACS LSRFortessa purchased from BD).
  • the equilibrium dissociation constant of CD3 antibody binding to human CD3e/g antigen was determined by kinetic binding method using the Biacore 8K (GE) system.
  • NHS N-hydroxysuccinimide
  • EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • Fc anti-human IgG
  • the anti-human IgG (Fc) antibody can bind to the Fc-terminal capture antibody of the CD3 antibody, and then add a series of concentrations of antigen to obtain the binding and dissociation curves of the antibody and the antigen.
  • the corresponding kinetic constants are calculated through Biacore Evaluation software. See below for details. surface.

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Abstract

本公开涉及靶向CD3的抗体及其应用。具体而言，本公开提供特异性结合至CD3的抗体或抗原结合片段、编码所述抗体或抗原结合片段的核酸、包含所述核酸的核酸载体、包含所述核酸或核酸载体的宿主细胞、包含前述物质的药物组合物、其制备方法以及应用。

Description

靶向CD3的抗体及其应用 技术领域
本申请涉及生物医药领域，尤其涉及一种靶向CD3的抗体或其抗原结合片段、及其制备方法和应用。
背景技术
TCR/CD3复合物是人和其他哺乳动物中T细胞功能和特异性的主要调节者，在抗原识别和细胞内信号转导途径方面起着非常重要的作用。其中，CD3是由四种不同的链(CD3ε链、CD3γ链、CD3ζ链和CD3δ链)组成的蛋白复合物，其包含三个二聚体，分别是CD3ε/γ(或称CD3e/g)、CD3ε/δ(或称CD3e/d)和CD3ζ/ζ(或称CD3z/z)，它们的胞内区均含有启动信号传递所必需的免疫受体酪氨酸激活基序(Immunoreceptor tyrosine-based activation motif，ITAM)。TCR是共价连接的α和β链的异二聚体(“TCRαβ”)，主要识别MHC分子呈递的抗原，但其胞内区域非常短，需要和CD3形成TCR/CD3复合物才能将刺激信号传入细胞内(Dong,D.,Zheng,L.,Lin,J.等.Nature 573,546–552(2019))。
目前，以CD3为靶点的抗体药物已广泛应用于临床。1986年，第一个被FDA批准上市的单克隆抗体药物就是CD3的单抗Muromonab(也称OKT3)，用于治疗器官移植后的急性排斥反应。之后，其它靶向CD3的单克隆抗体Otelixizumab、Teplizumab、Visilizumab等也相继被开发用于治疗克罗恩氏病、溃疡性结肠炎和I型糖尿病等。近年来，由于CD3在T细胞的活化、增殖等过程中的重要作用，靶向人CD3和肿瘤相关抗原(TAA)的双特异性或多特异性抗体也已成为抗肿瘤治疗中的研究热点。
靶向CD3的抗体(在本公开中与“CD3抗体”、“抗CD3抗体”、“特异性结合至CD3的抗体”可互换地使用)开发的难点在于难以得 到较好的免疫应答以及血清滴度，造成融合后阳性克隆数目少。目前市售可得的CD3抗体包括OKT3。使用OKT3治疗结束后T细胞功能通常在一周内恢复正常，但OKT3在起始使用时的副作用也是相当大的，包括OKT3流感样症候群、细胞因子风暴等。此外OKT3与食蟹猴(cynomolgus)来源的CD3不结合，对其在临床前安全性评价研究中选择灵长类动物食蟹猴带来了不便。
本公开提供新的抗CD3抗体，其能够结合人和食蟹猴CD3，副作用低，为包括癌症和自身免疫疾病等在内的疾病提供了新的潜在治疗方案。本公开的抗体或其抗原结合片段具有优异的生物学活性(例如，高亲和力)。其能够与人CD3e/g(hCD3e/g)、人CD3e/d(hCD3e/d)和食蟹猴CD3e/g(cCD3e/g)相结合，从而使得临床前安全评估研究中可以选择灵长类动物食蟹猴(cynomolgus,cyno)的疾病模型进行药理毒理实验，为临床前药理、毒理等研究带来极大的便利。此外，本公开的抗体或其抗原结合片段能够有效地激活人T细胞系Jurkat细胞的NFAT下游信号通路，有利于发挥后续的生物学活性。
发明内容
本公开涉及特异性结合至CD3的抗体或其抗原结合片段及其应用。
在第一方面，本公开涉及一种特异性结合至CD3的抗体或其抗原结合片段，其重链包含CDR1、CDR2和CDR3，其中：
(a)重链CDR1包含与NYYMH(SEQ ID NO:1)、DNYIH(SEQ ID NO:2)、DYTVN(SEQ ID NO:3)或DYTIH(SEQ ID NO:4)具有至少80％同一性的序列；
(b)重链CDR2包含与WTYPGNNNIKYNEKFKG(SEQ ID NO:18)、WIYPGSVNIKYNEKFKD(SEQ ID NO:6)或YINPFNSYTKYNQKFKD(SEQ ID NO:22)具有至少80％同一性的序列；以及
(c)重链CDR3包含与DGYGYYFFDY(SEQ ID NO:8)、DISRYYFDY(SEQ ID NO:9)、SVSTY(SEQ ID NO:10)或SVSIY(SEQ ID NO:11)具有至少80％同一性的序列。
在一些实施方案中，所述抗体或其抗原结合片段包含：
(a)重链CDR1，其包含选自以下的序列：NYYMH(SEQ ID NO:1)、DNYIH(SEQ ID NO:2)、DYTVN(SEQ ID NO:3)、DYTIH(SEQ ID NO:4)或者与SEQ ID NO:1-4中的任一个相比具有1个氨基酸添加、置换和/或缺失的序列；
(b)重链CDR2，其包含选自以下的序列：WTYPGX 1X 2X 3IKYNEKFKG(SEQ ID NO:5)，WIYPGSVNIKYNEKFKD(SEQ ID NO:6)或与SEQ ID NO:6相差不多于总计3个氨基酸添加、置换和/或缺失的序列，或者YINPFX 4X 5YTKYNQKFKD(SEQ ID NO:7)；其中X 1、X 2和X 3各自独立地为N或Q，X 4是N或S，X 5是S或D；
(c)重链CDR3，其包含选自以下的序列：DGYGYYFFDY(SEQ ID NO:8)、DISRYYFDY(SEQ ID NO:9)、SVSTY(SEQ ID NO:10)、SVSIY(SEQ ID NO:11)或者与SEQ ID NO:8-11中的任一个相差不多于总计2个氨基酸添加、置换和/或缺失的序列；
(d)轻链CDR1，其包含选自以下的序列：KSSQSLLNX 6RTRKNYLA(SEQ ID NO:12)或者KSSQSLLDX 7DX 8KTYLN(SEQ ID NO:13)；其中X 6是N、Q或S，X 7是S、A或G，X 8是G或A；
(e)轻链CDR2，其包含选自以下的序列：WASTRES(SEQ ID NO:14)或LVSKLNS(SEQ ID NO:15)；以及
(f)轻链CDR3，其包含选自以下的序列：KQSX 9X 10LRT(SEQ ID NO:16)或者WQGTHFPRT(SEQ ID NO:17)；其中X 9是Y或F，以及X 10是T或I。
在一些实施方案中，所述抗体或其抗原结合片段包含：
(1)重链CDR1，其包含以下序列或由其组成：NYYMH(SEQ  ID NO:1)；重链CDR2，其包含以下序列或由其组成：WTYPGX 1X 2X 3IKYNEKFKG(SEQ ID NO:5)，其中X 1、X 2和X 3各自独立地为N或Q；重链CDR3，其包含以下序列或由其组成：DGYGYYFFDY(SEQ ID NO:8)；轻链CDR1，其包含以下序列或由其组成：KSSQSLLNX 6RTRKNYLA(SEQ ID NO:12)，其中X 6是N或Q；轻链CDR2，其包含以下序列或由其组成：WASTRES(SEQ ID NO:14)；以及轻链CDR3，其包含以下序列或由其组成：KQSYTLRT(SEQ ID NO:31)；或
(2)重链CDR1，其包含以下序列或由其组成：DNYIH(SEQ ID NO:2)；重链CDR2，其包含以下序列或由其组成：WIYPGSVNIKYNEKFKD(SEQ ID NO:6)；重链CDR3，其包含以下序列或由其组成：DISRYYFDY(SEQ ID NO:9)；轻链CDR1，其包含以下序列或由其组成：KSSQSLLNSRTRKNYLA(SEQ ID NO:26)；轻链CDR2，其包含以下序列或由其组成：WASTRES(SEQ ID NO:14)；以及轻链CDR3，其包含以下序列或由其组成：KQSFILRT(SEQ ID NO:32)；或
(3)重链CDR1，其包含以下序列或由其组成：DYTVN(SEQ ID NO:3)；重链CDR2，其包含以下序列或由其组成：YINPFNSYTKYNQKFKD(SEQ ID NO:22)；重链CDR3，其包含以下序列或由其组成：SVSTY(SEQ ID NO:10)；轻链CDR1，其包含以下序列或由其组成：KSSQSLLDX 7DX 8KTYLN(SEQ ID NO:13)，其中X 7是S，X 8是G或A；轻链CDR2，其包含以下序列或由其组成：LVSKLNS(SEQ ID NO:15)；以及轻链CDR3，其包含以下序列或由其组成：WQGTHFPRT(SEQ ID NO:17)；或
(4)重链CDR1，其包含以下序列或由其组成：DYTIH(SEQ ID NO:4)；重链CDR2，其包含以下序列或由其组成：YINPFSDYTKYNQKFKD(SEQ ID NO:23)；重链CDR3，其包含以下序列或由其组成：SVSIY(SEQ ID NO:11)；轻链CDR1，其包含以下序列或由其组成：KSSQSLLDX 7DX 8KTYLN(SEQ ID NO:13)， 其中X 7是G或A，X 8是G；轻链CDR2，其包含以下序列或由其组成：LVSKLNS(SEQ ID NO:15)；以及轻链CDR3，其包含以下序列或由其组成：WQGTHFPRT(SEQ ID NO:17)。
在一些实施方案中，所述重链CDR2包含选自以下的序列：WTYPGNNNIKYNEKFKG(SEQ ID NO:18)、WTYPGQNNIKYNEKFKG(SEQ ID NO:19)、WTYPGNQNIKYNEKFKG(SEQ ID NO:20)、WTYPGQNQIKYNEKFKG(SEQ ID NO:21)、WIYPGSVNIKYNEKFKD(SEQ ID NO:6)、YINPFNSYTKYNQKFKD(SEQ ID NO:22)和YINPFSDYTKYNQKFKD(SEQ ID NO:23)。在一些实施方案中，所述轻链CDR1包含选自以下的序列：KSSQSLLNNRTRKNYLA(SEQ ID NO:24)、KSSQSLLNQRTRKNYLA(SEQ ID NO:25)、KSSQSLLNSRTRKNYLA(SEQ ID NO:26)、KSSQSLLDSDGKTYLN(SEQ ID NO:27)、KSSQSLLDSDAKTYLN(SEQ ID NO:28)、KSSQSLLDGDGKTYLN(SEQ ID NO:29)和KSSQSLLDADGKTYLN(SEQ ID NO:30)。在一些实施方案中，所述轻链CDR3包含选自以下的序列：KQSYTLRT(SEQ ID NO:31)、KQSFILRT(SEQ ID NO:32)或WQGTHFPRT(SEQ ID NO:17)。
在一些实施方案中，所述抗体或其抗原结合片段包含：
(1)重链CDR1，其包含以下序列或由其组成：NYYMH(SEQ ID NO:1)；重链CDR2，其包含以下序列或由其组成：WTYPGNNNIKYNEKFKG(SEQ ID NO:18)；重链CDR3，其包含以下序列或由其组成：DGYGYYFFDY(SEQ ID NO:8)；轻链CDR1，其包含以下序列或由其组成：KSSQSLLNNRTRKNYLA(SEQ ID NO:24)；轻链CDR2，其包含以下序列或由其组成：WASTRES(SEQ ID NO:14)；以及轻链CDR3，其包含以下序列或由其组成：KQSYTLRT(SEQ ID NO:31)；或
(2)重链CDR1，其包含以下序列或由其组成：NYYMH (SEQ ID NO:1)；重链CDR2，其包含以下序列或由其组成：WTYPGNNNIKYNEKFKG(SEQ ID NO:18)；重链CDR3，其包含以下序列或由其组成：DGYGYYFFDY(SEQ ID NO:8)；轻链CDR1，其包含以下序列或由其组成：KSSQSLLNQRTRKNYLA(SEQ ID NO:25)；轻链CDR2，其包含以下序列或由其组成：WASTRES(SEQ ID NO:14)；以及轻链CDR3，其包含以下序列或由其组成：KQSYTLRT(SEQ ID NO:31)；或
(3)重链CDR1，其包含以下序列或由其组成：NYYMH(SEQ ID NO:1)；重链CDR2，其包含以下序列或由其组成：WTYPGQNNIKYNEKFKG(SEQ ID NO:19)；重链CDR3，其包含以下序列或由其组成：DGYGYYFFDY(SEQ ID NO:8)；轻链CDR1，其包含以下序列或由其组成：KSSQSLLNNRTRKNYLA(SEQ ID NO:24)；轻链CDR2，其包含以下序列或由其组成：WASTRES(SEQ ID NO:14)；以及轻链CDR3，其包含以下序列或由其组成：KQSYTLRT(SEQ ID NO:31)；或
(4)重链CDR1，其包含以下序列或由其组成：NYYMH(SEQ ID NO:1)；重链CDR2，其包含以下序列或由其组成：WTYPGQNNIKYNEKFKG(SEQ ID NO:19)；重链CDR3，其包含以下序列或由其组成：DGYGYYFFDY(SEQ ID NO:8)；轻链CDR1，其包含以下序列或由其组成：KSSQSLLNQRTRKNYLA(SEQ ID NO:25)；轻链CDR2，其包含以下序列或由其组成：WASTRES(SEQ ID NO:14)；以及轻链CDR3，其包含以下序列或由其组成：KQSYTLRT(SEQ ID NO:31)；或
(5)重链CDR1，其包含以下序列或由其组成：NYYMH(SEQ ID NO:1)；重链CDR2，其包含以下序列或由其组成：WTYPGNQNIKYNEKFKG(SEQ ID NO:20)；重链CDR3，其包含以下序列或由其组成：DGYGYYFFDY(SEQ ID NO:8)；轻链CDR1，其包含以下序列或由其组成：KSSQSLLNNRTRKNYLA(SEQ ID NO:24)；轻链CDR2，其包含以下序列或由其组成：WASTRES (SEQ ID NO:14)；以及轻链CDR3，其包含以下序列或由其组成：KQSYTLRT(SEQ ID NO:31)；或
(6)重链CDR1，其包含以下序列或由其组成：NYYMH(SEQ ID NO:1)；重链CDR2，其包含以下序列或由其组成：WTYPGNQNIKYNEKFKG(SEQ ID NO:20)；重链CDR3，其包含以下序列或由其组成：DGYGYYFFDY(SEQ ID NO:8)；轻链CDR1，其包含以下序列或由其组成：KSSQSLLNQRTRKNYLA(SEQ ID NO:25)；轻链CDR2，其包含以下序列或由其组成：WASTRES(SEQ ID NO:14)；以及轻链CDR3，其包含以下序列或由其组成：KQSYTLRT(SEQ ID NO:31)；或
(7)重链CDR1，其包含以下序列或由其组成：NYYMH(SEQ ID NO:1)；重链CDR2，其包含以下序列或由其组成：WTYPGQNQIKYNEKFKG(SEQ ID NO:21)；重链CDR3，其包含以下序列或由其组成：DGYGYYFFDY(SEQ ID NO:8)；轻链CDR1，其包含以下序列或由其组成：KSSQSLLNNRTRKNYLA(SEQ ID NO:24)；轻链CDR2，其包含以下序列或由其组成：WASTRES(SEQ ID NO:14)；以及轻链CDR3，其包含以下序列或由其组成：KQSYTLRT(SEQ ID NO:31)；或
(8)重链CDR1，其包含以下序列或由其组成：NYYMH(SEQ ID NO:1)；重链CDR2，其包含以下序列或由其组成：WTYPGQNQIKYNEKFKG(SEQ ID NO:21)；重链CDR3，其包含以下序列或由其组成：DGYGYYFFDY(SEQ ID NO:8)；轻链CDR1，其包含以下序列或由其组成：KSSQSLLNQRTRKNYLA(SEQ ID NO:25)；轻链CDR2，其包含以下序列或由其组成：WASTRES(SEQ ID NO:14)；以及轻链CDR3，其包含以下序列或由其组成：KQSYTLRT(SEQ ID NO:31)；或
(9)重链CDR1，其包含以下序列或由其组成：DYTVN(SEQ ID NO:3)；重链CDR2，其包含以下序列或由其组成：YINPFNSYTKYNQKFKD(SEQ ID NO:22)；重链CDR3，其包含以 下序列或由其组成：SVSTY(SEQ ID NO:10)；轻链CDR1，其包含以下序列或由其组成：KSSQSLLDSDGKTYLN(SEQ ID NO:27)；轻链CDR2，其包含以下序列或由其组成：LVSKLNS(SEQ ID NO:15)；以及轻链CDR3，其包含以下序列或由其组成：WQGTHFPRT(SEQ ID NO:17)；或
(10)重链CDR1，其包含以下序列或由其组成：DYTVN(SEQ ID NO:3)；重链CDR2，其包含以下序列或由其组成：YINPFNSYTKYNQKFKD(SEQ ID NO:22)；重链CDR3，其包含以下序列或由其组成：SVSTY(SEQ ID NO:10)；轻链CDR1，其包含以下序列或由其组成：KSSQSLLDSDAKTYLN(SEQ ID NO:28)；轻链CDR2，其包含以下序列或由其组成：LVSKLNS(SEQ ID NO:15)；以及轻链CDR3，其包含以下序列或由其组成：WQGTHFPRT(SEQ ID NO:17)；或
(11)重链CDR1，其包含以下序列或由其组成：DYTIH(SEQ ID NO:4)；重链CDR2，其包含以下序列或由其组成：YINPFSDYTKYNQKFKD(SEQ ID NO:23)；重链CDR3，其包含以下序列或由其组成：SVSIY(SEQ ID NO:11)；轻链CDR1，其包含以下序列或由其组成：KSSQSLLDADGKTYLN(SEQ ID NO:30)；轻链CDR2，其包含以下序列或由其组成：LVSKLNS(SEQ ID NO:15)；以及轻链CDR3，其包含以下序列或由其组成：WQGTHFPRT(SEQ ID NO:17)；或
(12)重链CDR1，其包含以下序列或由其组成：DYTIH(SEQ ID NO:4)；重链CDR2，其包含以下序列或由其组成：YINPFSDYTKYNQKFKD(SEQ ID NO:23)；重链CDR3，其包含以下序列或由其组成：SVSIY(SEQ ID NO:11)；轻链CDR1，其包含以下序列或由其组成：KSSQSLLDGDGKTYLN(SEQ ID NO:29)；轻链CDR2，其包含以下序列或由其组成：LVSKLNS(SEQ ID NO:15)；以及轻链CDR3，其包含以下序列或由其组成：WQGTHFPRT(SEQ ID NO:17)。
在一些实施方案中，所述抗体或其抗原结合片段包含含有以下序列的重链可变区：
Figure PCTCN2022083004-appb-000001
Figure PCTCN2022083004-appb-000002
Figure PCTCN2022083004-appb-000003
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYTVNWVKQAPGQRLEWIGYINPFNSYTKYNQKFKDRATITADTSASTAYMELSSLRSEDTAVYFCANSVSTYWGQGTTVTVSS(SEQ ID NO:57)或者与前述序列具有至少85％同一性的序列。
在一些实施方案中，所述抗体或其抗原结合片段包含含有以下序列的轻链可变区：
Figure PCTCN2022083004-appb-000004
Figure PCTCN2022083004-appb-000005
Figure PCTCN2022083004-appb-000006
Figure PCTCN2022083004-appb-000007
DVVMTQTPLTLSVTIGQPASISCKSSQSLLDADGKTYLNWLLQRPGQSPKRLIQLVSKLNSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPRTFGGGTKLEIK(SEQ ID NO:82)或者与前述序列具有至少85％同一性的序列。
在一些实施方案中，所述抗体或其抗原结合片段包含以下重链可变区及轻链可变区的组：
(1)选自SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35或SEQ ID NO:36的氨基酸序列的重链可变区；以及选自SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60或SEQ ID NO:61的氨基酸序列的轻链可变区；或
(2)选自SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39或SEQ ID NO:40的氨基酸序列的重链可变区；以及选自SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64或SEQ ID NO:65的氨基酸序列的轻链可变区；或
(3)选自SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43或 SEQ ID NO:44的氨基酸序列的重链可变区；以及选自SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68或SEQ ID NO:69的氨基酸序列的轻链可变区；或
(4)选自SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47或SEQ ID NO:48的氨基酸序列的重链可变区；以及选自SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72或SEQ ID NO:73的氨基酸序列的轻链可变区；或
(5)选自SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52或SEQ ID NO:53的氨基酸序列的重链可变区；以及选自SEQ ID NO:74、SEQ ID NO:75或SEQ ID NO:76的氨基酸序列的轻链可变区；或
(6)选自SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56或SEQ ID NO:57的氨基酸序列的重链可变区；以及选自SEQ ID NO:77、SEQ ID NO:78、SEQ ID NO:79或SEQ ID NO:80的氨基酸序列的轻链可变区。
在一些实施方案中，所述抗体或其抗原结合片段包含以下(1)至(18)任一项的重链可变区及轻链可变区的组：
(1)氨基酸序列为SEQ ID NO:38的重链可变区，以及氨基酸序列为SEQ ID NO:62的轻链可变区；或
(2)氨基酸序列为SEQ ID NO:38的重链可变区，以及氨基酸序列为SEQ ID NO:63的轻链可变区；或
(3)氨基酸序列为SEQ ID NO:38的重链可变区，以及氨基酸序列为SEQ ID NO:64的轻链可变区；或
(4)氨基酸序列为SEQ ID NO:39的重链可变区，以及氨基酸序列为SEQ ID NO:63的轻链可变区；或
(5)氨基酸序列为SEQ ID NO:39的重链可变区，以及氨基酸序列为SEQ ID NO:64的轻链可变区；或
(6)氨基酸序列为SEQ ID NO:39的重链可变区，以及氨基酸序列为SEQ ID NO:65的轻链可变区；或
(7)氨基酸序列为SEQ ID NO:46的重链可变区，以及氨基酸序列为SEQ ID NO:71的轻链可变区；或
(8)氨基酸序列为SEQ ID NO:46的重链可变区，以及氨基酸序列为SEQ ID NO:72的轻链可变区；或
(9)氨基酸序列为SEQ ID NO:47的重链可变区，以及氨基酸序列为SEQ ID NO:70的轻链可变区；或
(10)氨基酸序列为SEQ ID NO:47的重链可变区，以及氨基酸序列为SEQ ID NO:72的轻链可变区；或
(11)氨基酸序列为SEQ ID NO:48的重链可变区，以及氨基酸序列为SEQ ID NO:70的轻链可变区；或
(12)氨基酸序列为SEQ ID NO:48的重链可变区，以及氨基酸序列为SEQ ID NO:71的轻链可变区；或
(13)氨基酸序列为SEQ ID NO:50的重链可变区，以及氨基酸序列为SEQ ID NO:76的轻链可变区；或
(14)氨基酸序列为SEQ ID NO:55的重链可变区，以及氨基酸序列为SEQ ID NO:79的轻链可变区；或
(15)氨基酸序列为SEQ ID NO:55的重链可变区，以及氨基酸序列为SEQ ID NO:80的轻链可变区；或
(16)氨基酸序列为SEQ ID NO:56的重链可变区，以及氨基酸序列为SEQ ID NO:79的轻链可变区；或
(17)氨基酸序列为SEQ ID NO:56的重链可变区，以及氨基酸序列为SEQ ID NO:80的轻链可变区；或
(18)氨基酸序列为SEQ ID NO:57的重链可变区，以及氨基酸序列为SEQ ID NO:79的轻链可变区。
在一些实施方案中，所述抗体或其抗原结合片段是人抗体、人源化抗体、嵌合抗体、单克隆抗体、多克隆抗体、重组抗体、单链抗体、双抗体、三链抗体、四链抗体、Fab片段、F(ab') 2片段、scFv片段、Fv片段、Fab’片段、结构域抗体。在一些实施方案中，所述抗体或其抗原结合片段是IgA抗体、IgD抗体、IgE抗体、IgM抗体、 IgG1抗体、IgG2抗体、IgG3抗体或IgG4抗体。
在一些实施方案中，所述抗体或其抗原结合片段结合人CD3和/或食蟹猴CD3。
在一些实施方案中，所述抗体或其抗原结合片段结合至CD3e/d和/或CD3e/g。
在一些实施方案中，所述抗体或其抗原结合片段(i)显著激活人总T细胞，上调CD25的表达和/或诱导IFN-γ的释放；和/或(ii)以小于4×10 -8M的K D值与人CD3结合。
在第二方面，本公开涉及一种抗体或其抗原结合片段，其与如第一方面所述的抗体或抗原结合片段竞争或交叉阻断与CD3尤其是CD3e/d或CD3e/g的结合。
在第三方面，本公开涉及一种经分离的核酸，其编码如第一方面或第二方面所述的抗体或其抗原结合片段。
在第四方面，本公开涉及一种核酸载体，其包含如第三方面所述的经分离的核酸。
在第五方面，本公开涉及一种宿主细胞，其包含如第三方面所述的经分离的核酸或如第四方面所述的核酸载体。
在第六方面，本公开涉及一种药物组合物，其包含如第一方面或第二方面所述的抗体或其抗原结合片段、如第三方面所述的经分离的核酸或如第四方面所述的核酸载体，以及药学上可接受的载体。
在第七方面，本公开涉及一种产生如第一方面或第二方面所述的抗体或其抗原结合片段的方法，其包括将如第五方面所述的宿主细胞在允许它表达所述抗体或其抗原结合片段的条件下孵育。
在第八方面，本公开涉及如第一方面或第二方面中任一个所述的抗体或其抗原结合片段、如第三方面所述的经分离的核酸、如第四方面所述的核酸载体或者如第六方面所述的药物组合物在制备用于抑制免疫应答或活化T细胞的药物中的用途。
在第九方面，本公开涉及如第一方面或第二方面中任一个所述的抗体或其抗原结合片段、如第三方面所述的经分离的核酸、如第四方面所述的核酸载体或者如第六方面所述的药物组合物在制备用 于治疗癌症或自身免疫疾病的药物中的用途。
在第十方面，本公开涉及如第一方面或第二方面中任一个所述的抗体或其抗原结合片段、如第三方面所述的经分离的核酸、如第四方面所述的核酸载体或者如第六方面所述的药物组合物，其用于抑制免疫应答或活化T细胞，或者用于治疗癌症或自身免疫疾病。
在第十一方面，本公开涉一种抑制免疫应答或活化T细胞，或者治疗癌症或自身免疫疾病的方法，其包括给予有需要的个体抑制有效量、活化有效量或治疗有效量的如第一方面或第二方面中任一个所述的抗体或其抗原结合片段、如第三方面所述的经分离的核酸、如第四方面所述的核酸载体或者如第六方面所述的药物组合物。
在一些实施方案中，前文所述癌症选自：黑素瘤(例如转移性恶性黑素瘤)、肾癌、前列腺癌、乳腺癌、结直肠癌、肺癌、鼻咽癌、口腔癌、肝癌、骨癌、胰腺癌、皮肤癌、头颈部癌、子宫癌、卵巢癌、直肠癌、胃癌、睾丸癌、输卵管癌、子宫内膜癌、子宫颈癌、阴道癌、外阴癌、霍奇金淋巴瘤、非霍奇金淋巴瘤、食道癌、小肠癌、大肠癌、内分泌系统癌症、甲状腺癌、甲状旁腺癌症、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病(包括急性髓性白血病、慢性髓性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞白血病)、膀胱癌、肾盂癌、中枢神经系统肿瘤、神经胶质瘤、脊髓轴肿瘤、脑干胶质瘤、垂体腺瘤、卡波济氏肉瘤、表皮样癌、鳞状细胞癌、T细胞淋巴瘤和它们的组合。
在一些实施方案中，前文所述自身免疫疾病选自：风湿关节炎、多发性硬化、干燥综合征、胰岛素依赖型糖尿病、自身免疫甲状腺炎、反应性关节炎、强直性脊柱炎、硬皮病、多发性肌炎、皮肌炎、牛皮癣、韦格纳氏肉芽肿病、克罗恩氏病、溃疡性结肠炎、狼疮例如系统性红斑狼疮、动脉粥样硬化、慢性阻塞性肺病、肝硬化、肾移植纤维化、肾移植肾病、肺纤维化以及它们的组合。
本公开中更详细地描述这些方面和其他方面。提供的所述方面中的每一者可以包括本文提供的多种实施方案。因此预计，可以在描述的每个方面包括涉及一种要素或要素组合的每一个实施方案， 并且明确考虑到上述方面和实施方案的全部此类组合。
附图说明
图1显示通过ELISA检测的小鼠血清中针对免疫原的血清效价的结果图。
图2显示通过ELISA检测的CD3抗体与人CD3e/g的结合的结果图。
图3显示通过ELISA检测的CD3抗体与食蟹猴CD3e/g结合的结果图。
图4显示通过ELISA检测的CD3抗体与人CD3e/d结合的结果图。
图5(a)-(e)显示通过FACS检测的CD3抗体与Jurkat细胞的结合的结果图。
图6(a)-(h)显示通过FACS检测的CD3抗体对CD25表达的影响。
图7(a)-(f)显示通过FACS检测的CD3抗体对IFN-γ分泌的影响。发明详述
除非本文另外定义，否则与本申请相关使用的科学和技术术语应当具有本领域普通技术人员通常理解的含义。另外，除非上下文所要求，单数术语应当包括复数并且复数术语应当包括单数。
通常，与本文所述的细胞和组织培养、分子生物学、免疫学、微生物学、遗传学和蛋白质和核酸化学及杂交相连使用的名称及其技术是本领域熟知和通常使用的那些。本申请的方法和技术通常根据本领域熟知和如本说明书通篇范围内引用及讨论的多种普通和更具体参考文献中所述的常规方法进行，除非另外说明。见，例如，Sambrook等人，Molecular Cloning：A Laboratory Manual，第3版，Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.(2001)，Ausubel等人，Current Protocols in Molecular Biology，Greene Publishing Associates(1992)，以及Harlow和Lane，Antibodies：A Laboratory Manual Cold Spring Harbor Laboratory Press， Cold Spring Harbor，N.Y.(1990)，所述文献通过引用并入本文。酶促反应和纯化技术根据制造商的说明书进行，如本领域中通常所完成或如本文所述。通常，与本文所述的分析化学、合成有机化学和医学和药物化学相连使用的术语及其实验室方法和技术是本领域熟知和通常使用的那些。标准技术可以用于化学合成、化学分析、药物制备、配制和递送及患者治疗。
应当理解本公开不限于本文所述的特定方法学、方案和试剂并且因而它们可以变动。本文所用的术语目的仅在于描述特定实施方案，并且不意在限制本公开的范围。
除了在操作实施例中或其中另外说明，表述本文所用的成分或反应条件的量的全部数字应当理解为在全部情况下受术语“约”修饰。与百分数相连使用时，术语“约”可以意指±5％，例如，1％、2％、3％或4％。
定义
本文所用的术语仅以描述具体的实施方案为目的而不意图限制本发明。除非上下文另有明确指示，开放式的表述“包括”和“包含”解释为还可以含有没有述及的结构组成部分或方法步骤，但需要注意的是，该开放式的表述也涵盖仅由所述的组分和方法步骤组成的情形(即涵盖了封闭式表述“由……组成”的情形)。
如全文所用，范围用作描述该范围内的每个数值和所有数值的简写形式。范围内的任何数值、尤其是整数值都可选做该范围的终点。例如，范围“至少80％同一性”用作描述该范围内的所有数值，例如80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％和100％，并且包括所有子范围，例如至少85％、至少90％、至少95％等。
如本文所用的“抗体”是指单体以及多聚体。可使用完整抗体(包括多聚体)或携带抗体的抗原结合片段的抗体片段。所述抗体可以是人抗体、人源化抗体、嵌合抗体、单克隆抗体、多克隆抗体或重组抗体。抗原结合片段可以通过重组DNA技术或通过酶切割或化学切 割完整抗体而产生。
“Fab片段”是具有V L、V H、C L和C H结构域的单价片段；“F(ab') 2片段”是具有在铰链区由二硫键连接的两个Fab片段的双价片段。
单链抗体(scFv)是其中V L和V H区通过接头(例如，氨基酸残基的合成性序列)连接以形成连续蛋白质链的抗体，其中所述接头足够地长以允许该蛋白质链自身回折并形成单价抗原结合位点(见，例如，Bird等人，Science 242:423-26(1988)和Huston等人，1988，Proc Natl Acad Sci USA 85:5879-83(1988))。双抗体是包含两条多肽链的双价抗体，其中每条多肽链包含由接头连接的V H和V L结构域，所述接头太短以致于不能允许相同链上的两个结构域之间配对，因而允许每个结构域与另一条多肽链上的互补结构域配对(见，例如，Holliger等人，1993，Proc Natl Acad Sci USA 90:6444-48(1993)，和Poljak等人，Structure 2:1121-23(1994))。如果双抗体的两条多肽链是相同的，则因它们配对产生的双抗体将具有两个相同的抗原结合位点。具有不同序列的多肽链可以用来产生具有两个不同抗原结合位点的双抗体。类似地，三抗体(或称“三链抗体”)和四抗体(或称“四链抗体”)是分别包含三条和四条可以是相同或不同的多肽链并且分别形成三个和四个可以是相同或不同的抗原结合位点的抗体。
给定抗体的互补性决定区(CDR)和构架区(FR)可以使用由Kabat等人在Sequences of Proteins of Immunological Interest，第5版，美国卫生和公众服务部，PHS，NIH，NIH出版编号91-3242，1991中描述的体系标识。根据需要，CDR也可以再定义根据替代性命名方案，如Chothia的那种(见Chothia&Lesk，1987，J.Mol.Biol. 196:901-917；Chothia等人，1989，Nature  342:878-883或Honegger&Pluckthun，2001，J.Mol.Biol. 309：657-670)。一个或多个CDR可以共价或非共价地并入分子中以使该分子变成抗原结合蛋白例如抗体。抗原结合蛋白可以将CDR并入作为更大多肽链的部分，可以将CDR共价地连接至另一条多肽链，或可以非共价地并入CDR。CDR允许抗原结合蛋白与特定目的抗原特异性结合。
在本领域中可以通过多种方法来定义抗体的CDR，用于鉴定抗体分子内可变区及抗体可变区氨基酸序列内的CDR的方法和技术也是本领域熟知的，并且可以用于识别在此公开的特定抗体可变区氨基酸序列内的CDR。在本申请中，所列CDR的氨基酸序列均是按照Kabat定义规则所示出的(本申请的权利要求中也是按照Kabat定义规则所示出的序列)。本领域技术人员应当理解的是，除非另有规定，否则术语给定抗体或其区(例如可变区)的“CDR”及“互补决定区”应了解为涵盖如通过本公开描述的上述已知方案中的任何一种界定的互补决定区。虽然本公开中请求保护的范围是基于Kabat定义规则所示出的序列，但是根据其他CDR的定义规则所对应的氨基酸序列也应当落在本发明的保护范围中。
本文所述“抗体或其抗原结合片段”还可以包括抗体重链恒定区和/或抗体轻链恒定区。例如，所述的抗体重链恒定区为小鼠源抗体重链恒定区或人源抗体重链恒定区；所述的抗体轻链恒定区为小鼠源轻链抗体恒定区或人源抗体轻链恒定区。更优选地，所述的抗体重链恒定区为人源抗体重链恒定区，例如人源IgG1、IgG2a、IgG2b、IgG2c、IgG3或IgG4抗体的重链恒定区；所述的抗体轻链恒定区为人源抗体轻链κ或λ链恒定区。
术语“人源化抗体”包括具有人种系免疫球蛋白序列的可变区和恒定区的抗体，其中已将衍生自另一种系的CDR序列接枝到人骨架序列上。
术语“核酸”包括单链和双链核苷酸聚合物。构成多核苷酸的核苷酸可以是核糖核苷酸或脱氧核糖核苷酸或任一类型核苷酸的修饰形式。所述修饰包括碱基修饰如溴尿核苷和肌苷衍生物，核糖修饰如2',3'-双脱氧核糖，和核苷酸间键修饰如硫代磷酸酯、二硫代磷酸酯、硒代磷酸酯、二硒代磷酸酯、硫代苯胺磷酸酯和磷酰胺酯。
术语“载体”意指用来转移蛋白编码信息至宿主细胞中的任何分子或实体(例如，核酸、质粒、噬菌体或病毒)。
术语“宿主细胞”意指已经用核酸序列转化或能够用核酸序列转化并且因而表达目的基因的细胞。该术语包括亲本细胞的子代，无 论说是子代在形态或在遗传构成方面是否与原始亲本细胞相同，只要存在目的基因即可。宿主细胞可以是原核生物(例如，大肠杆菌)，或其可以是真核生物(例如，单细胞真核生物(例如，酵母或其他真菌)、植物细胞(例如，烟草或番茄植物细胞)、动物细胞(例如，人细胞、猴细胞、仓鼠细胞、大鼠细胞、小鼠细胞、或昆虫细胞))或杂交瘤。
术语“同一性”指两个或更多个多肽分子或两个或更多个核酸分子的序列之间的关系，如通过比对和比较序列所测定。通常而言，同一性是指在考虑需要被引入以达成两个序列的最优比对的空位的数目和各空位的长度的情况下由两个氨基酸或核酸序列共有的相同位置的数目或百分比。当氨基酸序列被描述为与另一氨基酸序列至少85％或至少90％或至少95％同一时，氨基酸序列的差异可在于保守性置换(包括其中所有置换都是保守性置换)。
如本文所用，“保守性置换”在本领域中被认为是用一个氨基酸置换具有相似性质的另一个氨基酸。示例性的保守性置换在本领域中是公知的(参见例如，WO 97/09433，第10页，1997年3月13日公开；Lehninger，Biochemistry，第二版；Worth Publishers,Inc.NY:NY(1975)，第71-77页；Lewin,Genes IV,Oxford University Press,NY and Cell Press,Cambridge,MA(1990)，第8页)。
天然存在的氨基酸可以基于常见侧链特性划分成以下类组：
1)疏水性：正亮氨酸(Nle)、Met、Ala、Val、Leu；Ile；
2)中性亲水性：Cys、Ser、Thr、Asn；Gln；
3)酸性：Asp、Glu；
4)碱性：His、Lys、Arg；
5)影响链取向的残基：Gly、Pro；和
6)芳香性：Trp、Tyr、Phe。
保守性置换可以包括这些类组之一的成员与同一类组的另一个成员交换。保守氨基酸置换可以包括通常通过化学肽合成而不借助生物系统中合成而掺入的非天然氨基酸残基。这些包括肽模拟物和氨基酸部分的其他逆转或颠倒形式。非保守性置换可以包括以上类 组之一的成员与来自另一个类组的成员交换。可以将此类置换的残基导入与人抗体同源的抗体区域或分子的非同源区内。
“有效量”通常是足以降低症状的严重性和/或频率、消除症状和/或潜在病因、防止症状和/或其潜在病因出现和/或改善或治疗因疾病(例如，癌症或自身免疫疾病)所致或与之相关的损伤的量。在一些实施方案中，有效量是治疗有效量或预防有效量。“治疗有效量”是足够以任何方式纠正疾病状态(例如，癌症或自身免疫疾病)或症状、与所述疾病状态相关的特定状态或症状或者防止、阻碍、迟滞或逆转疾病状态或与疾病相关的任何其他不利症状进展的量。待使用的本公开抗体或其抗原结合蛋白、编码所述抗体或抗原结合片段的核酸、包含所述核酸的核酸载体、包含所述核酸或核酸载体的宿主细胞或包含前述物质的药物组合物的治疗有效量将取决于例如治疗背景和目标。本领域技术人员会理解，治疗的适宜剂量水平将部分地根据所递送的分子、正在使用的抗体或其抗原结合蛋白的适应症、施用途径和患者的体格大小(体重、体表面或器官尺寸)和/或状况(年龄和总体健康)变动。在某些实施方案中，临床医生可以调节剂量并且调整施用途径以获得最佳治疗效果。
术语“受试者”、“患者”和“个体”在本文可互换使用，是指温血动物，例如哺乳动物。该术语包括但不限于家畜、啮齿动物(例如大鼠和小鼠)、灵长类动物和人。优选该术语是指人。
如本文中所使用的，术语“EC 50”是指半数最大效应浓度，即能引起50％最大效应的浓度。
术语“CD3抗体”、“抗CD3抗体”、“特异性结合至CD3的抗体”可互换地使用，是指能够特异性结合至人或其他哺乳动物CD3的抗体。在一些实施方案中，所述抗体能以小于1×10 -4、小于1×10 -5、小于1×10 -6、小于1×10 -7、小于1×10 -8、小于1×10 -9、小于1×10 -10或小于1×10 -11M的K D值特异性结合CD3抗原。术语“CD3e/g抗体”是指能够特异性结合至人或其他哺乳动物CD3e/g亚基的抗体。在一些实施方案中，CD3e/g抗原具有如SEQ ID NO:83和/或SEQ ID NO:84所示的序列。在一些实施方案中，所述抗体能以小于1×10 -4、 小于1×10 -5、小于1×10 -6、小于1×10 -7、小于1×10 -8、小于1×10 -9、小于1×10 -10或小于1×10 -11M的K D值特异性结合CD3e/g抗原。术语“CD3e/d抗体”是指能够特异性结合至人或其他哺乳动物CD3e/d亚基的抗体。在一些实施方案中，所述抗体能以小于1×10 -4、小于1×10 -5、小于1×10 -6、小于1×10 -7、小于1×10 -8、小于1×10 -9、小于1×10 -10或小于1×10 -11M的K D值特异性结合CD3e/d抗原。在一些优选的实施方案中，所述抗体能以小于1×10 -6、小于1×10 -7、小于1×10 -8、小于1×10 -9、小于1×10 -10或小于1×10 -11M的K D值特异性结合CD3e/g抗原和/或CD3e/d抗原。
术语“K D”为平衡解离常数，是表征抗体亲和力的常用参数。其计算公示为K D＝[抗体][抗原]/[抗体-抗原]，表示处于平衡状态时抗体-抗原复合物的解离程度，K D越大说明解离越多，代表抗体和抗原之间的亲和力越弱，K D越小说明解离越少，代表抗体和抗原间亲和力越强。在一些优选的实施方案中，前文所述抗体以小于4×10 -8M的K D值与人CD3结合。
如本文所用，在类似“显著激活总T细胞、上调CD25的表达和/或诱导IFN-γ的释放”的表述中，“显著”可以指具有至少约50％、至少约60％、至少约70％、至少约80％、至少约85％、至少约90％或至少约95％的细胞被激活；或者上调CD25的表达量至少约50％、至少约60％、至少约70％、至少约80％、至少约85％、至少约90％或至少约95％；或者诱导IFN-γ的释放量至少为约5000pg/mL、约6000pg/mL、约7000pg/mL、约8000pg/mL、约9000pg/mL、约10000pg/mL、约11000pg/mL、约12000pg/mL、约13000pg/mL、约14000pg/mL、约15000pg/mL、约16000pg/mL、约17000pg/mL、约18000pg/mL、约19000pg/mL或约20000pg/mL。在一些实施方案中，“显著”可以指激活总T细胞、上调CD25的表达量和/或诱导IFN-γ的释放量为阴性对照即同型对照或空白对照的至少约1.5倍、至少约2倍、至少约2.5倍、至少约3倍、至少约3.5倍、至少约4倍或更多倍。在一些优选的实施方案中，“显著”可以指激活总T细胞、上调CD25的表达量和/或诱导IFN-γ的释放量为阴性对照即同型对 照或空白对照的至少约3倍。
术语“同型对照(Isotype control)”是指一种与试验抗体保持相似特性但缺乏特异性靶标的抗体。在流式实验中，同型对照作为阴性对照，可以区分特定抗体信号与非特异性背景染色，能够排除干扰因素以确保实验结果的准确性。
抗体
本公开涉及特异性结合至CD3(例如CD3e，尤其是CD3e/d和/或CD3e/g)的抗体或其抗原结合片段。在一些实施方案中，所述CD3是人CD3和/或食蟹猴CD3。
通常，本公开的抗体或其抗原结合片段一般包含如本文所述的一个或多个CDR(例如，1、2、3、4、5或6个CDR)。
在一些实施方案中，重链CDR1包含与SEQ ID NO:1-4中的任一个具有至少80％同一性(例如至少85％、90％、95％、96％、97％、98％、99％或100％同一性)的序列或与其相差不多于总计3个(例如1个、2个和3个)氨基酸添加、置换和/或缺失的序列。在一些实施方案中，重链CDR2包含与SEQ ID NO:5-7和18-23中的任一个具有至少80％同一性(例如至少85％、90％、95％、96％、97％、98％、99％或100％同一性)的序列或与其相差不多于总计3个(例如1个、2个和3个)氨基酸添加、置换和/或缺失的序列。在一些实施方案中，重链CDR3包含与SEQ ID NO:8-11中的任一个具有至少80％同一性(例如至少85％、90％、95％、96％、97％、98％、99％或100％同一性)的序列或与其相差不多于总计3个(例如1个、2个和3个)氨基酸添加、置换和/或缺失的序列。
在一些实施方案中，轻链CDR1包含与SEQ ID NO:12-13和24-30中的任一个具有至少80％同一性(例如至少85％、90％、95％、96％、97％、98％、99％或100％同一性)的序列或与其相差不多于总计3个(例如1个、2个和3个)氨基酸添加、置换和/或缺失的序列。在一些实施方案中，轻链CDR2包含与SEQ ID NO:14-15中的任一个具有至少80％同一性(例如至少85％、90％、95％、96％、97％、 98％、99％或100％同一性)的序列或与其相差不多于总计3个(例如1个、2个和3个)氨基酸添加、置换和/或缺失的序列。在一些实施方案中，轻链CDR3包含与SEQ ID NO:16-17和31-32中的任一个具有至少80％同一性(例如至少85％、90％、95％、96％、97％、98％、99％或100％同一性)的序列或与其相差不多于总计3个(例如1个、2个和3个)氨基酸添加、置换和/或缺失的序列。
在一些实施方案中，所述置换为保守性置换。
在一些实施方案中，本公开的抗体或其抗原结合片段包含重链可变区和/或轻链可变区。在一些实施方案中，所述重链可变区包含选自SEQ ID NO:33-57的氨基酸序列或与前述序列中的任一个具有至少85％同一性(例如至少90％、95％、96％、97％、98％、99％或100％同一性)的序列。在一些实施方案中，所述轻链可变区包含选自SEQ ID NO:58-82的氨基酸序列或者与前述序列中的任一个具有至少85％同一性(例如至少90％、95％、96％、97％、98％、99％或100％同一性)的序列。
在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:33所示的重链可变区和如SEQ ID NO:58所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:33所示的重链可变区和如SEQ ID NO:59所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:33所示的重链可变区和如SEQ ID NO:60所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:33所示的重链可变区和如SEQ ID NO:61所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:34所示的重链可变区和如SEQ ID NO:58所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:34所示的重链可变区和如SEQ ID NO:59所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:34所示的重链可变区和如SEQ ID NO:60所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原 结合片段具有如SEQ ID NO:34所示的重链可变区和如SEQ ID NO:61所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:35所示的重链可变区和如SEQ ID NO:58所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:35所示的重链可变区和如SEQ ID NO:59所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:35所示的重链可变区和如SEQ ID NO:60所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:35所示的重链可变区和如SEQ ID NO:61所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:36所示的重链可变区和如SEQ ID NO:58所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:36所示的重链可变区和如SEQ ID NO:59所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:36所示的重链可变区和如SEQ ID NO:60所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:36所示的重链可变区和如SEQ ID NO:61所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:37所示的重链可变区和如SEQ ID NO:62所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:37所示的重链可变区和如SEQ ID NO:63所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:37所示的重链可变区和如SEQ ID NO:64所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:37所示的重链可变区和如SEQ ID NO:65所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:38所示的重链可变区和如SEQ ID NO:62所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:38所示的重链可变区和如SEQ ID NO:63所示的轻链可变区；在一些优选的实施方案中，所述 抗体或其抗原结合片段具有如SEQ ID NO:38所示的重链可变区和如SEQ ID NO:64所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:38所示的重链可变区和如SEQ ID NO:65所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:39所示的重链可变区和如SEQ ID NO:62所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:39所示的重链可变区和如SEQ ID NO:63所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:39所示的重链可变区和如SEQ ID NO:64所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:39所示的重链可变区和如SEQ ID NO:65所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:40所示的重链可变区和如SEQ ID NO:62所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:40所示的重链可变区和如SEQ ID NO:63所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:40所示的重链可变区和如SEQ ID NO:64所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:40所示的重链可变区和如SEQ ID NO:65所示的轻链可变区。
在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:41所示的重链可变区和如SEQ ID NO:66所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:41所示的重链可变区和如SEQ ID NO:67所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:41所示的重链可变区和如SEQ ID NO:68所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:41所示的重链可变区和如SEQ ID NO:69所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:42所示的重链可变区和如SEQ ID NO:66所示 的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:42所示的重链可变区和如SEQ ID NO:67所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:42所示的重链可变区和如SEQ ID NO:68所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:42所示的重链可变区和如SEQ ID NO:69所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:43所示的重链可变区和如SEQ ID NO:66所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:43所示的重链可变区和如SEQ ID NO:67所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:43所示的重链可变区和如SEQ ID NO:68所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:43所示的重链可变区和如SEQ ID NO:69所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:44所示的重链可变区和如SEQ ID NO:66所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:44所示的重链可变区和如SEQ ID NO:67所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:44所示的重链可变区和如SEQ ID NO:68所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:44所示的重链可变区和如SEQ ID NO:69所示的轻链可变区。
在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:45所示的重链可变区和如SEQ ID NO:70所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:45所示的重链可变区和如SEQ ID NO:71所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:45所示的重链可变区和如SEQ ID NO:72所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段 具有如SEQ ID NO:45所示的重链可变区和如SEQ ID NO:73所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:46所示的重链可变区和如SEQ ID NO:70所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:46所示的重链可变区和如SEQ ID NO:71所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:46所示的重链可变区和如SEQ ID NO:72所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:46所示的重链可变区和如SEQ ID NO:73所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:47所示的重链可变区和如SEQ ID NO:70所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:47所示的重链可变区和如SEQ ID NO:71所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:47所示的重链可变区和如SEQ ID NO:72所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:47所示的重链可变区和如SEQ ID NO:73所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:48所示的重链可变区和如SEQ ID NO:70所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:48所示的重链可变区和如SEQ ID NO:71所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:48所示的重链可变区和如SEQ ID NO:72所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:48所示的重链可变区和如SEQ ID NO:73所示的轻链可变区。
在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:49所示的重链可变区和如SEQ ID NO:74所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:49所示的重链可变区和如SEQ ID NO:75所示的轻链 可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:49所示的重链可变区和如SEQ ID NO:76所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:50所示的重链可变区和如SEQ ID NO:74所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:50所示的重链可变区和如SEQ ID NO:75所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:50所示的重链可变区和如SEQ ID NO:76所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:51所示的重链可变区和如SEQ ID NO:74所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:51所示的重链可变区和如SEQ ID NO:75所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:51所示的重链可变区和如SEQ ID NO:76所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:52所示的重链可变区和如SEQ ID NO:74所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:52所示的重链可变区和如SEQ ID NO:75所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:52所示的重链可变区和如SEQ ID NO:76所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:53所示的重链可变区和如SEQ ID NO:74所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:53所示的重链可变区和如SEQ ID NO:75所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:53所示的重链可变区和如SEQ ID NO:76所示的轻链可变区。
在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:54所示的重链可变区和如SEQ ID NO:77所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有 如SEQ ID NO:54所示的重链可变区和如SEQ ID NO:78所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:54所示的重链可变区和如SEQ ID NO:79所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:54所示的重链可变区和如SEQ ID NO:80所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:55所示的重链可变区和如SEQ ID NO:77所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:55所示的重链可变区和如SEQ ID NO:78所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:55所示的重链可变区和如SEQ ID NO:79所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:55所示的重链可变区和如SEQ ID NO:80所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:56所示的重链可变区和如SEQ ID NO:77所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:56所示的重链可变区和如SEQ ID NO:78所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:56所示的重链可变区和如SEQ ID NO:79所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:56所示的重链可变区和如SEQ ID NO:80所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:57所示的重链可变区和如SEQ ID NO:77所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:57所示的重链可变区和如SEQ ID NO:78所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:57所示的重链可变区和如SEQ ID NO:79所示的轻链可变区；在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:57所示的重链可变区和如SEQ ID NO:80所示的轻链可变区。
在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:85所示的重链可变区和如SEQ ID NO:82所示的轻链可变区。在一些优选的实施方案中，所述抗体或其抗原结合片段具有如SEQ ID NO:89所示的重链可变区和如SEQ ID NO:81所示的轻链可变区。
在一些实施方案中，本公开的抗体或其抗原结合片段(i)显著激活人总T细胞，上调CD25的表达和/或诱导IFN-γ的释放；和/或(ii)以小于4×10 -8(例如小于1×10 -8、小于1×10 -9、小于1×10 -10或小于1×10 -11)M的K D值与人CD3(例如CD3e，尤其是CD3e/d和/或CD3e/g)结合。
另外，与本文公开的抗体或其抗原结合片段竞争或交叉阻断本文公开的抗体与CD3的结合，或自身由本文公开的抗体交叉阻断而免于结合CD3的抗体，也可用于本发明中。在一些情况下，这些竞争性抗体、交叉阻断性抗体或经交叉阻断的抗体具有与本文公开的抗体或其抗原结合片段的表位交叉和/或重叠的表位。在一些情况下，这些竞争性抗体、交叉阻断性抗体或经交叉阻断的抗体具有与本文公开的抗体或其抗原结合片段的表位相同的表位。
竞争性抗体、交叉阻断性抗体和经交叉阻断的抗体可使用本领域中已知的任何适合方法来鉴定，包括竞争ELISA或
Figure PCTCN2022083004-appb-000008
测定，其中竞争性或交叉阻断性抗体与人CD3的结合阻止本文公开的抗体的结合，或反之亦然。
在一些实施方案中，竞争性抗体或经交叉阻断的抗体被竞争或交叉阻断达到大于80％、大于85％、大于90％或大于95％。在一些实施方案中，竞争性抗体、交叉阻断性抗体或经交叉阻断的抗体是嵌合的、完全人，或是人源化的。
此外，本文公开的抗体或其抗原结合片段还可用于构建一种嵌合抗原受体或基因修饰的细胞，所述嵌合抗原受体包含如前文所述的本公开的抗体或其抗原结合片段；所述的基因修饰的细胞优选为真核细胞，更优选为分离的人细胞，进一步更优选为免疫细胞如T细胞，或NK细胞如NK92细胞系。
术语“嵌合抗原受体”或“CAR”指：包含能够结合抗原的胞外域(胞外结合结构域)、铰链结构域、跨膜结构域(跨膜区)和使胞质信号传到结构域的多肽(即胞内信号域)。铰链结构域可以被认为是用于向细胞外抗原结合区提供柔性的一部分。胞内信号域指经由确定的信号传导途径通过产生第二信使而将信息传递到细胞内以调节细胞活性的蛋白质、或通过相应于此类信使而作为效应子发挥作用的蛋白质，产生可以促进CAR的细胞(例如CART细胞)的免疫效应子功能的信号。胞内信号域包含信号传导结构域，还可以包括源自共刺激分子的共刺激胞内结构域。
术语“免疫细胞”指可以引发免疫应答的细胞，“免疫细胞”及其语法上的其他形式可以指任何来源的免疫细胞。“免疫细胞”包括例如衍生自在骨髓中产生的造血干细胞(HSC)的白血细胞(白细胞)、淋巴细胞(T细胞、B细胞、自然杀伤(NK)细胞和骨髓来源的细胞(嗜中性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞、单核细胞、巨噬细胞、树突状细胞)。术语“免疫细胞”也可以是人或非人的。例如，免疫细胞可以是来自血液的，如自体的T细胞、异体T细胞、自体NK细胞、异体NK细胞，也可以来源自细胞系，如利用EBV病毒感染来制备NK细胞系，从胚胎干细胞和iPSC诱导分化来的NK细胞以及NK92细胞系等。
本文公开的抗体或其抗原结合片段还可用于构建一种抗体药物偶联物，其包含细胞毒性剂，以及如前文所述的本公开的抗体或其抗原结合片段。在一些实施方案中，本文所述的抗体或抗原结合片段可以与治疗剂缀合，包含抗体或其抗原结合片段的抗体-药物缀合物可以共价或非共价结合治疗剂。在一些实施方案中，治疗剂是细胞毒性剂或细胞生长抑制剂(例如，细胞松弛素B、短杆菌肽D、溴化乙锭、依米丁、丝裂霉素、依托泊苷、替诺泊苷、长春新碱、长春碱、秋水仙碱、阿霉素、柔红霉素、二羟基蒽醌、美登木素生物碱(如DM-1和DM-4)、二酮、丝氨酸、丝裂霉素D、1-脱氢睾丸激素、糖皮质激素、普鲁卡因、丁卡因、利多卡因、普萘洛尔、嘌呤霉素、表柔比星和环磷酰胺及其类似物)。
药物组合物
本公开提供了包含本公开的抗体或其抗原结合片段的药物组合物。这些药物组合物包含治疗有效量的抗体或其抗原结合片段和一种或多种另外的组分，如生理上可接受的载剂、赋形剂或稀释剂。在一些实施方案中，这些另外的组分可以包括缓冲液、碳水化合物、多元醇、氨基酸、螯合剂、稳定剂、和/或防腐剂等。
此外，药物组合物可以包括治疗有效量的抗体或其抗原结合片段和一种或多种其他治疗剂作为活性成分。对于组合疗法，上述抗体或其抗原结合片段、上述抗体药物偶联物等和/或另外的治疗或诊断剂可以各自作为单一药剂，在适合于执行预期治疗或诊断的任何时间范围内进行施用。因此，这些单一药剂可以基本上同时(即作为单一制剂或在数分钟或数小时内)或以按顺序连续施用。
在一些实施方案中，所述其他治疗剂可以包含一种或多种选自以下的抑制剂：B-Raf抑制剂、EGFR抑制剂、MEK抑制剂、ERK抑制剂、K-Ras抑制剂、c-Met抑制剂、间变性淋巴瘤激酶(ALK)抑制剂、磷脂酰肌醇3激酶(PI3K)抑制剂、Akt抑制剂、mTOR抑制剂、PI3K/mTOR双重抑制剂、Bruton酪氨酸激酶(BTK)抑制剂、以及异柠檬酸脱氢酶1(IDH1)和/或异柠檬酸脱氢酶2(IDH2)抑制剂。在一些实施方案中，所述其他治疗剂是吲哚胺2,3-二加氧酶-1(IDO1)抑制剂(例如依帕卡司他)。
在一些实施方案中，其他治疗剂可以包含一种或多种选自以下的抑制剂：HER3抑制剂、LSD 1抑制剂、MDM2抑制剂、BCL2抑制剂、CHK1抑制剂、活化hedgehog信号通路抑制剂、以及选择性降解雌激素受体的药物。
在一些实施方案中，所述其他治疗剂可以包含一种或多种选自以下的治疗剂：曲贝替定、纳布紫杉醇、Trebananib、帕唑帕尼、西地尼布、帕博西尼、依维莫司、氟嘧啶、IFL、瑞格非尼、Reolysin、力比泰、塞瑞替尼、索坦、替西罗莫司、阿西替尼、依维莫司、索 拉非尼、Votrient、帕唑帕尼、IMA-901、AGS-003、卡博替尼、长春氟宁、Hsp90抑制剂、Ad-GM-CSF、替莫唑胺、IL-2、IFNa、长春碱、酞胺哌啶、达卡巴嗪、环磷酰胺、来那度胺、氮杂胞苷、来那度胺、硼替佐米、氨柔比星、卡非佐米、普拉曲沙和enzastaurin。
在一些实施方案中，其他治疗剂可以包含一种或多种选自以下的治疗剂：佐剂、TLR激动剂、肿瘤坏死因子(TNF)α、IL-1、HMGB1、IL-10拮抗剂、IL-4拮抗剂、IL-13拮抗剂、IL-17拮抗剂、HVEM拮抗剂、ICOS激动剂、靶向CX3CL1的治疗剂、靶向CXCL9的治疗剂、靶向CXCL10的治疗剂、靶向CCL5的治疗剂、LFA-1激动剂、ICAM1激动剂和选择素激动剂。
药物组合物可依据已知的方法进行施用。施用途径为例如口服、经静脉内、腹膜内、脑内(实质内)、脑室内、肌内、眼内、动脉内、肝门内或病灶内途径注射；通过持续释放系统或通过植入装置施用。在某些实施方案中，组合物可以通过推注注射施用或通过输注连续地施用或通过植入装置来施用。
制备方法
本公开的抗体或其抗原结合片段可以通过本领域已知的用于抗体制备的方法(例如，通过化学合成或通过重组表达技术)进行制备。
治疗用抗体例如单克隆抗体可由多种技术和途径进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等。通过杂交瘤技术制备单抗仍然是目前治疗用单抗制备方法的主流。
传统的杂交瘤制备技术由Kohler和Milstein在40年前建立(Kohler和Milstein 1975，Nature 256:495)，现在已广泛应用于科研、诊断、治疗等许多相关的单克隆抗体的制备和生产之中。其基本方法虽然延用至今，但在许多方面都有所变化、改进和创新，包括不同品系动物如转基因动物的使用、电融合技术的引入、高效筛 选技术设备如ClonePix设备的应用等，使杂交瘤技术的应用更多样化和更加高效。从常规动物如小鼠等制备的单抗，可以通过常规分子生物学方法克隆抗体重链可变区和轻链可变区基因，可变区基因可以嫁接到人源抗体恒定区基因从而形成人鼠嵌合抗体(Cabilly等人的美国专利号4,816,567)，以大大降低人体使用时的免疫原性。更进一步，鼠源抗体可变区的CDR结构域可以嫁接到人源抗体架构上，从而使鼠源抗体成分降低到5％以下，大大增加了抗体在人体内使用的安全性。这一途径得到抗体称为人源化抗体，并且是目前抗体药物市场的主要产品(Winter的美国专利号5,225,539和Queen等人的美国专利号5,530,101、5,585,089、5,693,762和6,180,370)。
抗体的重组表达需要构建含有编码该抗体的核酸的表达载体。所述核酸的制备方法为本领域常规的制备方法，例如可以包括以下的步骤：通过基因克隆技术获得编码上述抗体的核酸分子，或者通过人工全序列合成的方法得到编码上述抗体的核酸分子。
一旦已经获得编码抗体的核酸，就可以通过重组DNA技术产生用于产生该抗体的载体。所述载体可通过本领域常规方法获得，例如，将本申请所述的核酸分子连接于适宜的表达载体上构建而成。所述的表达载体为本领域常规的各种载体，只要其能够容载前述核酸分子即可。所述载体包含真核细胞表达载体和/或原核细胞表达载体。
通过常规技术将表达载体转移到宿主细胞，并且然后通过常规技术培养转染的细胞以产生本公开的抗体。所述宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地复制，且所携带所述的核酸可被有效表达即可。所述宿主细胞可为原核细 胞(例如大肠杆菌细胞)和/或真核细胞(例如，HEK293细胞或CHO细胞)。
本发明的应用
本公开的抗体或其抗原结合片段、经分离的核酸、核酸载体、宿主细胞、抗体药物偶联物和药物组合物可用于治疗多种病症，包括例如各种形式的癌症、感染、自身免疫性或炎性病症和/或纤维化病症。本公开的抗体或其抗原结合片段、经分离的核酸、核酸载体、宿主细胞、抗体药物偶联物和药物组合物也可用于抑制免疫应答或活化T细胞。
在一些实施方案中，提供了本公开的抗体或其抗原结合片段、经分离的核酸、核酸载体、宿主细胞、抗体药物偶联物和药物组合物在制备用于抑制免疫应答或活化T细胞的药物或试剂盒中的用途。在一些实施方案中，提供了本公开的抗体或其抗原结合片段、经分离的核酸、核酸载体、宿主细胞、抗体药物偶联物和药物组合物在制备用于预防、治疗或缓解疾病的药物或试剂盒中的用途。在一些实施方案中，所述疾病为癌症或自身免疫疾病。
在一些实施方案中，所述癌症包括但不限于：黑素瘤、肾癌、前列腺癌、乳腺癌、结直肠癌、肺癌、鼻咽癌、口腔癌、肝癌、骨癌、胰腺癌、皮肤癌、头颈部癌、子宫癌、卵巢癌、直肠癌、胃癌、睾丸癌、输卵管癌、子宫内膜癌、子宫颈癌、阴道癌、外阴癌、霍奇金淋巴瘤、非霍奇金淋巴瘤、食道癌、小肠癌、大肠癌、内分泌系统癌症、甲状腺癌、甲状旁腺癌症、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病、膀胱癌、肾盂癌、中枢神经系统肿瘤、神经胶质瘤、脊髓轴肿瘤、脑干胶质瘤、垂体腺瘤、卡波济氏肉瘤、表皮样癌、鳞状细胞癌或T细胞淋巴瘤。
在一些实施方案中，所述自身免疫疾病包括但不限于：风湿关节炎、多发性硬化、干燥综合征、胰岛素依赖型糖尿病、自身免疫甲状腺炎、反应性关节炎、强直性脊柱炎、硬皮病、多发性肌炎、 皮肌炎、牛皮癣、韦格纳氏肉芽肿病、克罗恩氏病、溃疡性结肠炎、狼疮例如系统性红斑狼疮、动脉粥样硬化、慢性阻塞性肺病、肝硬化、肾移植纤维化、肾移植肾病或肺纤维化。
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。如果下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例
实施例1 CD3抗体的制备
(一)免疫原的制备
CD3e/g是一个可溶性配体，我们设计了CD3e/g蛋白与人恒定区的产物—CD3e/g重组蛋白作为免疫原，CD3e/g蛋白的具体序列见表1。
表1免疫原序列
Figure PCTCN2022083004-appb-000009
(二)杂交瘤细胞的制备和抗体筛选
采用上述免疫原免疫6～8周龄SJL小鼠(购于上海斯莱克公司)，小鼠在SPF条件下饲养。初次免疫时，免疫原用弗氏完全佐剂乳化后尾静脉端注射0.25毫升，即每只小鼠注射50微克免疫原。加强免疫时，免疫原用弗氏不完全佐剂乳化后尾静脉端注射0.25毫升，即每只小鼠注射50微克免疫原。初次免疫与第一次加强免疫之间间隔2周，以后每次加强免疫之间间隔3周。每次加强免疫1周后采血，用ELISA检测血清中免疫原的抗体效价和特异性，结果如图1和表2所示。由图1和表2可知，经CD3e/g重组蛋白免疫后的小鼠血清对免疫原均有不同程度的结合，呈现抗原抗体反应，其中最高稀释度在一千左右。其中空白对照为1％(w/w)BSA，其中批次指第二次加强免疫后第七天的小鼠血清，表中的数据为OD 450nm值。
表2通过ELISA检测的CD3e/g重组蛋白免疫后小鼠血清的抗体效价
Figure PCTCN2022083004-appb-000010
免疫步骤完成前，将所选择的每只小鼠最后一次免疫腹腔注射100微克CD3e/g重组蛋白，3天后处死小鼠，收集脾细胞。加入NH 4OH至终浓度1％(w/w)，裂解脾细胞中掺杂的红细胞，获得脾细胞悬液。用DMEM基础培养基1000转每分钟离心清洗细胞3次，然后按活细胞数目5:1比率与小鼠骨髓瘤细胞SP2/0(购自ATCC#CRL-1581)混合，采用高效电融合方法(参见METHODS IN ENZYMOLOGY,VOL.220)进行细胞融合。融合后的细胞稀释到含20％胎牛血清、1×HAT的DMEM培养基中，所述百分比为质量百分比。然后按1×10 5/200微升每孔加入到96孔细胞培养板中，放入5％CO 2、37℃培养箱中，所述百分比为体积百分比。14天后 分别用包被抗原CD3e/g的ELISA板筛选细胞融合板上清，将ELISA结果中OD值大于2的阳性克隆扩增到24孔板，在含10％(w/w)HT胎牛血清的DMEM(购自Invitrogen公司，目录号11995-065)在37℃，5％(v/v)CO 2条件下扩大培养。培养3天后取24孔板中扩大培养的培养液进行离心，收集上清液，对上清液进行抗体亚型分析，用ELISA确定对抗原的结合活性。
根据24孔板筛选结果，挑选ELISA检测中OD 450nm>2的杂交瘤细胞为符合条件的阳性克隆，选择符合条件的杂交瘤细胞用有限稀释法在96孔板进行亚克隆，在含10％(w/w)FBS的DMEM培养基(购自Invitrogen公司)中37℃，5％(v/v)CO 2条件下培养。亚克隆后10天用ELISA进行初步筛选，挑选单个阳性单克隆扩增到24孔板继续培养。3天后用ELISA确定抗原结合阳性并用CD3e/g受体配体结合实验评估生物活性(评估标准为ELISA实验中OD 450nm值>2)。
根据24孔板样品检测结果，挑选克隆，并于含10％(w/w)FBS的DMEM培养基(购自Invitrogen公司)中在37℃，5％(v/v)CO 2条件下将挑选出的克隆进行扩大培养，液氮冻存即得本公开杂交瘤细胞，并可用于后续的抗体生产和纯化。
实施例2 CD3抗体的生产和纯化
将实施例1所得的杂交瘤细胞接种到T-75细胞培养瓶并用生产培养基(Hybridoma serum free medium，购自Invitrogen公司)驯化传代3代。待其生长状态良好，接种细胞培养转瓶。每个2升的培养转瓶中加入500毫升生产培养基，接种细胞密度为1.0×10 5/毫升。将培养转瓶置于37℃培养箱中的转瓶机上，转速3rpm。连续旋转培养14天后，收集细胞培养液，过滤去除细胞，并用0.45微米的滤膜过滤至培养上清液澄清。澄清的培养上清液可马上进行纯化或-30℃冻存。
澄清的杂交瘤细胞的培养上清液(300mL)中的抗体用2mL蛋 白A柱(购自GE Healthcare)纯化。蛋白A柱先用平衡缓冲液(PBS磷酸缓冲液，pH7.2)平衡，然后将澄清的培养上清液上样到蛋白A柱，控制流速在3mL/分钟。上样完毕后用平衡缓冲液清洗蛋白A柱，平衡缓冲液的体积为4倍蛋白A柱柱床体积。用洗脱液(0.1M甘氨盐酸缓冲液，pH 2.5)洗脱结合在蛋白A柱上的CD3e/g抗体，用紫外检测器监测洗脱情况(A 280紫外吸收峰)。收集洗脱的抗体，加入10％1.0M Tris-HCl缓冲液中和pH，所述百分比为体积百分比，然后立即用PBS磷酸缓冲液透析过夜，第二天换液1次并继续透析3小时。收集透析后的CD3e/g抗体，用0.22微米的滤器进行无菌过滤，无菌保存，即得纯化的CD3e/g抗体。
实施例3 CD3抗体的抗原结合活性
采用酶联免疫吸附实验(ELISA)检测抗原抗体结合位点。
对实施例2所得的纯化的CD3e/g抗体进行与CD3e/g(序列见表1)和CD3e/d蛋白(购自Sinobiological，CAT：CT038-H2508H-B)进行结合反应。
将人CD3e/g蛋白、食蟹猴CD3e/g蛋白(序列见表1)或人CD3e/d蛋白用PBS稀释到终浓度5.0μg/mL，然后以100μl每孔加到96孔ELISA板。用塑料膜封好4℃孵育过夜，第二天用洗板液[PBS+0.01％(v/v)Tween 20]洗板2次，加入封闭液[PBS+0.01％(v/v)Tween 20+1％(w/w)BSA]室温封闭2小时。倒掉封闭液，加入实施例2所得的纯化的CD3e/g抗体100μl每孔。37℃孵育2小时后，用洗板液[PBS+0.01％(v/v)Tween20]洗板3次。加入HRP(辣根过氧化物酶)标记的二抗(购自Sigma)，37℃孵育2小时后，用洗板液[PBS+0.01％(v/v)Tween20]洗板3次。加入TMB底物100μl每孔，室温孵育30分钟后，加入终止液(1.0N HCl)100μl每孔。用ELISA读板机(SpectraMax 384plus，购自Molecular Device)读取A 450nm数值，图2和表3显示了ELISA检测CD3e/g抗体与hCD3e/g的结合活性，图3和表4显示了ELISA检测CD3e/g抗体与cyno CD3e/g的结合活性，其中IgG对照为小鼠IgG，表中的 数据为OD 450nm值。其中，HIT3a购自BD pharmigen，目录号为555336；SP34购自BD pharmigen，目录号为551916。OKT3购自Biolegend。
由图2和图3、表3和表4中可以看出，本公开的抗体可以与人CD3e/g(hCD3e/g)结合，并且本公开的抗体与人CD3e/g的结合亲和力较OKT3更佳。同时本公开的抗体可以与食蟹猴CD3(cyno CD3e/g)结合，而OKT3不具有与食蟹猴CD3e/g的结合亲和力或者无显著结合。
由图4和表5可以看出，本公开的抗体还可以与人CD3e/d(hCD3e/d)结合，并且本公开的抗体与人CD3e/d的结合亲和力较SP34更佳。
表3通过ELISA检测的CD3e/g抗体与hCD3e/g的结合
Figure PCTCN2022083004-appb-000011
表4通过ELISA检测的CD3e/g抗体与cyno CD3e/g的结合
Figure PCTCN2022083004-appb-000012
Figure PCTCN2022083004-appb-000013
表5通过ELISA检测的CD3e/g抗体与hCD3e/d的结合
Figure PCTCN2022083004-appb-000014
实施例4抗体的人源化改造
应用鼠源抗人CD3抗体的重链和轻链可变区(VH和VL)序列来检索IMGT数据库中相应的人类抗体重轻链可变区序列。应用MOE软件，通过比对鼠源抗体重链和轻链可变区和检索出的人类抗体重轻链可变区基因序列，分别挑选与鼠源抗体同源性高的重链和轻链可变区种系基因作为模板，将鼠源抗体的CDR分别接枝到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。根据对选择的人抗体重链可变区和轻链可变区的结构同鼠源抗体结构分析以保证原有鼠源抗体的亲和力，将骨架序列中关键氨基酸回复突变为鼠源抗体对应的氨基酸，即得到人源化抗CD3单克隆抗体。其中CDR区氨基酸残基的确定由Kabat编号系统 确定并注释。
上述鼠源抗体的轻重链可变区与人源抗体的轻重链恒定区连接后形成嵌合抗体，mAb001抗体对应的嵌合抗体命名为CAb001，其他抗体类推。
3.1鼠抗mAb001的人源化
3.1.1 mAb001人源化方案一
(1)mAb001人源化构架选择
鼠源抗体mAb001的人源化轻链模板为IGKV1-39 IGKV1-39*01 X59315 V-KAPPA，人源化重链模板为IGHV1-8 IGHV1-8*01 M99637 VH，经人源化后得到人源化抗体hAb001，人源化可变区序列如下：
>hAb001 VH1-CDR接枝(SEQ ID NO:33)
Figure PCTCN2022083004-appb-000015
>hAb001 VL1-CDR接枝(SEQ ID NO:58)
Figure PCTCN2022083004-appb-000016
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，下划线为CDR序列，无下划线为FR序列，
(2)hAb001回复突变设计如下表：
Figure PCTCN2022083004-appb-000017
注：如Q3V表示依照氨基酸序列自然顺序编号，将3位Q突变回V。接枝的代表鼠抗体CDR植入人种系FR区序列。
(3)hAb001人源化序列组合如下表：
Figure PCTCN2022083004-appb-000018
注：该表表示各种突变组合所得的序列。如hAb001-6表示，在人源化的抗体hAb001-6上有轻链mAb001VL_hum2、重链mAb001VH_hum2的四个回复突变。其它类推。
(4)hAb001人源化具体序列如下：
>mAb001VL_hum1(SEQ ID NO:58)
Figure PCTCN2022083004-appb-000019
>mAb001VL_hum2(SEQ ID NO:59)
Figure PCTCN2022083004-appb-000020
>mAb001VL_hum3(SEQ ID NO:60)
Figure PCTCN2022083004-appb-000021
>mAb001VL_hum4(SEQ ID NO:61)
Figure PCTCN2022083004-appb-000022
>mAb001VH_hum1(SEQ ID NO:33)
Figure PCTCN2022083004-appb-000023
Figure PCTCN2022083004-appb-000024
>mAb001VH_hum2(SEQ ID NO:34)
Figure PCTCN2022083004-appb-000025
>mAb001VH_hum3(SEQ ID NO:35)
Figure PCTCN2022083004-appb-000026
>mAb001VH_hum4(SEQ ID NO:36)
Figure PCTCN2022083004-appb-000027
3.1.2 mAb001人源化方案二
(1)mAb001人源化构架选择
鼠源抗体mAb001的人源化轻链模板为IGKV4-1 IGKV4-1*01 Z0023 V-KAPPA，人源化重链模板为IGHV1-3 IGHV1-3*01 X62109 VH，经人源化后得到人源化抗体hAb001，人源化可变区序列如下：
>hAb001 VH2-CDR接枝(SEQ ID NO:37)
Figure PCTCN2022083004-appb-000028
>hAb001 VL2-CDR接枝(SEQ ID NO:62)
Figure PCTCN2022083004-appb-000029
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，下划线为CDR序列，无下划线为FR序列，
(2)hAb001回复突变设计如下表：
Figure PCTCN2022083004-appb-000030
Figure PCTCN2022083004-appb-000031
注：如S69T表示依照氨基酸序列自然顺序编号，将69位S突变回T。接枝的代表鼠抗体CDR植入人种系FR区序列。
(3)hAb001人源化序列组合如下表：
Figure PCTCN2022083004-appb-000032
注：该表表示各种突变组合所得的序列。如hAb001-22表示，在人源化的抗体hAb001-22上有轻链mAb001VL_hum6、重链mAb001VH_hum6的三个回复突变。其它类推。
(4)hAb001人源化具体序列如下：
>mAb001VL_hum5(SEQ ID NO:62)
Figure PCTCN2022083004-appb-000033
>mAb001VL_hum6(SEQ ID NO:63)
Figure PCTCN2022083004-appb-000034
>mAb001VL_hum7(SEQ ID NO:64)
Figure PCTCN2022083004-appb-000035
Figure PCTCN2022083004-appb-000036
>mAb001VL_hum8(SEQ ID NO:65)
Figure PCTCN2022083004-appb-000037
>mAb001VH_hum5(SEQ ID NO:37)
Figure PCTCN2022083004-appb-000038
>mAb001VH_hum6(SEQ ID NO:38)
Figure PCTCN2022083004-appb-000039
>mAb001 VH_hum7(SEQ ID NO:39)
Figure PCTCN2022083004-appb-000040
>mAb001VH_hum8(SEQ ID NO:40)
Figure PCTCN2022083004-appb-000041
3.2鼠抗mAb007的人源化
3.2.1鼠抗mAb007的人源化方案一
(1)mAb007人源化构架选择
鼠源抗体mAb007的人源化轻链模板为IGKV1-27 IGKV1-27*01 X63398 V-KAPPA F，人源化重链模板为IGHV1-8 IGHV1-8*01 M99637 VH F，经人源化后得到人源化抗体hAb007，人源化可变区序列如下：
>hAb007 VH1-CDR接枝(SEQ ID NO:41)
Figure PCTCN2022083004-appb-000042
Figure PCTCN2022083004-appb-000043
>hAb007 VL1-CDR接枝(SEQ ID NO:66)
Figure PCTCN2022083004-appb-000044
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，下划线为CDR序列，无下划线为FR序列，
(2)hAb007回复突变设计如下表：
Figure PCTCN2022083004-appb-000045
注：如A49S表示依照氨基酸序列自然顺序编号，将49位A突变回S。接枝的代表鼠抗体CDR植入人种系FR区序列。
(3)hAb007人源化序列组合如下表：
Figure PCTCN2022083004-appb-000046
Figure PCTCN2022083004-appb-000047
注：该表表示各种突变组合所得的序列。如hAb007-5表示，在人源化的抗体hAb007-5上有轻链mAb007VL_hum1、重链mAb007VH_hum2的四个回复突变。其它类推。
(4)hAb007人源化具体序列如下：
>mAb007VL_hum1(SEQ ID NO:66)
Figure PCTCN2022083004-appb-000048
>mAb007VL_hum2(SEQ ID NO:67)
Figure PCTCN2022083004-appb-000049
>mAb007VL_hum3(SEQ ID NO:68)
Figure PCTCN2022083004-appb-000050
>mAb007VL_hum4(SEQ ID NO:69)
Figure PCTCN2022083004-appb-000051
>mAb007VH_hum1(SEQ ID NO:41)
Figure PCTCN2022083004-appb-000052
>mAb007VH_hum2(SEQ ID NO:42)
Figure PCTCN2022083004-appb-000053
>mAb007VH_hum3(SEQ ID NO:43)
Figure PCTCN2022083004-appb-000054
>mAb007VH_hum4(SEQ ID NO:44)
Figure PCTCN2022083004-appb-000055
3.2.2鼠抗mAb007的人源化方案二
(1)mAb007人源化构架选择
鼠源抗体mAb007的人源化轻链模板为IGKV4-1 IGKV4-1*01 Z0023 V-KAPPA，人源化重链模板为IGHV1-3 IGHV1-3*01 X62109 VH，经人源化后得到人源化抗体hAb007，人源化可变区序列如下：
>hAb007 VH2-CDR接枝(SEQ ID NO:45)
Figure PCTCN2022083004-appb-000056
>hAb007 VL2-CDR接枝(SEQ ID NO:70)
Figure PCTCN2022083004-appb-000057
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，下划线为CDR序列，无下划线为FR序列，
(2)hAb007回复突变设计如下表：
Figure PCTCN2022083004-appb-000058
Figure PCTCN2022083004-appb-000059
注：如S69T表示依照氨基酸序列自然顺序编号，将69位S突变回T。接枝的代表鼠抗体CDR植入人种系FR区序列。
(3)hAb007人源化序列组合如下表：
Figure PCTCN2022083004-appb-000060
注：该表表示各种突变组合所得的序列。如hAb007-22表示，在人源化的抗体hAb007-22上有轻链mAb007VL_hum6、重链mAb007VH_hum6的三个回复突变。其它类推。
(4)hAb007人源化具体序列如下：
>mAb007VL_hum5(SEQ ID NO:70)
Figure PCTCN2022083004-appb-000061
>mAb007VL_hum6(SEQ ID NO:71)
Figure PCTCN2022083004-appb-000062
>mAb007VL_hum7(SEQ ID NO:72)
Figure PCTCN2022083004-appb-000063
>mAb007VL_hum8(SEQ ID NO:73)
Figure PCTCN2022083004-appb-000064
Figure PCTCN2022083004-appb-000065
>mAb007VH_hum5(SEQ ID NO:45)
Figure PCTCN2022083004-appb-000066
>mAb007VH_hum6(SEQ ID NO:46)
Figure PCTCN2022083004-appb-000067
>mAb007VH_hum7(SEQ ID NO:47)
Figure PCTCN2022083004-appb-000068
>mAb007VH_hum8(SEQ ID NO:48)
Figure PCTCN2022083004-appb-000069
3.3鼠抗mAb008的人源化
3.3.1鼠抗mAb008的人源化方案一
(1)mAb008人源化构架选择
鼠源抗体mAb008的人源化轻链模板为IIGKV2-30 IGKV2-30*01 X63403 V-KAPPA F，人源化重链模板为IGHV1-8 IGHV1-8*01 M99637 VH F，经人源化后得到人源化抗体hAb008，人源化可变区序列如下：
>hAb008 VH-CDR接枝(SEQ ID NO:49)
Figure PCTCN2022083004-appb-000070
>hAb008 VL-CDR接枝(SEQ ID NO:74)
Figure PCTCN2022083004-appb-000071
Figure PCTCN2022083004-appb-000072
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，下划线为CDR序列，无下划线为FR序列，
(2)hAb008回复突变设计如下表：
Figure PCTCN2022083004-appb-000073
注：如F41L表示依照氨基酸序列自然顺序编号，将41位F突变回L。接枝的代表鼠抗体CDR植入人种系FR区序列。
(3)hAb008人源化序列组合如下表：
Figure PCTCN2022083004-appb-000074
注：该表表示各种突变组合所得的序列。如hAb008-5表示，在人源化的抗体hAb008-5上有轻链mAb008VL_hum2、重链mAb007VH_hum2的三个回复突变。其它类推。
(4)hAb008人源化具体序列如下：
>mAb008VL_hum1(SEQ ID NO:74)
Figure PCTCN2022083004-appb-000075
>mAb008VL_hum2(SEQ ID NO:75)
Figure PCTCN2022083004-appb-000076
>mAb008VL_hum3(SEQ ID NO:76)
Figure PCTCN2022083004-appb-000077
>mAb008VH_hum1(SEQ ID NO:49)
Figure PCTCN2022083004-appb-000078
>mAb008VH_hum2(SEQ ID NO:50)
Figure PCTCN2022083004-appb-000079
>mAb008VH_hum3(SEQ ID NO:51)
Figure PCTCN2022083004-appb-000080
>mAb008VH_hum4(SEQ ID NO:52)
Figure PCTCN2022083004-appb-000081
>mAb008VH_hum5(SEQ ID NO:53)
Figure PCTCN2022083004-appb-000082
3.3.2杂交瘤鼠抗mAb008的人源化方案二
(1)mAb008人源化构架选择
鼠源抗体mAb008的人源化轻链模板为IIGKV2-30 IGKV2-30*01 X63403 V-KAPPA F，人源化重链模板为IGHV1-3 IGHV1-3*01 X62109 VH，经人源化后得到人源化抗体hAb008，人源化可变区序列如下：
>hAb008 VH2-CDR接枝(SEQ ID NO:54)
Figure PCTCN2022083004-appb-000083
>hAb008 VL2-CDR接枝(SEQ ID NO:77)
Figure PCTCN2022083004-appb-000084
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，下划线为CDR序列，无下划线为FR序列，
(2)hAb008回复突变设计如下表：
Figure PCTCN2022083004-appb-000085
注：如Y54Q表示依照氨基酸序列自然顺序编号，将54位Y突变回Q。接枝的代表鼠抗体CDR植入人种系FR区序列。
(3)hAb008人源化序列组合如下表：
Figure PCTCN2022083004-appb-000086
Figure PCTCN2022083004-appb-000087
注：该表表示各种突变组合所得的序列。如hAb008-22表示，在人源化的抗体hAb008-22上有轻链mAb008VL_hum6、重链mAb007VH_hum7的六个回复突变。其它类推。
(4)hAb008人源化具体序列如下：
>mAb008VL_hum4(SEQ ID NO:77)
Figure PCTCN2022083004-appb-000088
>mAb008VL_hum5(SEQ ID NO:78)
Figure PCTCN2022083004-appb-000089
>mAb008VL_hum6(SEQ ID NO:79)
Figure PCTCN2022083004-appb-000090
>mAb008VL_hum7(SEQ ID NO:80)
Figure PCTCN2022083004-appb-000091
>mAb008VH_hum6(SEQ ID NO:54)
Figure PCTCN2022083004-appb-000092
>mAb008VH_hum7(SEQ ID NO:55)
Figure PCTCN2022083004-appb-000093
Figure PCTCN2022083004-appb-000094
>mAb008VH_hum8(SEQ ID NO:56)
Figure PCTCN2022083004-appb-000095
>mAb008VH_hum9(SEQ ID NO:57)
Figure PCTCN2022083004-appb-000096
3.3.3杂交瘤鼠抗mAb008嵌合抗体的热点突变
对抗体mAb008热点进行点突变。轻链mAb008抗体有1个可突变位点，轻链的将34位G突变回A，产生热点突变嵌合抗体CAb008DA。
>mAb008VL DA(SEQ ID NO:81)
Figure PCTCN2022083004-appb-000097
>mAb008VH或CAb008VH(SEQ ID NO:89)
Figure PCTCN2022083004-appb-000098
>mAb008VL或CAb008VL(SEQ ID NO:90)
Figure PCTCN2022083004-appb-000099
3.4杂交瘤鼠抗mAb009嵌合抗体的热点突变
对抗体mAb009热点进行点突变。轻链mAb009抗体有2个可突变位点，轻链的将32位G突变回A，产生热点突变嵌合抗体CAb009DADG
>mAb009VL DADG(SEQ ID NO:82)
Figure PCTCN2022083004-appb-000100
Figure PCTCN2022083004-appb-000101
>mAb009VH或CAb009VH(SEQ ID NO:85)
Figure PCTCN2022083004-appb-000102
>mAb009VL或CAb009VL(SEQ ID NO:86)
Figure PCTCN2022083004-appb-000103
本公开所测试抗体的序列小结如下表：
表6测试抗体的序列编号
Figure PCTCN2022083004-appb-000104
Figure PCTCN2022083004-appb-000105
Figure PCTCN2022083004-appb-000106
>CAb001 VH(SEQ ID NO:87)
Figure PCTCN2022083004-appb-000107
>CAb001 VL(SEQ ID NO:88)
Figure PCTCN2022083004-appb-000108
>CAb007 VH(SEQ ID NO:91)
Figure PCTCN2022083004-appb-000109
>CAb007 VL(SEQ ID NO:92)
Figure PCTCN2022083004-appb-000110
实施例5 CD3抗体的人源化或热点突变变体对Jurkat细胞的结合
将Jurkat细胞在T-175细胞培养瓶中扩大培养至3×10 6/ml，离心吸尽培养基，用PBS缓冲液(购自Invitrogen)洗涤2次，进行细胞计数后将细胞用PBS缓冲液稀释至2×10 6细胞每毫升，加入1％BSA封闭液，所述百分比为质量百分比，冰上孵育30分钟，然后用PBS缓冲液离心洗涤2次。将收集的细胞用FACS缓冲液(PBS+1％BSA，所述百分比为质量百分比)悬浮至2×10 6细胞/mL，按每孔100微升加入到96孔FACS反应板中，加入实施例4所得的纯化的CD3抗体待测样品，每孔100微升，4℃孵育2小时。用FACS缓冲液离心洗涤2次，加入每孔100微升荧光(Alexa 488)标记的二抗(购自Jackson)，4℃孵育1小时。用FACS缓冲液离心洗涤3次，后用100微升FACS缓冲液悬浮细胞，用BD FACS LSRFortessa(购自BD公司)检测和分析结果。结果如图5所示，其中IgG对照为人IgG1(hIgG)。
实施例6抗体对人总T细胞的激活的影响
利用人总T细胞的分离试剂盒(EasySep TMDirect Human T Cell Isolation Kit,购自Stemcell,目录号19661)提取人总T细胞。用完全培养基洗涤3次。将T细胞与稀释好的CD3抗体混合均匀于96孔板后37℃培养3天。3天后收集培养上清，用ELISA试剂盒(IFN gamma Human Uncoated ELISA Kit,购自Invitrogen公司，目录号88-7316-88)检测培养上清的人IFN-γ水平。培养细胞用PBS洗涤一遍后加入标记的抗人CD25流式抗体(PE-Cy7 Mouse Anti-Human CD25，购自BD Pharmingen,目录号557741)避光染色30分钟后用PBS洗涤两次。使用FACS(BD FACS LSRFortessa购自BD)对人T细胞的CD25的表达进行分析。
结果如图6和图7所示，所述CD3抗体的人源化变体及热点突变变体均能显著激活人总T细胞，具体表现为CD25的上调表达以 及IFN-γ的释放。
实施例7抗体对人CD3e/g抗原的亲和力测定
使用Biacore 8K(GE)系统通过动力学结合方法确定CD3抗体结合人CD3e/g抗原(购于ACRO，目录号CDG-H52W5)的平衡解离常数。通过捕获法，首先用NHS(N-羟基琥珀酰亚胺)和EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)活化CM5芯片的羧基化的葡聚糖表面，后加入抗人IgG(Fc)抗体，其自由氨基可与活化的羧基形成酰胺键，从而固定于芯片表面。抗人IgG(Fc)抗体可结合CD3抗体的Fc端捕获抗体，再加入一系列浓度的抗原，得到抗体与抗原的结合和解离曲线，通过Biacore Evaluation软件计算获得相应的动力学常数，具体见下表。
表7：通过Biacore检测的CD3e/g抗体与hCD3e/g的K D
Figure PCTCN2022083004-appb-000111
Figure PCTCN2022083004-appb-000112
应理解，在阅读了本公开的上述内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (26)

  1. 一种特异性结合至CD3的抗体或其抗原结合片段，其重链包含CDR1、CDR2和CDR3，其中：
    (a)重链CDR1包含与SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3或SEQ ID NO:4具有至少80％同一性的序列；
    (b)重链CDR2包含与SEQ ID NO:18、SEQ ID NO:6或SEQ ID NO:22具有至少80％同一性的序列；以及
    (c)重链CDR3包含与SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10或SEQ ID NO:11具有至少80％同一性的序列。
  2. 如权利要求1所述的抗体或其抗原结合片段，其包含：
    (a)重链CDR1，其包含选自以下的序列：SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4或者与SEQ ID NO:1-4中的任一个相比具有1个氨基酸添加、置换和/或缺失的序列；
    (b)重链CDR2，其包含选自以下的序列：WTYPGX 1X 2X 3IKYNEKFKG(SEQ ID NO:5)，SEQ ID NO:6或与SEQ ID NO:6相差不多于总计3个氨基酸添加、置换和/或缺失的序列，或者YINPFX 4X 5YTKYNQKFKD(SEQ ID NO:7)；其中X 1、X 2和X 3各自独立地为N或Q，X 4是N或S，X 5是S或D；
    (c)重链CDR3，其包含选自以下的序列：SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11或者与SEQ ID NO:8-11中的任一个相差不多于总计2个氨基酸添加、置换和/或缺失的序列；
    (d)轻链CDR1，其包含选自以下的序列：KSSQSLLNX 6RTRKNYLA(SEQ ID NO:12)或者KSSQSLLDX 7DX 8KTYLN(SEQ ID NO:13)；其中X 6是N、Q或S，X 7是S、A或G，X 8是G或A；
    (e)轻链CDR2，其包含选自以下的序列：SEQ ID NO:14或SEQ ID NO:15；以及
    (f)轻链CDR3，其包含选自以下的序列：KQSX 9X 10LRT(SEQ ID NO:16)或SEQ ID NO:17；其中X 9是Y或F，以及X 10是T或I。
  3. 如权利要求1所述的抗体或其抗原结合片段，其包含：
    (1)重链CDR1，其包含以下序列或由其组成：SEQ ID NO:1；重链CDR2，其包含以下序列或由其组成：WTYPGX 1X 2X 3IKYNEKFKG(SEQ ID NO:5)，其中X 1、X 2和X 3各自独立地为N或Q；重链CDR3，其包含以下序列或由其组成：SEQ ID NO:8；轻链CDR1，其包含以下序列或由其组成：KSSQSLLNX 6RTRKNYLA(SEQ ID NO:12)，其中X 6是N或Q；轻链CDR2，其包含以下序列或由其组成：SEQ ID NO:14；以及轻链CDR3，其包含以下序列或由其组成：SEQ ID NO:31；或
    (2)重链CDR1，其包含以下序列或由其组成：SEQ ID NO:2；重链CDR2，其包含以下序列或由其组成：SEQ ID NO:6；重链CDR3，其包含以下序列或由其组成：SEQ ID NO:9；轻链CDR1，其包含以下序列或由其组成：SEQ ID NO:26；轻链CDR2，其包含以下序列或由其组成：SEQ ID NO:14；以及轻链CDR3，其包含以下序列或由其组成：SEQ ID NO:32；或
    (3)重链CDR1，其包含以下序列或由其组成：SEQ ID NO:3；重链CDR2，其包含以下序列或由其组成：SEQ ID NO:22；重链CDR3，其包含以下序列或由其组成：SEQ ID NO:10；轻链CDR1，其包含以下序列或由其组成：KSSQSLLDX 7DX 8KTYLN(SEQ ID NO:13)，其中X 7是S，X 8是G或A；轻链CDR2，其包含以下序列或由其组成：SEQ ID NO:15；以及轻链CDR3，其包含以下序列或由其组成：SEQ ID NO:17；或
    (4)重链CDR1，其包含以下序列或由其组成：SEQ ID NO:4；重链CDR2，其包含以下序列或由其组成：SEQ ID NO:23；重链CDR3，其包含以下序列或由其组成：SEQ ID NO:11；轻链CDR1，其包含以下序列或由其组成：KSSQSLLDX 7DX 8KTYLN(SEQ ID NO:13)，其中X 7是G或A，X 8是G；轻链CDR2，其包含以下序列或由其组成：SEQ ID NO:15；以及轻链CDR3，其包含以下序列或由其组成：SEQ ID NO:17。
  4. 如权利要求2所述的抗体或其抗原结合片段，其特征在于：
    所述重链CDR2包含选自以下的序列：SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:6、SEQ ID NO:22或SEQ ID NO:23；和/或
    所述轻链CDR1包含选自以下的序列：SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29或SEQ ID NO:30；和/或
    所述轻链CDR3包含选自以下的序列：SEQ ID NO:31、SEQ ID NO:32或SEQ ID NO:17。
  5. 如权利要求1-4任一项所述的抗体或其抗原结合片段，其包含以下(1)至(12)任一项所述的CDR：
    (1)重链CDR1，其包含以下序列或由其组成：SEQ ID NO:1；重链CDR2，其包含以下序列或由其组成：SEQ ID NO:18；重链CDR3，其包含以下序列或由其组成：SEQ ID NO:8；轻链CDR1，其包含以下序列或由其组成：SEQ ID NO:24；轻链CDR2，其包含以下序列或由其组成：SEQ ID NO:14；以及轻链CDR3，其包含以下序列或由其组成：SEQ ID NO:31；或
    (2)重链CDR1，其包含以下序列或由其组成：SEQ ID NO:1；重链CDR2，其包含以下序列或由其组成：SEQ ID NO:18；重链CDR3，其包含以下序列或由其组成：SEQ ID NO:8；轻链CDR1，其包含以下序列或由其组成：SEQ ID NO:25；轻链CDR2，其包含以下序列或由其组成：SEQ ID NO:14；以及轻链CDR3，其包含以下序列或由其组成：SEQ ID NO:31；或
    (3)重链CDR1，其包含以下序列或由其组成：SEQ ID NO:1；重链CDR2，其包含以下序列或由其组成：SEQ ID NO:19；重链CDR3，其包含以下序列或由其组成：SEQ ID NO:8；轻链CDR1，其包含以下序列或由其组成：SEQ ID NO:24；轻链CDR2，其包含以下序列或由其组成：SEQ ID NO:14；以及轻链CDR3，其包含以下序列或由其组成：SEQ ID NO:31；或
    (4)重链CDR1，其包含以下序列或由其组成：SEQ ID NO:1；重链CDR2，其包含以下序列或由其组成：SEQ ID NO:19；重链CDR3，其包含以下序列或由其组成：SEQ ID NO:8；轻链CDR1，其包含以下序列或由其组成：SEQ ID NO:25；轻链CDR2，其包含以下序列或由其组成：SEQ ID NO:14；以及轻链CDR3，其包含以下序列或由其组成：SEQ ID NO:31；或
    (5)重链CDR1，其包含以下序列或由其组成：SEQ ID NO:1；重链CDR2，其包含以下序列或由其组成：SEQ ID NO:20；重链CDR3，其包含以下序列或由其组成：SEQ ID NO:8；轻链CDR1，其包含以下序列或由其组成：SEQ ID NO:24；轻链CDR2，其包含以下序列或由其组成：SEQ ID NO:14；以及轻链CDR3，其包含以下序列或由其组成：SEQ ID NO:31；或
    (6)重链CDR1，其包含以下序列或由其组成：SEQ ID NO:1；重链CDR2，其包含以下序列或由其组成：SEQ ID NO:20；重链CDR3，其包含以下序列或由其组成：SEQ ID NO:8；轻链CDR1，其包含以下序列或由其组成：SEQ ID NO:25；轻链CDR2，其包含以下序列或由其组成：SEQ ID NO:14；以及轻链CDR3，其包含以下序列或由其组成：SEQ ID NO:31；或
    (7)重链CDR1，其包含以下序列或由其组成：SEQ ID NO:1；重链CDR2，其包含以下序列或由其组成：SEQ ID NO:21；重链CDR3，其包含以下序列或由其组成：SEQ ID NO:8；轻链CDR1，其包含以下序列或由其组成：SEQ ID NO:24；轻链CDR2，其包含以下序列或由其组成：SEQ ID NO:14；以及轻链CDR3，其包含以下序列或由其组成：SEQ ID NO:31；或
    (8)重链CDR1，其包含以下序列或由其组成：SEQ ID NO:1；重链CDR2，其包含以下序列或由其组成：SEQ ID NO:21；重链CDR3，其包含以下序列或由其组成：SEQ ID NO:8；轻链CDR1，其包含以下序列或由其组成：SEQ ID NO:25；轻链CDR2，其包含以下序列或由其组成：SEQ ID NO:14；以及轻链CDR3，其包含以 下序列或由其组成：SEQ ID NO:31；或
    (9)重链CDR1，其包含以下序列或由其组成：SEQ ID NO:3；重链CDR2，其包含以下序列或由其组成：SEQ ID NO:22；重链CDR3，其包含以下序列或由其组成：SEQ ID NO:10；轻链CDR1，其包含以下序列或由其组成：SEQ ID NO:27；轻链CDR2，其包含以下序列或由其组成：SEQ ID NO:15；以及轻链CDR3，其包含以下序列或由其组成：SEQ ID NO:17；或
    (10)重链CDR1，其包含以下序列或由其组成：SEQ ID NO:3；重链CDR2，其包含以下序列或由其组成：SEQ ID NO:22；重链CDR3，其包含以下序列或由其组成：SEQ ID NO:10；轻链CDR1，其包含以下序列或由其组成：SEQ ID NO:28；轻链CDR2，其包含以下序列或由其组成：SEQ ID NO:15；以及轻链CDR3，其包含以下序列或由其组成：SEQ ID NO:17；或
    (11)重链CDR1，其包含以下序列或由其组成：SEQ ID NO:4；重链CDR2，其包含以下序列或由其组成：SEQ ID NO:23；重链CDR3，其包含以下序列或由其组成：SEQ ID NO:11；轻链CDR1，其包含以下序列或由其组成：SEQ ID NO:30；轻链CDR2，其包含以下序列或由其组成：SEQ ID NO:15；以及轻链CDR3，其包含以下序列或由其组成：SEQ ID NO:17；或
    (12)重链CDR1，其包含以下序列或由其组成：SEQ ID NO:4；重链CDR2，其包含以下序列或由其组成：SEQ ID NO:23；重链CDR3，其包含以下序列或由其组成：SEQ ID NO:11；轻链CDR1，其包含以下序列或由其组成：SEQ ID NO:29；轻链CDR2，其包含以下序列或由其组成：SEQ ID NO:15；以及轻链CDR3，其包含以下序列或由其组成：SEQ ID NO:17。
  6. 如前述权利要求任一项所述的抗体或其抗原结合片段，其特征在于所述抗体或其抗原结合片段包含含有选自SEQ ID NO:33-57的氨基酸序列或与前述序列具有至少85％同一性的氨基酸序列的重链可变区。
  7. 如前述权利要求任一项所述的抗体或其抗原结合片段，其特征在于所述抗体或其抗原结合片段包含含有选自SEQ ID NO:58-82的氨基酸序列或者与前述序列具有至少85％同一性的氨基酸序列的轻链可变区。
  8. 如前述权利要求任一项所述的抗体或其抗原结合片段，其特征在于所述抗体或其抗原结合片段包含以下重链可变区及轻链可变区的组：
    (1)选自SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35或SEQ ID NO:36的氨基酸序列的重链可变区；以及选自SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60或SEQ ID NO:61的氨基酸序列的轻链可变区；或
    (2)选自SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39或SEQ ID NO:40的氨基酸序列的重链可变区；以及选自SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64或SEQ ID NO:65的氨基酸序列的轻链可变区；或
    (3)选自SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43或SEQ ID NO:44的氨基酸序列的重链可变区；以及选自SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68或SEQ ID NO:69的氨基酸序列的轻链可变区；或
    (4)选自SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47或SEQ ID NO:48的氨基酸序列的重链可变区；以及选自SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72或SEQ ID NO:73的氨基酸序列的轻链可变区；或
    (5)选自SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52或SEQ ID NO:53的氨基酸序列的重链可变区；以及选自SEQ ID NO:74、SEQ ID NO:75或SEQ ID NO:76的氨基酸序列的轻链可变区；或
    (6)选自SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56或SEQ ID NO:57的氨基酸序列的重链可变区；以及选自SEQ ID  NO:77、SEQ ID NO:78、SEQ ID NO:79或SEQ ID NO:80的氨基酸序列的轻链可变区。
  9. 如权利要求8所述的抗体或其抗原结合片段，其特征在于所述抗体或其抗原结合片段包含以下(1)至(18)任一项的重链可变区及轻链可变区的组：
    (1)氨基酸序列为SEQ ID NO:38的重链可变区，以及氨基酸序列为SEQ ID NO:62的轻链可变区；或
    (2)氨基酸序列为SEQ ID NO:38的重链可变区，以及氨基酸序列为SEQ ID NO:63的轻链可变区；或
    (3)氨基酸序列为SEQ ID NO:38的重链可变区，以及氨基酸序列为SEQ ID NO:64的轻链可变区；或
    (4)氨基酸序列为SEQ ID NO:39的重链可变区，以及氨基酸序列为SEQ ID NO:63的轻链可变区；或
    (5)氨基酸序列为SEQ ID NO:39的重链可变区，以及氨基酸序列为SEQ ID NO:64的轻链可变区；或
    (6)氨基酸序列为SEQ ID NO:39的重链可变区，以及氨基酸序列为SEQ ID NO:65的轻链可变区；或
    (7)氨基酸序列为SEQ ID NO:46的重链可变区，以及氨基酸序列为SEQ ID NO:71的轻链可变区；或
    (8)氨基酸序列为SEQ ID NO:46的重链可变区，以及氨基酸序列为SEQ ID NO:72的轻链可变区；或
    (9)氨基酸序列为SEQ ID NO:47的重链可变区，以及氨基酸序列为SEQ ID NO:70的轻链可变区；或
    (10)氨基酸序列为SEQ ID NO:47的重链可变区，以及氨基酸序列为SEQ ID NO:72的轻链可变区；或
    (11)氨基酸序列为SEQ ID NO:48的重链可变区，以及氨基酸序列为SEQ ID NO:70的轻链可变区；或
    (12)氨基酸序列为SEQ ID NO:48的重链可变区，以及氨基酸序列为SEQ ID NO:71的轻链可变区；或
    (13)氨基酸序列为SEQ ID NO:50的重链可变区，以及氨基酸序列为SEQ ID NO:76的轻链可变区；或
    (14)氨基酸序列为SEQ ID NO:55的重链可变区，以及氨基酸序列为SEQ ID NO:79的轻链可变区；或
    (15)氨基酸序列为SEQ ID NO:55的重链可变区，以及氨基酸序列为SEQ ID NO:80的轻链可变区；或
    (16)氨基酸序列为SEQ ID NO:56的重链可变区，以及氨基酸序列为SEQ ID NO:79的轻链可变区；或
    (17)氨基酸序列为SEQ ID NO:56的重链可变区，以及氨基酸序列为SEQ ID NO:80的轻链可变区；或
    (18)氨基酸序列为SEQ ID NO:57的重链可变区，以及氨基酸序列为SEQ ID NO:79的轻链可变区。
  10. 如权利要求1-9任一项所述的抗体或其抗原结合片段，其特征在于所述抗体或其抗原结合片段是人抗体、人源化抗体、嵌合抗体、单克隆抗体、多克隆抗体、重组抗体、单链抗体、双抗体、三链抗体、四链抗体、Fab片段、F(ab') 2片段、scFv片段、Fv片段、Fab’片段或结构域抗体。
  11. 如权利要求10所述的抗体或其抗原结合片段，其特征在于所述抗体或其抗原结合片段结合人CD3和/或食蟹猴CD3。
  12. 如权利要求11所述的抗体或其抗原结合片段，其结合至CD3e/d和/或CD3e/g。
  13. 如权利要求11所述的抗体或其抗原结合片段，其特征在于所述抗体或其抗原结合片段(i)显著激活总T细胞、上调CD25的表达和/或诱导IFN-γ的释放；和/或(ii)以小于4×10 -8M的K D值与人CD3结合。
  14. 一种抗体或其抗原结合片段，其与如权利要求1-13中任一项所述的抗体或抗原结合片段竞争或交叉阻断与CD3尤其是CD3e/d或CD3e/g的结合。
  15. 一种经分离的核酸，其编码如权利要求1-14中任一项所述 的抗体或其抗原结合片段。
  16. 一种核酸载体，其包含如权利要求15所述的经分离的核酸。
  17. 一种宿主细胞，其包含如权利要求15所述的经分离的核酸或如权利要求16所述的核酸载体。
  18. 一种药物组合物，其包含如权利要求1-14中任一项所述的抗体或其抗原结合片段、如权利要求15所述的经分离的核酸或如权利要求16所述的核酸载体，以及药学上可接受的载体。
  19. 一种产生如权利要求1-14中任一项所述的抗体或其抗原结合片段的方法，其包括将如权利要求17所述的宿主细胞在允许其表达所述抗体或其抗原结合片段的条件下进行孵育。
  20. 如权利要求1-14中任一项所述的抗体或其抗原结合片段、如权利要求15所述的经分离的核酸、如权利要求16所述的核酸载体或者如权利要求18所述的药物组合物在制备用于抑制免疫应答或活化T细胞的药物中的用途。
  21. 如权利要求1-14中任一项所述的抗体或其抗原结合片段、如权利要求15所述的经分离的核酸、如权利要求16所述的核酸载体或者如权利要求18所述的药物组合物在制备用于治疗癌症或自身免疫疾病的药物中的用途。
  22. 一种抑制免疫应答或活化T细胞的方法，其包括给予有需要的受试者治疗有效量的如权利要求1-14中任一项所述的抗体或其抗原结合片段、如权利要求15所述的经分离的核酸、如权利要求16所述的核酸载体或者如权利要求18所述的药物组合物。
  23. 一种治疗癌症或自身免疫疾病的方法，其包括给予有需要的受试者治疗有效量的如权利要求1-14中任一项所述的抗体或其抗原结合片段、如权利要求15所述的经分离的核酸、如权利要求16所述的核酸载体或者如权利要求18所述的药物组合物。
  24. 如权利要求21所述的用途或者如权利要求23所述的方法，其中所述癌症选自包括以下的组：黑素瘤、肾癌、前列腺癌、乳腺癌、结直肠癌、肺癌、鼻咽癌、口腔癌、肝癌、骨癌、胰腺癌、皮肤癌、头颈部癌、子宫癌、卵巢癌、直肠癌、胃癌、睾丸癌、输卵 管癌、子宫内膜癌、子宫颈癌、阴道癌、外阴癌、霍奇金淋巴瘤、非霍奇金淋巴瘤、食道癌、小肠癌、大肠癌、内分泌系统癌症、甲状腺癌、甲状旁腺癌症、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病、膀胱癌、肾盂癌、中枢神经系统肿瘤、神经胶质瘤、脊髓轴肿瘤、脑干胶质瘤、垂体腺瘤、卡波济氏肉瘤、表皮样癌、鳞状细胞癌和T细胞淋巴瘤。
  25. 如权利要求21所述的用途或者如权利要求23所述的方法，其中所述自身免疫疾病选自包括以下的组：风湿关节炎、多发性硬化、干燥综合征、胰岛素依赖型糖尿病、自身免疫甲状腺炎、反应性关节炎、强直性脊柱炎、硬皮病、多发性肌炎、皮肌炎、牛皮癣、韦格纳氏肉芽肿病、克罗恩氏病、溃疡性结肠炎、狼疮例如系统性红斑狼疮、动脉粥样硬化、慢性阻塞性肺病、肝硬化、肾移植纤维化、肾移植肾病和肺纤维化。
  26. 如权利要求22-25任一项所述的方法，其中所述如权利要求1-14中任一项所述的抗体或其抗原结合片段、如权利要求15所述的经分离的核酸、如权利要求16所述的核酸载体或者如权利要求18所述的药物组合物与另一治疗剂顺序或同时施用。
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CN103694350A (zh) * 2007-04-03 2014-04-02 安进研发（慕尼黑）股份有限公司 跨物种特异性CD3-ε结合结构域
US20140141020A1 (en) * 2011-06-14 2014-05-22 Mayo Foundation For Medical Education And Research Anti-cd3 therapies
CN110431231A (zh) * 2016-12-22 2019-11-08 第一三共株式会社 抗-cd3抗体和包含所述抗体的分子
CN111886250A (zh) * 2017-12-27 2020-11-03 特尼奥生物股份有限公司 CD3-δ/ε异二聚体特异性抗体
CN112533950A (zh) * 2018-06-29 2021-03-19 百奥赛图(北京)医药科技股份有限公司 抗CD3e抗体及其用途
CN112912397A (zh) * 2018-05-24 2021-06-04 詹森生物科技公司 抗cd3抗体及其用途
CN114174339A (zh) * 2019-06-07 2022-03-11 阿迪马布有限责任公司 高亲和力抗cd3抗体及其产生和使用方法
CN114276451A (zh) * 2020-09-27 2022-04-05 嘉和生物药业有限公司 靶向CD3e/g的抗体或其抗原结合片段、其制备和应用

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CN103694350A (zh) * 2007-04-03 2014-04-02 安进研发（慕尼黑）股份有限公司 跨物种特异性CD3-ε结合结构域
US20140141020A1 (en) * 2011-06-14 2014-05-22 Mayo Foundation For Medical Education And Research Anti-cd3 therapies
CN110431231A (zh) * 2016-12-22 2019-11-08 第一三共株式会社 抗-cd3抗体和包含所述抗体的分子
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