WO2023001303A1 - 药物组合物及用途 - Google Patents

药物组合物及用途 Download PDF

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WO2023001303A1
WO2023001303A1 PCT/CN2022/107522 CN2022107522W WO2023001303A1 WO 2023001303 A1 WO2023001303 A1 WO 2023001303A1 CN 2022107522 W CN2022107522 W CN 2022107522W WO 2023001303 A1 WO2023001303 A1 WO 2023001303A1
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seq
antibody
variable region
amino acid
chain variable
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PCT/CN2022/107522
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French (fr)
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王忠民
李百勇
夏瑜
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中山康方生物医药有限公司
康方药业有限公司
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Priority to AU2022314097A priority Critical patent/AU2022314097A1/en
Priority to IL310217A priority patent/IL310217A/en
Priority to CA3226242A priority patent/CA3226242A1/en
Priority to KR1020247006091A priority patent/KR20240038043A/ko
Publication of WO2023001303A1 publication Critical patent/WO2023001303A1/zh

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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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Definitions

  • the invention belongs to the field of medicine and relates to a pharmaceutical composition, which comprises an anti-TIGIT antibody or an antigen-binding fragment thereof, and an anti-CTLA4-anti-PD-1 bispecific antibody or an antigen-binding fragment thereof.
  • TIGIT T cell Ig and ITIM domain, also known as WUCAM, Vstm3, VSIG9 is a member of the poliovirus receptor (PVR)/Nectin family.
  • TIGIT consists of an extracellular immunoglobulin variable region (IgV) domain, a type I transmembrane domain and a classical immunoreceptor tyrosine inhibitory motif (ITIM) and immunoglobulin tyrosine tail (ITT) motifs intracellular domain composition.
  • TIGIT is highly expressed in lymphocytes, especially in effector and regulatory CD4+ T cells, follicular helper CD4+ T cells and effector CD8+ T cells, and natural killer (NK) cells (Yu X, Harden K, Gonzalez L C, et al al.The surface protein TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells[J].Nature immunology,2009,10(1):48).
  • CD155 also known as PVR, Necl5 or Tage4
  • CD112 also known as PVRL2/nectin 2
  • CD113 also known as PVRL3
  • TIGIT binding ligands Vanet L, Smyth M J. Balancing natural killer cell activation through paired receptors[J].Nature Reviews Immunology,2015,15(4):243-254
  • CD155 is the high-affinity ligand of TIGIT.
  • TIGIT binding ligands CD155 and CD112 can inhibit the killing effect of NK cells on cells with high expression of TIGIT (Stanietsky N, Simic H, Arapovic J, et al.
  • TIGIT acts as an immune checkpoint for NK cells
  • the inhibitory receptor TIGIT can lead to NK cell depletion during tumor development
  • anti-TIGIT monoclonal antibody can reverse NK cell depletion and be used for immunotherapy of various tumors (Zhang Q, Bi J, Zheng X, et al. Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-tumor immunity[J].Nature immunology,2018,19(7):723-732).
  • TIGIT blockade alone or in combination with PD-1 blockade plus CD96 blockade can significantly reduce the growth of B16 melanoma in wild-type and Cd155-/- mouse models (Li XY, Das I, Lepletier A, et al.. Cd155 loss enhances tumor suppression via combined host and tumor-intrinsic mechanisms. J Clin Invest 2018; 128:2613–25).
  • CD112R blockade alone or in combination with TIGIT blockade and/or PD-1 blockade, increases the cytokine-producing capacity of TILs in ovarian, endometrioma, and lung tumors (Whelan S, Ophir E, Kotturi MF, et al.. PVRIG and PVRL2 Are Induced in Cancer and Inhibit CD8 + T-cell Function. Cancer Immunol Res 2019; 7:257–68).
  • Anti-TIGIT antibody drugs as new immune checkpoint antibody drugs, have broad application prospects and can be used for immunotherapy of tumors.
  • Tiragolumab developed by Roche is already in Phase 3 clinical stage, and it is reported that TIGIT monoclonal antibody Tiragolumab combined with PD-L1 drug Tecentrip (atezolizumab) as first-line therapy is effective in the treatment of PD-L1 positive metastatic non-small cell tumors.
  • Tecentrip atezolizumab
  • TIGIT is used for the treatment of non-small cell lung cancer, small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, pancreatic cancer, cervical cancer, multiple myeloma, non-Hodgkin An important target for lymphoma, B lymphocytoma, and plasma cell carcinoma.
  • the transmembrane receptor PD-1 (programmed cell death-1) is a member of the CD28 gene family and is expressed in activated T cells, B cells and myeloid cells.
  • the ligands PDL1 (Programmed cell death 1 ligand 1, also referred to as PDL-1) and PDL2 (Programmed cell death 1 ligand 2, also referred to as PDL-2) of PD-1 belong to the B7 superfamily. Cells are expressed, including T cells, B cells and endothelial cells and epithelial cells, PDL2 is only expressed in antigen-presenting cells such as dendritic cells and macrophages.
  • the PD-1/PDL1 signaling pathway plays an important role in regulating immune tolerance, microbial infection and tumor immune escape.
  • the expression of PD-1 is mainly in immune cells such as T cells, and the ligand of PD-1, PDL1, is mainly highly expressed in many human tumor tissues.
  • Blocking the PD-1/PDL1 signaling pathway can activate suppressed T cells to attack cancer cells. Blocking PD-1/PDL1 signaling can promote the proliferation of tumor antigen-specific T cells, play a role in killing tumor cells, and then inhibit local tumor growth (Julie R et al., 2012, N Engl J Med.366:2455–2465 ).
  • An effective method is to regulate the expression of PD-1 by injecting anti-PD-1 antibody in vivo.
  • Cytotoxic T lymphocyte associated antigen-4 (cytotoxic T lymphocyte associated antigen 4, also referred to as CTLA4) has a very similar relationship with CD28 molecule in gene structure, chromosomal location, sequence homology and gene expression.
  • Receptor for molecule B7 mainly expressed on the surface of activated T cells. The combination of CTLA4 and B7 can inhibit the activation of mouse and human T cells and play a negative regulatory role in T cell activation.
  • CTLA4 antibody or anti-CTLA4 monoclonal antibody
  • CTLA4 ligand can prevent CTLA4 from binding to its natural ligand, thereby blocking CTLA4's transduction of T cell negative regulatory signals and enhancing T cell reactivity to various antigens, in this regard
  • CTLA4 monoclonal antibodies are currently in clinical trials or approved for the treatment of prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, malignant melanoma, etc. (Grosso JF., Jure-Kunkel MN., 2013 , Cancer Immun. 13:5.).
  • Interleukin 2 is produced by T cells and is a growth factor that regulates T cell subsets. It is also an important factor that regulates immune responses. It can promote the proliferation of activated B cells and participate in antibody responses, hematopoiesis and tumor surveillance. Recombinant human IL-2 has been approved by the U.S. FDA for the treatment of malignant tumors (including melanoma, kidney tumors, etc.), and is undergoing clinical research for the treatment of chronic viral infections (Chavez, A.R., et al., 2009, Ann N Y Acad Sci, 1182: p.14-27).
  • CTLA4 and CTLA4 antibody as an important factor affecting the function of T cells, interfere with the immune microenvironment of the body. In vitro and in vivo tests, CTLA4 antibody can specifically relieve CTLA4's immune suppression on the body, activate T cells, and induce IL-2 production. It has broad application prospects in gene therapy for anti-tumor and parasite diseases.
  • the inventors used a mammalian cell expression system to express recombinant human TIGIT as an antigen to immunize mice, and obtained hybridoma cells through the fusion of mouse spleen cells and myeloma cells.
  • the inventor obtained the hybridoma cell line LT019 (preservation number CCTCC NO: C2020208) by screening a large number of samples.
  • the hybridoma cell line LT019 can secrete and produce specific monoclonal antibodies (named 26B12) that specifically bind to human TIGIT, and the monoclonal antibodies can effectively bind to TIGIT, reducing TIGIT's ability to inhibit immunity.
  • the role of cells promote the activity of T cells, reverse the exhaustion of NK cells, and enhance the killing effect of immune cells on tumors.
  • the present inventors creatively produced humanized antibodies against human TIGIT (named 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4).
  • One aspect of the invention relates to anti-TIGIT antibodies or antigen-binding fragments thereof, wherein,
  • the anti-TIGIT antibody comprises HCDR1-HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:1 and LCDR1-LCDR3 contained in the light chain variable region shown in SEQ ID NO:6,
  • the heavy chain variable region of the antibody comprises amino acid sequences HCDR1-HCDR3 respectively shown in SEQ ID NOs: 3-5; and the light chain variable region of the antibody comprises the amino acid sequences respectively LCDR1-LCDR3 as shown in SEQ ID NOs:8-10.
  • the anti-TIGIT antibody or antigen-binding fragment thereof wherein the amino acid sequence of the heavy chain variable region of the antibody is selected from SEQ ID NO: 1, SEQ ID NO: 11. SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17; and
  • amino acid sequence of the light chain variable region of the antibody is selected from SEQ ID NO:6, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23 and SEQ ID NO:25.
  • the anti-TIGIT antibody or antigen-binding fragment thereof wherein,
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 6;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 19;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 19;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 13
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 21;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 13
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 23;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 21;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 23;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 25; or
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 25.
  • the anti-TIGIT antibody or antigen-binding fragment thereof wherein the antibody includes a non-CDR region, and the non-CDR region is from a species other than murine, For example from human antibodies.
  • the anti-TIGIT antibody or antigen-binding fragment thereof wherein the heavy chain constant region of the antibody is Ig gamma-1 chain C region (such as NCBI ACCESSION: P01857);
  • the light chain constant region is Ig kappa chain C region (eg NCBI ACCESSION: P01834).
  • the anti-TIGIT antibody or its antigen-binding fragment wherein, the anti-TIGIT antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single chain antibody, humanized antibody, chimeric antibody or diabody.
  • the anti-TIGIT antibody or antigen-binding fragment thereof wherein the antibody binds to TIGIT- mFc with a KD of less than 4E-10 or less than 4E-11; preferably, The KD is measured by a Fortebio molecular interaction instrument.
  • the anti-TIGIT antibody or antigen-binding fragment thereof wherein the antibody binds to TIGIT-mFc with an EC 50 of less than 1.5 nM, less than 1.2 nM or less than 1 nM; preferably , the EC 50 was measured by flow cytometry.
  • the anti-TIGIT antibody is a monoclonal antibody.
  • the anti-TIGIT antibody is a humanized antibody, a chimeric antibody, a multispecific antibody (such as a bispecific antibody).
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2 , Fd, Fv, dAb, Fab/c, complementarity determining region fragments, single chain antibodies (e.g., scFv ), humanized antibodies, chimeric antibodies or bispecific antibodies.
  • the anti-TIGIT antibody or antigen-binding fragment thereof wherein the antibody is an antibody produced by a hybridoma cell line LT019, and the hybridoma cell line LT019 is preserved in the China Type Culture Collection Center (CCTCC), the deposit number is CCTCC NO: C2020208.
  • CTCC China Type Culture Collection Center
  • Another aspect of the invention pertains to an isolated nucleic acid molecule encoding the anti-TIGIT antibody or antigen-binding fragment thereof of any one of the invention.
  • a further aspect of the invention relates to a vector comprising an isolated nucleic acid molecule of the invention.
  • a further aspect of the invention relates to a host cell comprising an isolated nucleic acid molecule of the invention, or a vector of the invention.
  • Another aspect of the present invention relates to the hybridoma cell line LT019, which is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: C2020208.
  • CTCC China Center for Type Culture Collection
  • Another aspect of the present invention relates to a conjugate, which includes an antibody and a coupling part, wherein the antibody is the anti-TIGIT antibody or an antigen-binding fragment thereof according to any one of the present invention, and the coupling part can be A label for detection; preferably, the coupling moiety is a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.
  • kits which includes any one of the anti-TIGIT antibodies or antigen-binding fragments of the present invention, or includes the conjugate of the present invention;
  • the kit further includes a second antibody that specifically recognizes the antibody; optionally, the second antibody also includes a detectable label, such as a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance or enzyme.
  • a detectable label such as a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance or enzyme.
  • Another aspect of the present invention relates to the use of any one of the antibodies of the present invention or the conjugate of the present invention in the preparation of a kit for detecting the presence or level of TIGIT in a sample.
  • Another aspect of the present invention relates to a pharmaceutical composition, which comprises any one of the anti-TIGIT antibody or its antigen-binding fragment of the present invention or the conjugate of the present invention; optionally, the pharmaceutical composition also A pharmaceutically acceptable carrier and/or excipient is included.
  • the pharmaceutical composition further comprises one or more anti-PD-1 antibodies, or one or more anti-PD-L1 antibodies, such as anti-CTLA4-anti- PD-1 bispecific antibody.
  • the pharmaceutical composition wherein, calculated according to the mass of the antibody, the mass ratio of the anti-TIGIT antibody or its antigen-binding fragment to the anti-PD-1 antibody or anti-PD-L1 antibody It is (1:5)-(5:1), for example: 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1 or 5:1 .
  • Yet another aspect of the present invention relates to a combination product (such as a kit) comprising a first product and a second product separately packaged, wherein,
  • the first product comprises the anti-TIGIT antibody or its antigen-binding fragment according to any one of the present invention, the conjugate of the present invention or the pharmaceutical composition according to any one of the present invention;
  • the second product comprises at least one anti-PD-1 antibody or at least one anti-PD-L1 antibody, such as an anti-CTLA4-anti-PD-1 bispecific antibody;
  • the combined product further comprises a third product packaged independently, the third product comprising one or more chemotherapeutic drugs,
  • the first product and the second product also independently comprise one or more pharmaceutically acceptable excipients;
  • the combined product further includes product instructions.
  • the combination product wherein, calculated according to the mass of the antibody, the mass ratio of the anti-TIGIT antibody or its antigen-binding fragment to the anti-PD-1 antibody or anti-PD-L1 antibody is (1:5)-(5:1), for example: 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1 or 5:1.
  • Another aspect of the present invention relates to the antibody described in any one of the present invention, the conjugate of the present invention, the pharmaceutical composition described in any one of the present invention or the combination product described in any one of the present invention. Use in the preparation of medicines for treating and/or preventing tumors.
  • the antibody according to any one of the present invention, the conjugate of the present invention, the pharmaceutical composition according to any one of the present invention or the combination product according to any one of the present invention which are used for treatment and/or or prevent tumors.
  • Another aspect of the present invention relates to a method for treating and/or preventing tumors, comprising administering to a subject in need an effective amount of the antibody of any one of the present invention, the conjugate of the present invention, the The steps of the pharmaceutical composition described in any one of the above or the combination product described in any one of the present invention.
  • the present invention relates to a method for preventing and/or treating tumors (especially malignant tumors) or infections or infectious diseases, comprising administering to a subject a therapeutically effective amount of an anti-TIGIT antibody, combined with anti-CTLA4-anti-PD-1 bispecific Antibodies, more preferably, further combined administration of one or more drugs (such as chemotherapeutics or growth inhibitors, targeted therapeutics, antibody-drug conjugates, antimetabolites, antibiotics, plant-based anticancer drugs and/or Or hormone drugs, doxorubicin, tamoxifen, megestrol, and/or asparaginase, preferably, the anti-TIGIT antibody, anti-CTLA4-anti-PD-1 bispecific antibody and tumor Chemotherapy drugs are given simultaneously or sequentially.
  • drugs such as chemotherapeutics or growth inhibitors, targeted therapeutics, antibody-drug conjugates, antimetabolites, antibiotics, plant-based anticancer drugs and/or Or hormone drugs, doxorubicin, tam
  • the chemotherapeutic agent or growth inhibitory agent is selected from platinum drugs (such as cisplatin, carboplatin or oxaliplatin), fluorouracil antineoplastic drugs, cyclophosphamide, pemetrexed, paclitaxel, Vinblastine, doxorubicin, goserelin, alkylating agent, anthracycline, antihormonal agent, aromatase inhibitor, antiandrogen, protein kinase inhibitor, lipid kinase inhibitor, antisense oligo Nucleotides, ribozymes, antimetabolites, topoisomerase inhibitors, cytotoxic or antitumor antibiotics, proteasome inhibitors, antimicrotubule agents, EGFR antagonists, retinoids, tyrosine kinase inhibitors, Histone deacetylase inhibitors and combinations thereof.
  • platinum drugs such as cisplatin, carboplatin or oxaliplatin
  • the targeted therapeutic agent is selected from B-raf inhibitors, MEK inhibitors, K-ras inhibitors, c-Met inhibitors, Alk inhibitors, phosphatidylinositol 3-kinase inhibitors, Akt inhibitors, mTOR inhibitors, bisphosphatidylinositol 3-kinase/mTOR inhibitors, and combinations thereof.
  • the antibody-drug conjugate comprises a drug selected from the group consisting of maytansine, monomethyl auristatin E, calicheamicin, esperamicin, and a radioisotope chelator.
  • the present invention relates to a method for preventing and/or treating tumors (especially malignant tumors), comprising administering to a subject a therapeutically effective amount of an anti-TIGIT antibody, combined with an anti-CTLA4-anti-PD-1 bispecific antibody, more preferably Preferably, one or more chemotherapeutic drugs are further administered in combination.
  • the anti-TIGIT antibody, anti-CTLA4-anti-PD-1 bispecific antibody and chemotherapeutic drugs are administered simultaneously or sequentially.
  • the tumor is selected from one or more of the following: pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, cervical cancer, multiple myeloma, non-Hodgkin Lymphoma, B lymphocytoma, plasma cell carcinoma, head and neck cancer, brain cancer, throat cancer, nasopharyngeal cancer, esophageal cancer, esophageal squamous cell carcinoma, thyroid cancer, mesothelioma, adenocarcinoma (such as pancreatic cancer, breast cancer ), lung cancer (such as small cell lung cancer, small cell lung cancer), breast cancer, liver cancer (such as hepatocellular carcinoma, hepatobiliary cancer), gastric cancer, gastrointestinal cancer, bowel cancer (such as colon cancer, colorectal cancer), biliary tract cancer (such as cholangiocarcinoma), kidney cancer, fallopian tube cancer, endometrial cancer, cervical cancer, bladder cancer, urot
  • the anti-CTLA4-anti-PD-1 bispecific antibody comprises:
  • the functional region of the first protein is an immunoglobulin
  • the functional region of the second protein is a single-chain antibody
  • the functional region of the first protein is a single-chain antibody
  • the functional region of the second protein is an immunoglobulin protein
  • the immunoglobulin which comprises HCDR1-HCDR3 in the heavy chain variable region shown in SEQ ID NO: 27 (preferably HCDR1-HCDR3 shown in SEQ ID NOs: 29-31, respectively), and SEQ ID NO LCDR1-LCDR3 in the light chain variable region shown in :28 (preferably LCDR1-LCDR3 shown in SEQ ID NOs:32-34 respectively); and said single chain antibody, which comprises SEQ ID NO:35 HCDR1-HCDR3 in the heavy chain variable region shown (preferably HCDR1-HCDR3 shown in SEQ ID NOs:37-39, respectively) and LCDR1-LCDR3 in the light chain variable region shown in SEQ ID NO:36 ( Preferably LCDR1-LCDR3 as shown in SEQ ID NOs:40-42 respectively);
  • the immunoglobulin which comprises HCDR1-HCDR3 in the heavy chain variable region shown in SEQ ID NO: 35 (preferably HCDR1-HCDR3 shown in SEQ ID NOs: 37-39 respectively) and SEQ ID NO: LCDR1-LCDR3 in the light chain variable region shown in 36 (preferably LCDR1-LCDR3 shown in SEQ ID NOs:40-42 respectively); and said single-chain antibody, which comprises SEQ ID NO:27 HCDR1-HCDR3 in the heavy chain variable region (preferably HCDR1-HCDR3 shown in SEQ ID NOs: 29-31, respectively), and LCDR1-LCDR3 in the light chain variable region shown in SEQ ID NO: 28 ( Preferred are LCDR1-LCDR3 as shown in SEQ ID NOs: 32-34, respectively).
  • the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention wherein,
  • the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO: 27, SEQ ID NO: 43 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, At least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, A sequence of 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology; and the amino acid sequence of the light chain variable region of the immunoglobulin is selected from SEQ ID NO: 28 , SEQ ID NO: 44 or at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at
  • the amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from SEQ ID NO:35, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 or at least 60 %, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, A sequence of at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology; and the immunized
  • the amino acid sequence of the light chain variable region of the globulin is selected from SEQ ID NO:36, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 or at least 60% thereof, at least 65%, at least 70%, at least 75%,
  • the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention wherein the bispecific antibody is selected from any one of the following (1)-(20):
  • the amino acid sequence of the heavy chain variable region of said immunoglobulin is as SEQ ID NO: 27 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence shown, and the amino acid sequence of the light chain variable region of the immunoglobulin is as SEQ ID NO: 28 or therewith At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, 92%,
  • the amino acid sequence of the heavy chain variable region of said immunoglobulin is as SEQ ID NO: 27 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence shown, and the amino acid sequence of the light chain variable region of the immunoglobulin is as SEQ ID NO: 28 or therewith At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, 92%,
  • the amino acid sequence of the heavy chain variable region of said immunoglobulin is as SEQ ID NO: 27 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence shown, and the amino acid sequence of the light chain variable region of the immunoglobulin is as SEQ ID NO: 28 or therewith At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, 92%,
  • the amino acid sequence of the heavy chain variable region of said immunoglobulin is as SEQ ID NO: 43 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence shown, and the amino acid sequence of the light chain variable region of the immunoglobulin is as SEQ ID NO: 44 or therewith At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, 92%
  • the amino acid sequence of the heavy chain variable region of said immunoglobulin is as SEQ ID NO: 43 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence shown, and the amino acid sequence of the light chain variable region of the immunoglobulin is as SEQ ID NO: 44 or therewith At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, 92%
  • the amino acid sequence of the heavy chain variable region of said immunoglobulin is as SEQ ID NO: 43 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence shown, and the amino acid sequence of the light chain variable region of the immunoglobulin is as SEQ ID NO: 44 or therewith At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, 92%
  • the amino acid sequence of the heavy chain variable region of said immunoglobulin is as SEQ ID NO: 35 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence shown, and the amino acid sequence of the light chain variable region of the immunoglobulin is as SEQ ID NO: 36 or therewith At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, 92%
  • the amino acid sequence of the heavy chain variable region of said immunoglobulin is as SEQ ID NO: 35 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence shown, and the amino acid sequence of the light chain variable region of the immunoglobulin is as SEQ ID NO: 36 or therewith At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, 92%
  • the amino acid sequence of the heavy chain variable region of said immunoglobulin is as SEQ ID NO: 45 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence shown, and the amino acid sequence of the light chain variable region of the immunoglobulin is as SEQ ID NO: 49 or therewith At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, 92%
  • the amino acid sequence of the heavy chain variable region of the immunoglobulin is as SEQ ID NO: 45 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence shown, and the amino acid sequence of the light chain variable region of the immunoglobulin is as SEQ ID NO: 49 or therewith At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, 92%
  • the amino acid sequence of the heavy chain variable region of the immunoglobulin is as SEQ ID NO: 46 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence shown, and the amino acid sequence of the light chain variable region of the immunoglobulin is as SEQ ID NO: 50 or therewith At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, 92%
  • the amino acid sequence of the heavy chain variable region of the immunoglobulin is as SEQ ID NO: 46 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence shown, and the amino acid sequence of the light chain variable region of the immunoglobulin is as SEQ ID NO: 50 or therewith At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, 92%
  • the amino acid sequence of the heavy chain variable region of the immunoglobulin is as SEQ ID NO: 27 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence shown, and the amino acid sequence of the light chain variable region of the immunoglobulin is as SEQ ID NO: 28 or therewith At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, 92%
  • the amino acid sequence of the heavy chain variable region of the immunoglobulin is as SEQ ID NO: 27 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence shown, and the amino acid sequence of the light chain variable region of the immunoglobulin is as SEQ ID NO: 28 or therewith At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, 92%
  • the amino acid sequence of the heavy chain variable region of the immunoglobulin is as SEQ ID NO: 43 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence shown, and the amino acid sequence of the light chain variable region of the immunoglobulin is as SEQ ID NO: 44 or therewith At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, 92%
  • the amino acid sequence of the heavy chain variable region of the immunoglobulin is as SEQ ID NO: 43 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence shown, and the amino acid sequence of the light chain variable region of the immunoglobulin is as SEQ ID NO: 44 or therewith At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, 92%
  • the amino acid sequence of the heavy chain variable region of the immunoglobulin is as SEQ ID NO: 47 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence shown, and the amino acid sequence of the light chain variable region of the immunoglobulin is as SEQ ID NO: 51 or therewith At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, 92%
  • the amino acid sequence of the heavy chain variable region of the immunoglobulin is as SEQ ID NO: 48 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence shown, and the amino acid sequence of the light chain variable region of the immunoglobulin is as SEQ ID NO: 52 or therewith At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, 92%
  • the amino acid sequence of the heavy chain variable region of the immunoglobulin is as SEQ ID NO: 47 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence shown, and the amino acid sequence of the light chain variable region of the immunoglobulin is as SEQ ID NO: 51 or therewith At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, 92%
  • the amino acid sequence of the heavy chain variable region of the immunoglobulin is as SEQ ID NO: 48 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the sequence shown, and the amino acid sequence of the light chain variable region of the immunoglobulin is as SEQ ID NO: 52 or therewith At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88 %, at least 89%, at least 90%, at least 91%, 92%
  • the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention wherein,
  • the amino acid sequence of the heavy chain of the immunoglobulin is as SEQ ID NO: 53 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83% %, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96% , at least 97%, at least 98% or at least 99% homology, and the amino acid sequence of its light chain is as SEQ ID NO: 54 or has at least 60%, at least 65%, at least 70%, at least 75% therewith %, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%,
  • the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention wherein the functional domain of the first protein is directly linked to the functional domain of the second protein or linked through a linking fragment; and/ Or the heavy chain variable region of the single-chain antibody is directly connected to the light chain variable region of the single-chain antibody or connected through a linking fragment.
  • the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention wherein the connecting fragment is (GGGGS)n, n is a positive integer; preferably, n is 1, 2, 3 , 4, 5 or 6.
  • the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention wherein the first protein functional domain and the second protein functional domain are independently 1, 2 or more than 2 .
  • the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention wherein the single-chain antibody (preferably the variable region of the heavy chain) is linked to the C-terminus of the heavy chain of the immunoglobulin.
  • the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention wherein,
  • the immunoglobulin is of human IgG1 subtype
  • the heavy chain constant region of the immunoglobulin has one of the following mutation combinations:
  • the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention wherein the bispecific antibody comprises:
  • the functional region of the first protein is immunoglobulin, and the functional region of the second protein is a single-chain antibody;
  • the amino acid sequence of the heavy chain of the immunoglobulin is as SEQ ID NO: 53 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83% %, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, at least 96% , at least 97%, at least 98% or at least 99% homology, and the amino acid sequence of its light chain is as SEQ ID NO: 54 or has at least 60%, at least 65%, at least 70%, at least 75% therewith %, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%,
  • the amino acid sequence of the heavy chain variable region of the single chain antibody is as SEQ ID NO: 48 or has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82% , at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%, 94%, at least 95%, At least 96%, at least 97%, at least 98% or at least 99% homology of the sequence shown, and the amino acid sequence of the light chain variable region of the single chain antibody is as SEQ ID NO: 52 or has at least 60% therewith , at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, 92%, 93%
  • the single-chain antibody is linked to the C-terminus of the heavy chain of the immunoglobulin;
  • the first protein functional region is connected to the second protein functional region through a first connecting segment; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody through a second Segment concatenation; said first concatenated fragment and said second concatenated fragment are the same or different;
  • amino acid sequences of the first connecting segment and the second connecting segment are independently selected from SEQ ID NO:55 and SEQ ID NO:56;
  • amino acid sequences of the first connecting fragment and the second connecting fragment are shown in SEQ ID NO:56.
  • the amino acid sequence of the heavy chain of the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention is shown in SEQ ID NOs: 57, and the amino acid sequence of the light chain is shown in SEQ ID NOs: 59.
  • the specific antibody structure is IgG-scFv, in which the IgG part is anti-PD1 antibody, and the scFv part is anti-CTLA4 antibody.
  • the HCDR1 sequence of the anti-PD1 antibody is shown in SEQ ID NO: 61
  • the HCDR2 sequence is shown in SEQ ID NO: 62
  • the HCDR3 sequence is shown in SEQ ID NO: 63
  • the VH sequence is shown in SEQ ID NO: 73
  • anti-PD1 The LCDR1 sequence of the antibody is shown in SEQ ID NO:70
  • the LCDR2 sequence is shown in SEQ ID NO:71
  • the LCDR3 sequence is shown in SEQ ID NO:72
  • the VL sequence is shown in SEQ ID NO:76
  • the HCDR1 sequence of the anti-CTLA4 antibody is shown in SEQ ID NO:64
  • the HCDR2 sequence is shown in SEQ ID NO:65
  • the HCDR3 sequence is shown in SEQ ID NO:66
  • the VH sequence is shown in SEQ ID NO:74.
  • Anti-CTLA4 antibody The LCDR1 sequence is shown in SEQ ID NO:67
  • the LCDR2 sequence is shown in SEQ ID NO:68
  • the LCDR3 sequence is shown in SEQ ID NO:69
  • the VL sequence is shown in SEQ ID NO:75.
  • the unit preparation comprises: 1-10000 mg (preferably 10-1000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150- 250mg or 200mg) of the anti-TIGIT antibody described in any aspect of the present invention and 1-10000mg (preferably 1-1000mg, preferably 50-500mg, 100-400mg, 150-300mg, 150-250mg, 200mg or 100mg) of the present invention
  • 1-10000mg preferably 1-1000mg, preferably 50-500mg, 100-400mg, 150-300mg, 150-250mg, 200mg or 100mg
  • the anti-CTLA4-anti-PD-1 bispecific antibody described in any aspect, and optionally one or more chemotherapeutic drugs described in the present invention such as platinum drugs and/or fluorouracil anti-tumor drugs); wherein , the anti-TIGIT antibody, the anti-CTLA4-anti-PD-1 bispecific antibody and the chemotherapeutic drug are separately packaged.
  • the present invention relates to a method for preventing or treating cancer or tumors, wherein one or more unit preparations of the present invention are administered to a subject in need, preferably, the anti-CTLA4-anti- PD-1 bispecific antibody, anti-TIGIT antibody, and chemotherapeutics were each administered separately.
  • a single pharmaceutical dosage unit preferably for the treatment of tumors, comprising 0.1-10000 mg (preferably 1-1000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100 mg) of any one of the anti-TIGIT antibodies of the present invention and 0.1-10000 mg (preferably 1-1000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100 mg) of any of the present invention
  • 0.1-10000 mg preferably 1-1000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100 mg
  • the anti-TIGIT antibody, the anti-CTLA4-anti-PD-1 bispecific antibody and/or the chemotherapeutic agent are in a form suitable for intravenous injection or intravenous infusion , preferably in liquid form.
  • an effective amount of the anti-TIGIT antibody described in any one of the present invention and/or the anti-CTLA4-anti-PD-1 bispecific antibody described in any one of the present invention is administered to the subject
  • the step of anti-antibodies is before or after surgical treatment, and/or before or after radiation treatment.
  • the single administration dose of the anti-TIGIT antibody described in any one of the present invention and/or the anti-CTLA4 anti-PD-1 bispecific antibody described in any one of the present invention 0.1-100 mg per kilogram of body weight, preferably 1-10 mg; or, a single administration of the anti-TIGIT antibody according to any one of the present invention and/or the anti-CTLA4 anti-PD-1 bispecific antibody according to any one of the present invention
  • the dose is 10-1000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg or 200 mg per subject,
  • it is administered twice daily to about every other day, or every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks. medicine once;
  • the administration method is intravenous drip or intravenous injection.
  • variable regions of the light and heavy chains determine the binding of antigens; the variable regions of each chain contain three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2, HCDR3 , the CDRs of the light chain (L) comprise LCDR1, LCDR2, LCDR3; which are named by Kabat et al., see Bethesda M.d., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 1991; 1-3:91-3242.
  • CDRs complementarity determining regions
  • the CDRs of the heavy chain (H) include HCDR1, HCDR2, HCDR3
  • the CDRs of the light chain (L) comprise LCDR1, LCDR2, LCDR3; which are named by Kabat et al., see Bethesda M.d., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 1991; 1-3:91-3242
  • CDRs can also be defined by the IMGT numbering system, see Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc.
  • IMGT/3Dstructure-DB and IMGT/DomainGapAlign a database and a tool for immunoglobulins or antibodies, T cell receptors , MHC, IgSF and MhcSF. Nucleic acids research 2009;38(suppl_1):D301-D307.
  • the amino acid sequence of the CDR region of the monoclonal antibody sequence is analyzed by technical means well known to those skilled in the art, for example, through the VBASE2 database according to the definition of IMGT.
  • the antibodies 26B12, 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 involved in the present invention have the same CDR:
  • amino acid sequences of the three CDR regions of the heavy chain variable region are as follows:
  • HCDR3 ARLDYGNYGGAMDY (SEQ ID NO: 5);
  • amino acid sequences of the three CDR regions of the light chain variable region are as follows:
  • LCDR1 QHVSTA (SEQ ID NO:8)
  • LCDR3 QQHYITPWT (SEQ ID NO: 10).
  • TIGIT when referring to the amino acid sequence of TIGIT (NCBI GenBank ID: NP_776160.2), it includes the full length of the TIGIT protein, or an extracellular immunoglobulin variable region (IgV) domain or a cell-containing Fragments of the outer immunoglobulin variable region (IgV) domain; also include fusion proteins of TIGIT, eg, fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG.
  • fusion proteins of TIGIT eg, fragments fused to Fc protein fragments (mFc or hFc) of mouse or human IgG.
  • mFc or hFc Fc protein fragments
  • TIGIT protein or “TIGIT” shall include all such sequences, including the indicated sequences as well as natural or artificial variants thereof. And, when describing the sequence fragment of TIGIT protein, it not only includes the sequence fragment, but also includes the corresponding sequence fragment in its natural or artificial variant.
  • EC 50 refers to the concentration for 50% of maximal effect, which refers to the concentration that can cause 50% of the maximal effect.
  • antibody refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having a "light” (L) chain and a “heavy” (H) chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also comprising a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region ( VH ) and a heavy chain constant region ( CH ).
  • the heavy chain constant region consists of 3 domains ( CH1 , CH2 and CH3 ).
  • Each light chain is composed of a light chain variable region (V L ) and a light chain constant region ( CL ).
  • the light chain constant region consists of one domain, CL .
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from amino-terminus to carboxy-terminus.
  • the variable regions ( VH and VL ) of each heavy/light chain pair form the antigen-binding site, respectively. Assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda Md (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196:901-917; Chothia et al.
  • IMGT/3Dstructure-DB and IMGT/DomainGapAlign a database and a tool for immunoglobulins or antibodies, Definition of T cell receptors, MHC, IgSF and MhcSF.” Nucleic acids research 2009; 38(suppl_1):D301-D307.
  • antibody is not limited to any particular method of producing antibodies. For example, it includes, inter alia, recombinant antibodies, monoclonal antibodies and polyclonal antibodies.
  • Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3, or IgG4 subtype
  • IgAl IgA2, IgD, IgE, or IgM antibodies.
  • the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for Specific binding to an antigen, which is also referred to as an "antigen-binding moiety".
  • an antigen-binding moiety which is also referred to as an "antigen-binding moiety”.
  • antigen-binding fragments include Fab, Fab', F(ab') 2 , Fd, Fv, dAb, and complementarity determining regions (CDRs) Fragments, single chain antibodies (eg, scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • CDRs complementarity determining regions
  • the term “Fd fragment” means an antibody fragment consisting of VH and CH1 domains;
  • the term “Fv fragment” means an antibody fragment consisting of VL and VH domains of a single arm of an antibody Antibody fragment;
  • the term “dAb fragment” means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544-546 (1989));
  • the term “Fab fragment” means an antibody fragment consisting of VL , VH , C Antibody fragments consisting of L and CH1 domains;
  • the term “ F (ab') 2 fragment” means an antibody fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region.
  • the antigen-binding fragment of an antibody is a single-chain antibody (e.g., a scFv) in which the VL and VH domains are paired to form a monovalent molecule via a linker that enables production as a single polypeptide chain (see, e.g., Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)).
  • scFv molecules may have the general structure: NH2 - VL -linker- VH -COOH or NH2 - VH -linker- VL -COOH.
  • Suitable prior art linkers consist of the repeated GGGGS amino acid sequence or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur.J. Immunol.31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001) Cancer Immunol.
  • the antigen-binding fragment of an antibody is a diabody, i.e., a diabody in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow expression on both ends of the same chain. Pairing between two domains forces the domain to pair with the complementary domain of another chain and creates two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444 -6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).
  • the antigen-binding fragment of an antibody is a "bispecific antibody", which refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) or antibody analog through a conjugation arm.
  • Linkages include, but are not limited to, chemical reactions, gene fusion, and enzymatic reactions.
  • Antigen-binding fragments of antibodies may be "multispecific antibodies” including, for example, trispecific antibodies, which are antibodies with three different antigen-binding specificities, and tetraspecific antibodies, which are antibodies with four different antigen-binding specificities. antibodies.
  • ankyrin repeat protein linked to IgG antibody, scFv-Fc antibody fragment or combination thereof, such as CN104341529A.
  • Anti-IL-17a fynomer combined with anti-IL-6R antibody, as in WO2015141862A1.
  • a given antibody (such as the monoclonal antibody 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3 provided by the present invention) can be obtained using conventional techniques known to those skilled in the art (for example, recombinant DNA technology or enzymatic or chemical fragmentation methods).
  • 26B12H1L4, and 26B12H4L4) antigen-binding fragments of antibodies (for example, the above-mentioned antibody fragments) were obtained, and the antigen-binding fragments of antibodies were screened for specificity in the same manner as for whole antibodies.
  • the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, except for natural mutations that may occur spontaneously, A population of identical antibody molecules.
  • mAbs are highly specific for a single epitope on an antigen.
  • polyclonal antibodies usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on antigens.
  • Monoclonal antibodies can usually be obtained using hybridoma technology first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256(5517): 495), but it can also be obtained by recombinant DNA technology (see US Patent 4,816,567).
  • humanized antibody refers to the replacement of all or part of the CDR regions of a human immunoglobulin (recipient antibody) with the CDR regions of a non-human antibody (donor antibody).
  • Antibodies or antibody fragments, wherein the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of corresponding non-human antibodies, or by amino acid residues of other antibodies, so as to further improve or optimize the performance of the antibody.
  • isolated means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolated or “isolated” do not exclude the admixture of artificial or synthetic substances, nor the presence of other impurities which do not affect the activity of the substance.
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector is called an expression vector.
  • a vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phage such as lambda phage or M13 phage and animal viruses.
  • artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • Phage such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, papillomaviruses, Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • papillomaviruses papillomaviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolar virus eg
  • the term "host cell” refers to cells that can be used to introduce vectors, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK 293 cells or human cells.
  • an antibody that specifically binds to an antigen refers to an antibody that is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Binds the antigen with an affinity (K D ) of 10 ⁇ 8 M, 10 ⁇ 9 M or 10 ⁇ 10 M or less.
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and antigen.
  • the antibody has a dissociation equilibrium constant (K D ) of less than about 10 ⁇ 5 M, such as less than about 10 ⁇ 6 M, 10 ⁇ 7 M, 10 ⁇ 8 M, 10 ⁇ 9 M, or 10 ⁇ 10 M or less.
  • Binds antigen eg, TIGIT protein).
  • KD can be determined using methods known to those skilled in the art, for example using a Fortebio Molecular Interaction Instrument.
  • amino acids are generally represented by single-letter and three-letter abbreviations known in the art.
  • alanine can be represented by A or Ala.
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient compatible with the subject and the active ingredient pharmacologically and/or physiologically, These are well known in the art (see e.g. Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers agent.
  • pH regulators include but not limited to phosphate buffer
  • surfactants include but not limited to cationic, anionic or nonionic surfactants such as Tween-80
  • ionic strength enhancers include but not limited to sodium chloride.
  • an effective amount refers to an amount sufficient to achieve, or at least partially achieve, the desired effect.
  • an effective amount for preventing a disease refers to an amount sufficient to prevent, arrest, or delay the occurrence of a disease (such as a tumor);
  • an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the occurrence of a disease in a patient Amount of disease and its complications.
  • hybridomas and offspring cells are used interchangeably, and when the terms “hybridoma” and “hybridoma cell line” are referred to, they also include subclones of hybridomas and offspring cells.
  • single drug dosage unit means at the time of the dosing regimen, (preferably per kg body weight of the subject) to be administered to the subject comprising the anti-CTLA4 and anti-PD-1 bispecific antibody of the present invention, A single dosage form of the anti-TIGIT antibody, such as an injection, eg in an ampoule.
  • the dosing regimen comprises, for example, administration of a single pharmaceutical dosage unit according to a dosing cycle of twice daily to about every other day, or every 3, 4, 5, 6 days , 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks administration once.
  • first such as the first protein functional region, the first connecting fragment
  • second such as the second protein functional region, the second connecting fragment
  • a “therapeutically effective amount” or “therapeutically effective dose” of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes regression of a disease that Disease regression is evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of impairment or disability resulting from disease affliction.
  • the ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predicting efficacy in humans, or by in vitro assays The activity of the drug was determined in the assay.
  • a “prophylactically effective amount" of a drug means that when administered alone or in combination with an antineoplastic agent to a subject at risk of developing cancer (e.g., a subject with a premalignant condition) or a subject at risk of recurrence of the cancer, Amount of any drug that prevents cancer from developing or coming back. In certain embodiments, the prophylactically effective amount completely prevents the occurrence or recurrence of cancer. "Inhibiting" the occurrence or recurrence of cancer means reducing the likelihood of the occurrence or recurrence of cancer, or preventing the occurrence or recurrence of cancer altogether.
  • the monoclonal antibody of the present invention can specifically bind to TIGIT well, and has extremely strong affinity, reduces the effect of TIGIT on inhibiting immune cells, promotes T cell activity, reverses NK cell exhaustion, and enhances the killing effect of immune cells on tumors.
  • Anti-TIGIT antibodies alone or in combination with anti-CTLA4-anti-PD-1 bispecific antibodies (and/or chemotherapy drugs) can effectively treat or prevent tumors, and anti-TIGIT antibodies combined with anti-CTLA4-anti-PD-1 bispecific antibodies can effectively inhibit tumors
  • the pharmacological effect of growth is better than that of anti-TIGIT antibody single drug or anti-CTLA4-anti-PD-1 bispecific antibody single drug, and has good application prospects and market value.
  • Figure 1 Detection results of binding activity of 26B12H1L1, 26B12H2L2, 26B12H2L3, 26B12H3L2 and RG6058 antibodies to TIGIT-mFc.
  • Figure 2 Detection results of binding activity of 26B12H3L3, 26B12H1L4, 26B12H4L1, 26B12H4L4 and RG6058 antibodies to TIGIT-mFc.
  • Figure 3 The activity detection results of 26B12H1L1, 26B12H2L2, 26B12H2L3, 26B12H3L2 and RG6058 antibodies competing with human CD155-hFc-Biotin for binding to TIGIT-mFc.
  • Figure 4 The activity detection results of 26B12H3L3, 26B12H1L4, 26B12H4L1, 26B12H4L4 and RG6058 antibodies competing with human CD155-hFc-Biotin for binding to TIGIT-mFc.
  • Figure 5 The results of detection of affinity constants between 26B12H3L3 and TIGIT-mFc.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 5nM, 1.67nM, 0.557nM, 0.185nM, and 0.06nM, respectively.
  • Figure 6 The results of detection of affinity constants between 26B12H1L1 and TIGIT-mFc.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 5nM, 1.67nM, 0.557nM, 0.185nM, and 0.06nM, respectively.
  • Figure 7 Graph of detection results of affinity constant between 26B12H2L2 and TIGIT-mFc.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 5nM, 1.67nM, 0.557nM, 0.185nM, and 0.06nM, respectively.
  • Figure 8 The results of detection of affinity constants between 26B12H2L3 and TIGIT-mFc.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 5nM, 1.67nM, 0.557nM, 0.185nM, and 0.06nM, respectively.
  • Figure 9 Graph of detection results of affinity constant between 26B12H3L2 and TIGIT-mFc.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 5nM, 1.67nM, 0.557nM, 0.185nM, and 0.06nM, respectively.
  • Figure 10 Graph of detection results of affinity constant between 26B12H4L4 and TIGIT-mFc.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 5nM, 1.67nM, 0.557nM, 0.185nM, and 0.06nM, respectively.
  • Figure 11 Graph of detection results of affinity constant between 26B12H1L4 and TIGIT-mFc.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 5nM, 1.67nM, 0.557nM, 0.185nM, and 0.06nM, respectively.
  • Figure 12 Graph of detection results of affinity constant between 26B12H4L1 and TIGIT-mFc.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 5nM, 1.67nM, 0.557nM, 0.185nM, and 0.06nM, respectively.
  • Figure 13 The detection results of the affinity constant between RG6058 and TIGIT-mFc.
  • concentrations of antibodies added to each pair of curves from top to bottom in the figure are 5nM, 1.67nM, 0.557nM, 0.185nM, and 0.06nM, respectively.
  • Figure 14 FACS detection of humanized antibodies 26B12H2L2 and RG6058 binding activity to 293T-TIGIT cell membrane surface antigen TIGIT.
  • Figure 15 FACS detection of the activity of humanized antibodies 26B12H2L2 and RG6058 competing with CD155 to bind to TIGIT on the cell membrane surface of 293T-TIGIT.
  • Figure 16 FACS detection of the activity of humanized antibodies 26B12H2L2 and RG6058 competing with CD112 to bind to TIGIT on the cell membrane surface of 293T-TIGIT.
  • Figure 17 Detection of the amount of secreted IL-2 after adding TIGIT antibody in Jurkat-TIGIT and HT1080-aCD3scFv cell systems.
  • Figure 18 Effect of hTIGIT-BALB/c transgenic mouse CT26 tumor model.
  • Figure 19 Changes in body weight of the CT26 tumor model of hTIGIT-BALB/c transgenic mice.
  • Figure 20 The effect of 26B12H2L2 combined with anti-CTLA4-anti-PD-1 bispecific antibody CP004(hG1TM) on the CT26 tumor model of BALB/c-hPD1/hTIGIT transgenic mice.
  • Figure 21 The combination of 26B12H2L2 and anti-CTLA4-anti-PD-1 bispecific antibody CP004(hG1TM) changes the body weight of BALB/c-hPD1/hTIGIT transgenic mouse CT26 tumor model.
  • Hybridoma cell line LT019 (TIGIT-26B12), which was deposited in the China Center for Type Culture Collection (CCTCC) on October 23, 2020, with the preservation number CCTCC NO: C2020208, and the preservation address is China. Wuhan. Wuhan University, zip code :430072.
  • BALB/c mice used were purchased from Guangdong Medical Experimental Animal Center.
  • the positive control antibody RG6058 used can refer to sequence 34 and sequence 36 in Chinese patent publication CN108290946 for its sequence.
  • the combined anti-CTLA4-anti-PD-1 bispecific antibody CP004(hG1TM) used was produced by Zhongshan Kangfang Biomedical Co., Ltd., and its sequence was derived from the published patent CN112300286A
  • For the antibody refer to the full-length sequence of the heavy chain of CP004 (hG1TM) (the amino acid sequence is shown in SEQ ID NOs:57, and the nucleotide sequence is shown in SEQ ID NO:58), the full-length sequence of the light chain (the amino acid sequence is shown in SEQ ID NO:58) NOs:59, nucleotide sequence as shown in SEQ ID NO:60),
  • CP004(hG1TM) structure is IgG-scFv, wherein IgG part is anti-PD1 antibody, scFv part is anti-CTLA4 antibody,
  • the HCDR1 sequence of the anti-PD1 antibody is shown in SEQ ID NO: 61
  • the HCDR2 sequence is shown in SEQ ID NO: 62
  • the HCDR3 sequence is shown in SEQ ID NO: 63
  • the VH sequence is shown in SEQ ID NO: 73
  • anti-PD1 The LCDR1 sequence of the antibody is shown in SEQ ID NO:70
  • the LCDR2 sequence is shown in SEQ ID NO:71
  • the LCDR3 sequence is shown in SEQ ID NO:72
  • the VL sequence is shown in SEQ ID NO:76
  • the HCDR1 sequence of the anti-CTLA4 antibody is shown in SEQ ID NO:64
  • the HCDR2 sequence is shown in SEQ ID NO:65
  • the HCDR3 sequence is shown in SEQ ID NO:66
  • the VH sequence is shown in SEQ ID NO:74.
  • Anti-CTLA4 antibody The LCDR1 sequence is shown in SEQ ID NO:67
  • the LCDR2 sequence is shown in SEQ ID NO:68
  • the LCDR3 sequence is shown in SEQ ID NO:69
  • the VL sequence is shown in SEQ ID NO:75
  • the antigen used to prepare the anti-TIGIT antibody is human TIGIT-mFc (TIGIT is Genbank ID: NP_776160.2, and the sequence of mFc is shown in SEQ ID NO: 77).
  • Splenocytes from immunized mice were fused with mouse myeloma cells to produce hybridoma cells.
  • human TIGIT-mFc as an antigen, hybridoma cells were screened by indirect ELISA method to obtain hybridoma cells that can secrete antibodies that specifically bind to TIGIT.
  • a stable hybridoma cell line was obtained by the limiting dilution method.
  • the above hybridoma cell lines were named hybridoma cell line LT019, and the monoclonal antibodies secreted by them were named 26B12.
  • Hybridoma cell line LT019 also known as TIGIT-26B12
  • CTCC China Center for Type Culture Collection
  • the LT019 cell line prepared above was cultured with CD medium (Chemical Defined Medium, containing 1% penicillin and streptomycin) under the condition of 5% CO 2 and 37°C. After 7 days, the cell culture supernatant was collected, subjected to high-speed centrifugation, vacuum-filtered with a microporous membrane, and purified using a HiTrap protein A HP column to obtain antibody 26B12.
  • CD medium Chemical Defined Medium, containing 1% penicillin and streptomycin
  • the mRNA was extracted from the LT019 cell line cultured in Example 1 according to the method of Cultured Cell Bacteria Total RNA Extraction Kit (Tiangen, Cat. No. DP430).
  • the PCR amplification product was directly cloned by TA, and the specific operation was performed according to the pEASY-T1 Cloning Kit (Transgen CT101) kit manual.
  • the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and the fragment length is 363bp.
  • the encoded amino acid sequence is shown in SEQ ID NO: 1, with a length of 121 amino acids.
  • the sequence of heavy chain HCDR1 is shown in SEQ ID NO:3, the sequence of HCDR2 is shown in SEQ ID NO:4, and the sequence of HCDR3 is shown in SEQ ID NO:5.
  • the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO: 7, and the length is 321bp.
  • amino acid sequence encoded by it is shown in SEQ ID NO: 6, with a length of 107 amino acids.
  • the sequence of the light chain LCDR1 is shown in SEQ ID NO:8, the sequence of LCDR2 is shown in SEQ ID NO:9, and the sequence of LCDR3 is shown in SEQ ID NO:10.
  • Example 3 Light chain and heavy chain design and preparation of humanized antibodies against human TIGIT
  • the antibody model was simulated by computer, and mutations were then designed according to the model to obtain the potential of antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4
  • Variable region sequence antibody constant region sequence, from NCBI database, heavy chain constant region is Ig gamma-1 chain C region, ACCESSION: P01857; light chain constant region is Ig kappa chain C region, ACCESSION: P01834).
  • the length of the nucleic acid sequence of the heavy chain variable region is 363bp, and the length of the encoded amino acid sequence is 121aa;
  • the nucleic acid sequences of the regions are all 321bp in length, and the encoded amino acid sequences are all 107aa in length.
  • HCDR1 The sequence of HCDR1 is shown in SEQ ID NO:3, the sequence of HCDR2 is shown in SEQ ID NO:4, and the sequence of HCDR3 is shown in SEQ ID NO:5;
  • the sequence of LCDR1 is shown in SEQ ID NO:8, the sequence of LCDR2 is shown in SEQ ID NO:9, and the sequence of LCDR3 is shown in SEQ ID NO:10.
  • Ig gamma-1chain C region is used for the heavy chain constant region, ACCESSION: P01857; the light chain constant region is used for Ig kappa chain C region, ACCESSION: P01834.
  • the corresponding light and heavy chain recombination plasmid design gene combination (pcDNA3.1-26B12H1/pcDNA3.1-26B12L1, pcDNA3.1-26B12H4/pcDNA3.1-26B12L1, pcDNA3.1-26B12H2/pcDNA3.1-26B12L2 , pcDNA3.1-26B12H3/pcDNA3.1-26B12L2, pcDNA3.1-26B12H2/pcDNA3.1-26B12L3, pcDNA3.1-26B12H3/pcDNA3.1-26B12L3, pcDNA3.1-26B12H1/pcDNA3.1-26B12L4, and pcDNA3 .1-26B12H4/pcDNA3.1-26B12L4) were co-transfected into 293F cells and the culture medium was collected for purification.
  • an endotoxin-free expression plasmid was prepared and the plasmid was transiently transfected into HEK293 cells for antibody expression. After culturing for 7 days, the cell culture fluid was collected, and protein A column was used for affinity purification to obtain humanized antibodies.
  • Embodiment 4 ELISA method measures the binding activity of antibody and antigen TIGIT-mFc
  • the plate was washed 4 times with PBST, and then TMB (Neogen, 308177) was added to develop color in the dark for 4 min, and stop solution was added to terminate the color reaction.
  • TMB Neogen, 308177
  • stop solution was added to terminate the color reaction.
  • the microplate plate into the microplate reader, select the light wavelength of 450nm to read the OD value of each well of the microplate plate. The data were analyzed and processed with SoftMax Pro 6.2.1 software.
  • Embodiment 5 competition ELISA method measures antibody and CD155-hFc-Biotin competition binding respectively activity of TIGIT-mFc
  • TIGIT-mFc 2 ⁇ g/mL was coated on the microtiter plate, and incubated overnight at 4°C. After the incubation, the antigen-coated ELISA plate was washed once with PBST, and then 1% BSA in PBST solution was used as the ELISA plate blocking solution for blocking for 2 hours. After the enzyme plate was blocked, the plate was washed 3 times with PBST.
  • Table 3 and Table 4 show the results of the competition between the antibody and CD155-hFc-Biotin for binding to TIGIT-mFc. Taking the antibody concentration as the abscissa and the absorbance value as the ordinate, curve fitting was performed to calculate the EC 50 of the antibody competing with CD155-hFc-Biotin for binding to TIGIT-mFc. The results are shown in Table 3 and Table 4, Figure 3 and Figure 4 below.
  • Table 4 Activity detection results of 26B12H3L3, 26B12H1L4, 26B12H4L1, 26B12H4L4 and RG6058 competing with CD155-hFc-Biotin for binding to TIGIT-mFc
  • 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 have the function of effectively competing with CD155-hFc-Biotin for binding to TIGIT-mFc.
  • Example 6 Determination of humanized antibody 26B12H3L3, 26B12H1L1, 26B12H2L2, 26B12H2L3, 26B12H3L2, 26B12H4L4, Kinetics of binding of 26B12H1L4, 26B12H4L1 and RG6058 to antigen TIGIT-mFc parameter
  • the sample dilution buffer is PBS, 0.02% Tween-20, 0.1% BSA, pH7.4. Immobilize TIGIT-mFc on the AMC sensor at a concentration of 3 ⁇ g/mL for 50 s, equilibrate the sensor in the buffer for 60 s, and bind the TIGIT-mFc immobilized on the sensor to the antibody at a concentration of 0.06-5 nM (triple dilution) , time 120s, protein dissociation in the buffer, time 300s.
  • the detection temperature is 37 degrees
  • the detection frequency is 0.3 Hz
  • the vibration rate of the sample plate is 1000 rpm.
  • the data were analyzed with a 1:1 model fit to obtain affinity constants.
  • TIGIT antibodies to TIGIT-mFc from strong to weak were: 26B12H2L2, 26B12H4L1, 26B12H1L1, RG6058, 26B12H4L4, 26B12H1L4, 26B12H2L3, 26B12H3L2, 26B12H3L3.
  • the affinity of humanized antibodies 26B12H2L2, 26B12H4L1, and 26B12H1L1 is stronger than that of the positive drug RG6058, while the affinity of 26B12H4L4 is comparable to that of the positive drug RG6058.
  • Example 7 FACS detection of binding activity of humanized antibodies 26B12H2L2 and RG6058 to 293T-TIGIT cell membrane surface antigen TIGIT
  • TIGIT vector plenti6.3-TIGITFL-BSD (TIGIT is GenbankID: NP_776160.2, commissioned GenScript Gene to optimize and synthesize the full-length cDNA sequence of human TIGIT, named TIGITFL, and cloned it into pUC57simple (provided by GenScript) vector, Obtain pUC57simple-TIGITFL plasmid.With the pUC57simple-TIGITFL plasmid synthesized by BamHI&XhoI double digestion, reclaim the TIGITFL target gene fragment and pass restriction site BamHI&XhoI subcloning to plenti6.3 expression vector, carrier pLenti6.3 is purchased from Invitrogen company) transfection 293T cells were screened to obtain the cell line 293T-TIGIT cells stably expressing TIGIT.
  • Collect 293T-TIGIT cells (DMEM+10% FBS), centrifuge for 5 minutes, remove the supernatant, resuspend, count and viability (P7, 95.79%), dilute the cells, add 30w cells to each well of a transparent pointed-bottom 96-well plate , add 200 ⁇ L 1% PBSA to each tube, centrifuge for 5 min, and remove the supernatant.
  • add 100 ⁇ L of antibody to each well final concentration is 300nM, 100nM, 33.3nM, 11.1nM, 3.7nM, 1.23nM, 0.41nM, 0.041nM, 0.0041nM), and design blank control and isotype control, and incubate on ice 60min.
  • Table 6 FACS detection of binding activity of humanized antibodies 26B12H2L2 and RG6058 to 293T-TIGIT cell membrane surface antigen TIGIT
  • the experimental results are shown in Table 6 and Figure 14.
  • the EC 50 of the positive control antibody RG6058 binding to the cell membrane surface antigen TIGIT was 1.257nM, while the EC 50 of the humanized antibody 26B12H2L2 binding to the cell membrane surface antigen TIGIT was 0.917nM.
  • the experimental results showed that the ability of the humanized antibody 26B12H2L2 to bind to the cell membrane surface antigen TIGIT was stronger than that of the positive control antibody RG6058.
  • Example 8 FACS detection of humanized antibodies 26B12H2L2 and RG6058 with CD155 or Competitive activity of human CD112 binding to 293T-TIGIT cell membrane surface antigen TIGIT
  • Experimental method Collect 293T-TIGIT cells, centrifuge for 5 minutes, remove the supernatant, resuspend, count and viability (94.95%), dilute the cells, add 30w cells to each well of a transparent pointed-bottom 96-well plate, add 200 ⁇ L 1 to each tube %PBSA, centrifuged for 5min, and removed the supernatant. Add 100 ⁇ L of antibody to each well according to the experimental design (final concentrations are 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.123 nM, 0.0123 nM), and design blank control and isotype control, and incubate on ice for 30 min.
  • CD155 final concentration of 10nM
  • CD112 final concentration of 30nM
  • Zhongshan Akefang Biomedical Co., Ltd. batch number: 20190726, where the GenBank number of CD112 is NP_001036189.1
  • incubate on ice in the dark for 60 minutes then add 200 ⁇ L of 1% PBSA to each tube, centrifuge for 5 minutes, remove the supernatant, and wash twice.
  • APC goat anti-mouse IgG purchased from Biolegend Company, lot number 405308 (minimal x-reactivity) antibody (300-fold dilution in PBSA) to each sample, and incubate on ice in the dark for 40 min; add 200 ⁇ L PBSA to each tube, and centrifuge 5min, remove the supernatant. Add 200 ⁇ L PBSA to resuspend the cells, transfer to the flow tube, and flow cytometer to detect the average fluorescence intensity of the cells at each concentration.
  • Table 7 FACS detection of activity of humanized antibodies 26B12H2L2 and RG6058 competing with CD155 to bind to 293T-TIGIT cell membrane surface antigen TIGIT
  • Table 8 FACS detection of activity of humanized antibodies 26B12H2L2 and RG6058 competing with CD112 to bind to 293T-TIGIT cell membrane surface antigen TIGIT
  • the EC 50 of the positive control antibody RG6058 competing for CD155 binding to TIGIT was 1.212nM, while the EC 50 of the humanized antibody 26B12H2L2 competing for CD155 binding to TIGIT was 1.049nM; the EC 50 of the positive control antibody RG6058 competing for CD112 binding to TIGIT was 1.224nM, while the EC 50 of humanized antibody 26B12H2L2 competing for CD112 binding to TIGIT was 1.140nM.
  • Example 9 Adding TIGIT to Jurkat-TIGIT and HT1080-aCD3scFv cell systems antibody mixed lymphatic reaction
  • TIGIT vector plenti6.3/V5-TIGITFL-BSD vector pLenti6.3 purchased from Invitrogen
  • vector pLenti6.3 purchased from Invitrogen
  • anti-CD3 antibody vector pCDH-aCD3scFv-puro Entrust GenScript to optimize and synthesize the cDNA sequence of anti-CD3scFv, and clone it into the pUC57simple (provided by GenScript) vector to obtain the pUC57simpl
  • Collect Jurkat-TIGIT and HT1080-aCD3scFv cells in the logarithmic growth phase In a 96-well plate, add 5W cells to each well of Jurkat-TIGIT and add 1W cells to each well of HT1080-aCD3scFv. Add diluted antibody (final concentration: 10nM, 50nM, 250nM) and anti-human CD28 antibody (purchased from R&D Company, product number: MAB342-500) (3 ⁇ g/mL), put it into the incubator and cultivate for 48h; Clear, IL-2 content was detected with IL-2 ELISA detection kit.
  • diluted antibody final concentration: 10nM, 50nM, 250nM
  • anti-human CD28 antibody purchased from R&D Company, product number: MAB342-500
  • Example 10 26B12H2L2 pairs inoculated with CT26 mice in hTigit-BALB/c transgenic mice Therapeutic effect of transplanted tumor in mice
  • mice Inoculate 500,000 CT26 on the back of hTigit-BALB/c transgenic mice (the mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., and the normal TIGIT gene of the mice was replaced by the human TIGIT gene).
  • Cells mouse colon cancer cell line, purchased from ATCC
  • the specific steps of the experiment were 25 million/ml CT26 cells were inoculated with 200 ⁇ l of each inoculated mouse to establish a mouse tumor model.
  • 26B12H2L2 has a strong pharmacological effect on the hTIGIT-BALB/c transgenic mouse CT26 tumor model; it has the potential to treat and/or prevent tumors, especially colon cancer.
  • 26B12H2L2 had no effect on the body weight of hTIGIT-BALB/c transgenic mice as a CT26 tumor model, indicating that the 26B12H2L2 antibody had no toxic side effects on the mice.
  • Example 11 Combination of anti-TIGIT antibody and anti-CTLA4-anti-PD-1 bispecific antibody for effective treatment
  • CT26 cells human colon cancer cells, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • inoculated subcutaneously in female BALB/c-hPD1/hTIGIT mice aged 5-7 weeks purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • the volume was randomly divided into 4 groups, 6 rats in each group.
  • the day of grouping was defined as day D0, and the administration began on day D0 of grouping.
  • the administration method of the combined administration group is as follows: the drugs are prepared separately and administered successively (there is no specific requirement on the order and interval of administration, one drug is given before another drug is administered). See Table 10 for modeling and specific administration methods. After administration, the length and width of the tumors in each group were measured, and the tumor volume was calculated.
  • Table 10 Dosing regimen of anti-TIGIT antibody combined with anti-CTLA4-anti-PD-1 bispecific antibody in the treatment of CT26 transplanted tumor BALB/c-hPD1/hTIGIT mouse model
  • the tumor-bearing mice had good tolerance to the test drug CP004(hG1TM) and 26B12H2L2 alone and in combination, and each group had no effect on the body weight of the tumor-bearing mice.

Abstract

一种药物组合物，其包含抗TIGIT抗体或其抗原结合片段，和抗CTLA4-抗PD-1双特异性抗体或其抗原结合片段。其中抗体的重链可变区包含氨基酸序列分别如SEQ ID NOs:3-5所示的HCDR1-HCDR3；轻链可变区包含氨基酸序列分别如SEQ ID NOs:8-10所示的LCDR1-LCDR3。

Description

药物组合物及用途 技术领域
本发明属于医药领域，涉及药物组合物，其包含抗TIGIT抗体或其抗原结合片段，和抗CTLA4-抗PD-1双特异性抗体或其抗原结合片段。
背景技术
TIGIT(T cell Ig and ITIM domain,又称为WUCAM，Vstm3，VSIG9)是脊髓灰质炎病毒受体(PVR)/Nectin家族的成员。TIGIT由细胞外免疫球蛋白可变区(IgV)结构域，I型跨膜结构域和具有经典免疫受体酪氨酸抑制基序(ITIM)和免疫球蛋白酪氨酸尾(ITT)基序的细胞内结构域组成。TIGIT在淋巴细胞特别是在效应和调节性CD4+T细胞、滤泡辅助CD4+T细胞和效应CD8+T细胞以及自然杀伤(NK)细胞中高表达(Yu X,Harden K,Gonzalez L C,et al.The surface protein TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells[J].Nature immunology,2009,10(1):48)。
CD155(又称为PVR、Necl5或Tage4)、CD112(又称为PVRL2/nectin 2)和CD113(又称为PVRL3)是TIGIT结合的配体(Martinet L,Smyth M J.Balancing natural killer cell activation through paired receptors[J].Nature Reviews Immunology,2015,15(4):243-254)，其中CD155是TIGIT的高亲和力配体。在NK细胞中，TIGIT结合配体CD155和CD112可以抑制NK细胞对TIGIT高表达细胞的杀伤作用(Stanietsky N,Simic H,Arapovic J,et al.The interaction of TIGIT with PVR and PVRL2inhibits human NK cell cytotoxicity[J].Proceedings of the National Academy of Sciences,2009,106(42):17858-17863)。而有报道发现在同时阻断PD-1和TIGIT时，可以增强CD8+T细胞的杀伤作用(Johnston R J,Comps-Agrar L,Hackney J,et al.The  immunoreceptor TIGIT regulates antitumor and antiviral CD8+T cell effector function[J].Cancer cell,2014,26(6):923-937)。在最新的研究中发现，TIGIT作为NK细胞的免疫检查点，肿瘤发展过程中抑制性受体TIGIT可导致NK细胞耗竭，并证明抗TIGIT单抗可逆转NK细胞耗竭并用于多种肿瘤的免疫治疗(Zhang Q,Bi J,Zheng X,et al.Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-tumor immunity[J].Nature immunology,2018,19(7):723-732)。
此外有报道，TIGIT阻断剂单独或与PD-1阻滞剂联合使用再加上CD96阻断剂，可以显著降低野生型和Cd155-/-小鼠模型中B16黑色素瘤的生长(Li X-Y,Das I,Lepletier A,et al..Cd155 loss enhances tumor suppression via combined host and tumor-intrinsic mechanisms.J Clin Invest 2018；128:2613–25)。CD112R阻断剂单独或与TIGIT阻断剂和/或PD-1阻断剂联合使用，能增加卵巢瘤、子宫内膜瘤和肺肿瘤中TIL产生细胞因子的能力(Whelan S,Ophir E,Kotturi MF,et al..PVRIG and PVRL2 Are Induced in Cancer and Inhibit CD8 +T-cell Function.Cancer Immunol Res 2019；7:257–68)。
抗TIGIT抗体药物作为新型免疫检查点抗体药物具有广泛的应用前景，可用于肿瘤的免疫治疗。罗氏制药(Roche)研发的Tiragolumab已处于临床3期阶段，并且据报道TIGIT单抗Tiragolumab联合PD-L1药物Tecentrip(阿特珠单抗Atezolizumab)作为一线疗法，在治疗PD-L1阳性转移性非小细胞肺癌(NSCLC)患者的2期临床研究中发现Tiragolumab与Tecentriq的组合耐受性良好，疾病进展风险下降43％，联用效果显著(Exit C.Roche to present first clinical data on novel anti-TIGIT cancer immunotherapy tiragolumab at ASCO[J])。已有的临床信息记录表明，TIGIT是用于治疗非小细胞肺癌、小细胞肺癌、乳腺癌、卵巢癌、结直肠癌、黑色素癌、胰腺癌、宫颈瘤、多发性骨髓瘤、非霍奇金淋巴瘤、B淋巴细胞瘤、浆细胞癌的重要靶点。
跨膜受体PD-1(程序性细胞死亡-1)是CD28基因家族成员之一，在活化的T细胞，B细胞以及骨髓系细胞都有表达。PD-1的配体PDL1(Programmed cell death 1 ligand 1，亦简称为PDL-1)和PDL2(Programmed cell death 1 ligand 2，亦简称为PDL-2)均属于B7超家族，其中PDL1在多种细胞都有表达，包括T细胞，B细胞以及内皮细胞和上皮细胞，PDL2则仅表达于抗原呈递细胞如树突状细胞和巨噬细胞。
PD-1/PDL1信号通路在调节免疫耐受、微生物感染及肿瘤免疫逃逸中发挥重要作用。PD-1的表达主要在T细胞等免疫细胞，而PD-1的配体PDL1主要在许多人类肿瘤组织呈高表达。阻断PD-1/PDL1信号通路可使被抑制的T细胞激活，进而攻击癌细胞。阻断PD-1/PDL1信号可以促进肿瘤抗原特异性T细胞的增殖，发挥杀伤肿瘤细胞的作用，进而抑制局部肿瘤生长(Julie R et al.,2012，N Engl J Med.366:2455–2465)。另外，高表达PDL1的肿瘤伴随着很难被检测到的癌症(Hamanishi et al.,2007,Proc.Natl.Acad.Sci.USA 104:3360-5)。一种实施有效的方法是通过体内注射抗PD-1抗体对PD-1的表达进行调控。由于PD-1抗体的广谱抗肿瘤前景和惊人的药效，业界普遍认为针对PD-1通路的抗体将带来多种肿瘤治疗的突破性进展：例如用于治疗非小细胞性肺癌，肾细胞癌，卵巢癌，黑色素瘤(Homet M.B.,Parisi G.,et al.,2015,Semin Oncol.42(3):466-473)，白血病以及贫血病(Held SA,Heine A,et al.,2013,Curr Cancer Drug Targets.13(7):768-74)。
细胞毒性T淋巴细胞相关抗原-4(cytotoxic T lymphocyte sociated antigen 4，亦简称为CTLA4)与CD28分子在基因结构、染色体定位、序列的同源性及基因表达具有十分相近的关系，都是共刺激分子B7的受体，主要表达于被激活T细胞表面。CTLA4与B7结合后能抑制小鼠和人T细胞的激活，在T细胞活化中起负调节作用。
CTLA4抗体(或抗CTLA4单克隆抗体)或CTLA4配体可以阻止CTLA4与其天然配体结合，从而封闭CTLA4对T细胞负性调节信号 的传导，增强T细胞对各种抗原的反应性，在这方面体内与体外研究结果基本一致。目前已有CTLA4单克隆抗体处于临床试验阶段或被批准用于治疗前列腺癌，膀胱癌，结肠直肠癌，胃肠道癌，肝癌，恶性黑色素瘤等(Grosso JF.,Jure-Kunkel MN.,2013,Cancer Immun.13:5.)。
白细胞介素2(IL-2)由T细胞产生，是调节T细胞亚群的生长因子，也是调控免疫应答的重要因子，并可促进活化B细胞增殖，参与抗体反应、造血和肿瘤监视。重组的人IL-2已经被美国FDA批准用于治疗恶性肿瘤(包括黑色素瘤、肾肿瘤等)，同时正在进行治疗慢性病毒感染的临床研究(Chavez,A.R.,et al.,2009,Ann N Y Acad Sci,1182:p.14-27)。CTLA4及CTLA4抗体作为T细胞功能状况的重要影响因素，干预机体免疫微环境。体外和体内试验中，CTLA4抗体可特异地解除CTLA4对机体免疫抑制，激活T细胞，诱导IL-2产生，在抗肿瘤及寄生虫等疾病的基因治疗中有广泛应用前景。
综上所述，开发更低毒性、更有效的治疗手段以及联合给药治疗方案具有巨大的临床意义。
发明内容
本发明人经过深入的研究和创造性的劳动，利用哺乳动物细胞表达系统表达出重组的人TIGIT作为抗原免疫小鼠，经小鼠脾脏细胞与骨髓瘤细胞融合获得杂交瘤细胞。发明人通过对大量样本的筛选，得到了杂交瘤细胞株LT019(保藏编号为CCTCC NO:C2020208)。
本发明人惊奇地发现，杂交瘤细胞株LT019分别能够分泌产生与人TIGIT特异性结合的特异性单克隆抗体(命名为26B12)，并且该单克隆抗体能够十分有效地结合TIGIT，降低TIGIT抑制免疫细胞的作用，促进T细胞活性，逆转NK细胞耗竭，增强免疫细胞对肿瘤的杀伤作用。进一步地，本发明人创造性地制得了抗人TIGIT的人源化抗体(命名为26B12H1L1、26B12H4L1、26B12H2L2、26B12H3L2、26B12H2L3、26B12H3L3、26B12H1L4和26B12H4L4)。
本发明人还惊奇地发现，本发明的抗体26B12H1L1、26B12H4L1、 26B12H2L2、26B12H3L2、26B12H2L3、26B12H3L3、26B12H1L4和26B12H4L4具有与TIGIT结合的活性，并且具有极强的亲和力；26B12H1L1、26B12H4L1、26B12H2L2、26B12H3L2、26B12H2L3、26B12H3L3、26B12H1L4和26B12H4L4可以有效地降低TIGIT的活性。
由此提供了下述发明：
本发明的一个方面涉及抗TIGIT抗体或其抗原结合片段，其中，
所述抗TIGIT抗体包含SEQ ID NO:1所示的重链可变区包含的HCDR1-HCDR3和SEQ ID NO:6所示的轻链可变区包含的LCDR1-LCDR3，
优选地，按照IMGT编号系统，所述抗体的重链可变区包含氨基酸序列分别如SEQ ID NOs:3-5所示的HCDR1-HCDR3；并且所述抗体的轻链可变区包含氨基酸序列分别如SEQ ID NOs:8-10所示的LCDR1-LCDR3。
在本发明的一个或多个实施方式中，所述的抗TIGIT抗体或其抗原结合片段，其中，所述抗体的重链可变区的氨基酸序列选自SEQ ID NO:1、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15和SEQ ID NO:17；并且
所述抗体的轻链可变区的氨基酸序列选自SEQ ID NO:6、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23和SEQ ID NO:25。
在本发明的一个或多个实施方式中，所述的抗TIGIT抗体或其抗原结合片段，其中，
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:6所示；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:11所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:19所示；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:17所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:19所示；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:13所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:21所示；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:13所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:23所示；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:15所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:21所示；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:15所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:23所示；
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:11所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:25所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:17所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:25所示。
在本发明的一个或多个实施方式中，所述的抗TIGIT抗体或其抗原结合片段，其中，所述的抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
在本发明的一个或多个实施方式中，所述的抗TIGIT抗体或其抗原结合片段，其中，所述抗体的重链恒定区为Ig gamma-1 chain C region(例如NCBI ACCESSION:P01857)；轻链恒定区为Ig kappa chain C region(例如NCBI ACCESSION:P01834)。
在本发明的一个或多个实施方式中，所述的抗TIGIT抗体或其抗原结合片段，其中，所述抗TIGIT抗体或其抗原结合片段选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体、人源化抗体、嵌合抗体或双抗体。
在本发明的一个或多个实施方式中，所述的抗TIGIT抗体或其抗原结合片段，其中，所述抗体以小于4E-10或小于4E-11的K D结合TIGIT-mFc；优选地，所述K D通过Fortebio分子相互作用仪测得。
在本发明的一个或多个实施方式中，所述的抗TIGIT抗体或其抗原结合片段，其中，所述抗体以小于1.5nM、小于1.2nM或小于1nM的EC 50结合TIGIT-mFc；优选地，所述EC 50通过流式细胞仪测得。
在本发明的一些实施方案中，所述的抗TIGIT抗体为单克隆抗体。
在本发明的一些实施方案中，所述的抗TIGIT抗体为人源化抗体、嵌合抗体、多特异性抗体(例如双特异性抗体)。
在本发明的一些实施方式中，所述抗原结合片段选自Fab、Fab'、F(ab') 2、Fd、Fv、dAb、Fab/c、互补决定区片段、单链抗体(例如，scFv)、人源化抗体、嵌合抗体或双特异性抗体。
在本发明的一个或多个实施方式中，所述的抗TIGIT抗体或其抗原结合片段，其中所述抗体是由杂交瘤细胞株LT019产生的抗体，所述杂交瘤细胞株LT019保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2020208。
本发明的另一方面涉及分离的核酸分子，其编码本发明中任一项所述的抗TIGIT抗体或其抗原结合片段。
本发明的再一方面涉及一种载体，其包含本发明的分离的核酸分子。
本发明的再一方面涉及一种宿主细胞，其包含本发明的分离的核酸分子，或者本发明的载体。
本发明的再一方面涉及杂交瘤细胞株LT019，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2020208。
本发明的再一方面涉及偶联物，其包括抗体以及偶联部分，其中，所述抗体为本发明中任一项所述的抗TIGIT抗体或其抗原结合片段，所述偶联部分为可检测的标记；优选地，所述偶联部分为放射性同位素、荧光物质、发光物质、有色物质或酶。
本发明的再一方面涉及试剂盒，其包括本发明中任一项所述的抗TIGIT抗体或其抗原结合片段，或者包括本发明的偶联物；
优选地，所述试剂盒还包括第二抗体，其特异性识别所述抗体；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、发光物质、有色物质或酶。
本发明的再一方面涉及本发明中任一项所述的抗体或者本发明的偶联物在制备试剂盒中的用途，所述试剂盒用于检测TIGIT在样品中的存在或其水平。
本发明的再一方面涉及一种药物组合物，其包含本发明中任一项所述的抗TIGIT抗体或其抗原结合片段或者本发明的偶联物；可选地，所述药物组合物还包括药学上可接受的载体和/或赋形剂。
在本发明的一个或多个实施方式中，所述的药物组合物，其还包含一种或多种抗PD-1抗体，或者一种或多种抗PD-L1抗体，例如抗CTLA4-抗PD-1双特异性抗体。
在本发明的一个或多个实施方式中，所述的药物组合物，其中，按照抗体的质量计算，抗TIGIT抗体或其抗原结合片段与抗PD-1抗体或抗PD-L1抗体的质量比为(1：5)-(5：1)，例如：1：5、1：4、1：3、1：2、1：1、2：1、3：1、4：1或5：1。
本发明的再一方面涉及一种组合产品(例如试剂盒)，其包含独立包装的第一产品和第二产品，其中，
所述第一产品包含本发明中任一项所述的抗TIGIT抗体或其抗原结合片段、本发明的偶联物或者本发明中任一项所述的药物组合物；
所述第二产品包含至少一种抗PD-1抗体或者至少一种抗PD-L1抗体，例如抗CTLA4-抗PD-1双特异性抗体；
优选地，所述组合产品还包含独立包装的第三产品，所述第三产品包含一种或多种化疗药物，
优选地，所述第一产品和所述第二产品还独立地包含一种或多种药学上可接受的辅料；
优选地，所述组合产品还包含产品说明书。
在本发明的一个或多个实施方式中，所述的组合产品，其中，按照抗体的质量计算，抗TIGIT抗体或其抗原结合片段与抗PD-1抗体或抗PD-L1抗体的质量比为(1：5)-(5：1)，例如：1：5、1：4、1：3、1：2、1：1、2：1、3：1、4：1或5：1。
本发明的再一方面涉及本发明中任一项所述的抗体、本发明的偶联物、本发明中任一项所述的药物组合物或者本发明中任一项所述的组合产品在制备治疗和/或预防肿瘤的药物中的用途。
根据本发明中任一项所述的抗体、本发明的偶联物、本发明中任一项所述的药物组合物或者本发明中任一项所述的组合产品，其用于治疗和/或预防肿瘤。
本发明的再一方面涉及一种治疗和/或预防肿瘤的方法，包括给予有需求的受试者以有效量的本发明中任一项所述的抗体、本发明的偶 联物、本发明中任一项所述的药物组合物或者本发明中任一项所述的组合产品的步骤。
本发明涉及一种预防和/或治疗肿瘤(特别是恶性肿瘤)或感染或传染病的方法，包括给受试者施用治疗有效量的抗TIGIT抗体，联合施用抗CTLA4-抗PD-1双特异性抗体，更优选地，进一步联合施用一种或多种药物(如化疗剂或生长抑制剂，靶向治疗剂，抗体-药物缀合物，抗代谢药物，抗生素，植物类抗癌药物和/或激素类药物，阿霉素类，他莫昔芬，甲地孕酮，和/或门冬酰胺酶，优选地，所述抗TIGIT抗体、抗CTLA4-抗PD-1双特异性抗体和肿瘤化疗药物同时或顺序施用。
在一些实施方案中，所述化疗剂或生长抑制剂选自铂类药物(如顺铂、卡铂或奥沙利铂)、氟尿嘧啶类抗肿瘤药、环磷酰胺、培美曲塞、紫杉醇、长春碱类、阿霉素类、戈舍瑞林、烷化剂、蒽环类、抗激素剂、芳香酶抑制剂、抗雄激素剂、蛋白激酶抑制剂、脂质激酶抑制剂、反义寡核苷酸、核酶、抗代谢物、拓扑异构酶抑制剂、细胞毒剂或抗肿瘤抗生素、蛋白酶体抑制剂、抗微管剂、EGFR拮抗剂、类视色素、酪氨酸激酶抑制剂、组蛋白脱乙酰酶抑制剂及其组合。
在一些实施方案中，所述靶向治疗剂选自B-raf抑制剂、MEK抑制剂、K-ras抑制剂、c-Met抑制剂、Alk抑制剂、磷脂酰肌醇3-激酶抑制剂、Akt抑制剂、mTOR抑制剂、双磷脂酰肌醇3-激酶/mTOR抑制剂及其组合。
在一些实施方案中，所述抗体-药物缀合物包含选自下组的药物：美登新碱、单甲基auristatin E、加利车霉素、esperamicin和放射性同位素螯合剂。
本发明涉及一种预防和/或治疗肿瘤(特别是恶性肿瘤)的方法，包括给受试者施用治疗有效量的抗TIGIT抗体，联合施用抗CTLA4-抗PD-1双特异性抗体，更优选地，进一步联合施用一种或多种化疗药物优选地，所述抗TIGIT抗体、抗CTLA4-抗PD-1双特异性抗体和化疗药物同时或顺序施用。
在本发明的一个或多个实施方案中，所述肿瘤选自如下的一种或 多种：胰腺癌、乳腺癌、卵巢癌、结直肠癌、宫颈瘤、多发性骨髓瘤、非霍奇金淋巴瘤、B淋巴细胞瘤、浆细胞癌，头和颈癌、脑癌、咽喉癌、鼻咽癌、食管癌、食管鳞癌、甲状腺癌、间皮瘤、腺癌(如胰腺癌、乳腺癌)、肺癌(如小细胞肺癌、小细胞肺癌)、乳腺癌、肝癌(如肝细胞癌、肝胆癌)、胃癌、胃肠道癌、肠癌(如结肠癌、结肠直肠癌)、胆道癌(如胆管癌)、肾癌、输卵管癌、子宫内膜癌、宫颈癌、膀胱癌、尿路上皮癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、骨髓瘤(如多发性骨髓瘤)、非霍奇金淋巴瘤、B淋巴细胞瘤、浆细胞癌、白血病、淋巴瘤、骨癌、骨肉瘤、软骨肉瘤、高度微卫星不稳定型(MSI-H)或错配修复缺陷型(dMMR)实体瘤。
在本发明的一个或多个实施方案中，所述抗CTLA4-抗PD-1双特异性抗体包括：
靶向PD-1的第一蛋白功能区，和
靶向CTLA4的第二蛋白功能区；
其中，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为单链抗体；或者，所述第一蛋白功能区为单链抗体，所述第二蛋白功能区为免疫球蛋白；
其中，
所述的免疫球蛋白，其包含SEQ ID NO:27所示的重链可变区中的HCDR1-HCDR3(优选分别如SEQ ID NOs:29－31所示的HCDR1－HCDR3)，和SEQ ID NO:28所示的轻链可变区中的LCDR1-LCDR3(优选分别如SEQ ID NOs:32－34所示的LCDR1－LCDR3)；和所述的单链抗体，其包含SEQ ID NO:35所示的重链可变区中的HCDR1-HCDR3(优选分别如SEQ ID NOs:37－39所示的HCDR1－HCDR3)和SEQ ID NO:36所示的轻链可变区中的LCDR1-LCDR3(优选分别如SEQ ID NOs:40－42所示的LCDR1－LCDR3)；
或者，
所述的免疫球蛋白，其包含SEQ ID NO:35所示的重链可变区中的HCDR1-HCDR3(优选分别如SEQ ID NOs:37－39所示的HCDR1－HCDR3)和SEQ ID NO:36所示的轻链可变区中的LCDR1-LCDR3(优选 分别如SEQ ID NOs:40－42所示的LCDR1－LCDR3)；和所述的单链抗体，其包含SEQ ID NO:27所示的重链可变区中的HCDR1-HCDR3(优选分别如SEQ ID NOs:29－31所示的HCDR1－HCDR3)，和SEQ ID NO:28所示的轻链可变区中的LCDR1-LCDR3(优选分别如SEQ ID NOs:32－34所示的LCDR1－LCDR3)。
在具体实施方案中，本发明所述的抗CTLA4-抗PD-1双特异性抗体，其中，
所述免疫球蛋白的重链可变区的氨基酸序列选自SEQ ID NO:27，SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列；并且所述免疫球蛋白的轻链可变区的氨基酸序列选自SEQ ID NO:28，SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列；和，所述单链抗体的重链可变区的氨基酸序列选自SEQ ID NO:35、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47，SEQ ID NO:48或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列；并且所述单链抗体的轻链可变区的氨基酸序列选自SEQ ID NO:36、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51，SEQ ID NO:52或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列；
或者，
所述免疫球蛋白的重链可变区的氨基酸序列选自SEQ ID NO:35、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47，SEQ ID NO:48或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列；并且所述免疫球蛋白的轻链可变区的氨基酸序列选自SEQ ID NO:36、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51，SEQ ID NO:52或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列；和，所述单链抗体的重链可变区的氨基酸序列选自SEQ ID NO:27，SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列；并且所述单链抗体的轻链可变区的氨基酸序列选自SEQ ID NO:28，SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列。
在具体实施方案中，本发明所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述双特异性抗体选自如下的(1)－(20)中的任一项：
(1)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:27或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:28或与其具有至少 60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:35或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:36或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
(2)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:27或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:28或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:45或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:49或与其具有至少60％，至少65％，至少70％，至少 75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
(3)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:27或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:28或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:46或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:50或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
(4)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:44或与其具有至少 60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:35或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:36或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
(5)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:45或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:49或与其具有至少60％，至少65％，至少70％，至少 75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
(6)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:46或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:50或与其具有至少60％，70％，80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％同源性的序列所示；
(7)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:35或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:36或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少 89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:27或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:28或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
(8)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:35或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:36或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％， 93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
(9)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:45或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:49或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:27或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:28或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
(10)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:45或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:49或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少 89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
(11)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:46或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:50或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:27或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:28或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％， 93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
(12)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:46或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:50或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
(13)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:27或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:28或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少 89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:47或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:51或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
(14)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:27或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:28或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:48或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:52或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％， 93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
(15)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:47或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:51或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
(16)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少 89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:48或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:52或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
(17)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:47或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:51或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:27或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:28或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％， 93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
(18)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:48或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:52或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:27或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:28或与其具有至少60％，70％，80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％同源性的序列所示；
(19)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:47或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:51或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重 链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
以及，
(20)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:48或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:52或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源 性的序列所示。
在具体实施方案中，本发明所述的抗CTLA4-抗PD-1双特异性抗体，其中，
所述免疫球蛋白的重链的氨基酸序列如SEQ ID NO:53或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且其轻链的氨基酸序列如SEQ ID NO:54或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示。
在具体实施方案中，本发明所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述第一蛋白功能区与所述第二蛋白功能区直接连接或者通过连接片段连接；和/或所述单链抗体的重链可变区与所述单链抗体的轻链可变区直接连接或者通过连接片段连接。
在具体实施方案中，本发明所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述连接片段为(GGGGS)n，n为正整数；优选地，n为1、2、3、4、5或6。
在具体实施方案中，本发明所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区独立地为1个、2个或者2个以上。
在具体实施方案中，本发明所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述单链抗体(优选重链可变区)连接在免疫球蛋白的重链的C末端。
在具体实施方案中，本发明所述的抗CTLA4-抗PD-1双特异性抗体，其中，
所述免疫球蛋白为人IgG1亚型；
其中，按照EU编号系统，所述免疫球蛋白的重链恒定区具有如下突 变组合之一：
L234A和L235A；或者
L234A和G237A；或者
L235A和G237A；或者
L234A、L235A、G237A。
在具体实施方案中，本发明所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述双特异性抗体包括：
靶向PD-1的第一蛋白功能区，和
靶向CTLA4的第二蛋白功能区；
所述第一蛋白功能区为1个，所述第二蛋白功能区为2个；
其中，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为单链抗体；
所述免疫球蛋白的重链的氨基酸序列如SEQ ID NO:53或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且其轻链的氨基酸序列如SEQ ID NO:54或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:48或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:52或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少 90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
所述单链抗体连接在免疫球蛋白的重链的C末端；
所述第一蛋白功能区与所述第二蛋白功能区通过第一连接片段连接；并且所述单链抗体的重链可变区与所述单链抗体的轻链可变区通过第二连接片段连接；所述第一连接片段和所述第二连接片段相同或不同；
优选地，所述第一连接片段和所述第二连接片段的氨基酸序列独立地选自SEQ ID NO:55和SEQ ID NO:56；
优选地，所述第一连接片段和所述第二连接片段的氨基酸序列均如SEQ ID NO:56所示。
在具体实施方案中，本发明所述的抗CTLA4-抗PD-1双特异性抗体的重链氨基酸序列如SEQ ID NOs:57所示，轻链氨基酸序列如SEQ ID NOs:59所示，双特异性抗体结构为IgG-scFv，其中IgG部分为抗PD1抗体，scFv部分为抗CTLA4抗体，
其中抗PD1抗体的HCDR1序列如SEQ ID NO:61所示，HCDR2序列如SEQ ID NO:62所示，HCDR3序列如SEQ ID NO:63所示，VH序列SEQ ID NO:73所示，抗PD1抗体的LCDR1序列如SEQ ID NO:70所示，LCDR2序列如SEQ ID NO:71所示，LCDR3序列如SEQ ID NO:72所示，VL序列SEQ ID NO:76所示，
抗CTLA4抗体的HCDR1序列如SEQ ID NO:64所示，HCDR2序列如SEQ ID NO:65所示，HCDR3序列如SEQ ID NO:66所示，VH序列SEQ ID NO:74所示，抗CTLA4抗体的LCDR1序列如SEQ ID NO:67所示，LCDR2序列如SEQ ID NO:68所示，LCDR3序列如SEQ ID NO:69所示，VL序列SEQ ID NO:75所示。
在本发明的另一个方面，涉及单位制剂，优选用于治疗肿瘤，其中，所述单位制剂包含：1～10000mg(优选10-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg或200mg)的本发明任一方面所述的抗TIGIT抗体和1～10000mg(优选1-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg、200mg或100mg)的本发明任一方面所述的抗CTLA4-抗PD-1双特异性抗体，和任选地一种或多种本发明所述的化疗药 (如铂类药物和/或氟尿嘧啶类抗肿瘤药)；其中，所述抗TIGIT抗体、所述抗CTLA4-抗PD-1双特异性抗体和化疗药分别单独包装。
本发明涉及用于预防或治疗癌症或肿瘤的方法，其中，向有需要的受试者给予一份或多份本发明所述的单位制剂，优选地，所述单位制剂中的抗CTLA4-抗PD-1双特异性抗体，抗TIGIT抗体和化疗药各自分开施用。
在本发明的另一个方面，涉及单次药物剂量单元，优选用于治疗肿瘤，其包含0.1-10000mg(优选1-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg、200mg或100mg)的本发明任一项所述的抗TIGIT抗体和0.1-10000mg(优选1-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg、200mg或100mg)的本发明任一项所述的抗CTLA4-抗PD-1双特异性抗体。
在本发明的一个或多个实施方案中，其中所述抗TIGIT抗体，所述抗CTLA4-抗PD-1双特异性抗体和/或所述化疗药为适于静脉注射或静脉滴注的形式，优选液体的形式。
在本发明的一个或多个实施方案中，其中给受试者施用有效量的本发明任一项所述抗TIGIT抗体和/或本发明任一项所述抗CTLA4-抗PD-1双特异性抗体的步骤为在手术治疗之前或之后，和/或在放射治疗之前或之后。
在本发明的一个或多个实施方案中，其中，本发明任一项所述抗TIGIT抗体和/或本发明任一项所述抗CTLA4抗PD-1双特异性抗体的单次给药剂量为每千克体重0.1-100mg，优选1-10mg；或者，本发明任一项所述抗TIGIT抗体和/或本发明任一项所述抗CTLA4抗PD-1双特异性抗体的单次给药剂量为每位受试者10-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg或200mg，
优选地，每天两次至约每隔一天一次给药，或每3天、4天、5天、6天、10天、1周、2周、3周、4周、5周或6周给药一次；
优选地，给药方式为静脉滴注或静脉注射。
轻链和重链的可变区决定抗原的结合；每条链的可变区均含有三个高变区，称互补决定区(CDR)(重链(H)的CDR包含HCDR1、HCDR2、HCDR3，轻链(L)的CDR包含LCDR1、LCDR2、LCDR3；其由Kabat等人命名，见Bethesda M.d.,Sequences of Proteins of  Immunological Interest,Fifth Edition,NIH Publication 1991；1-3:91-3242。在已知抗体重链和轻链可变区序列的情况下，目前有几种确定抗体CDR区的方法，包括Kabat，IMGT，Chothia和AbM编号系统。然而，每种关于抗体或其变体的CDR的定义的应用都将在本文定义和使用的术语的范围内。如果给定该抗体的可变区氨基酸序列，则本领域技术人员通常可确定特定CDR，而不依赖于该序列自身之外的任何实验数据。
优选地，CDR也可以由IMGT编号系统定义，请参见Ehrenmann,Francois,Quentin Kaas,and Marie-Paule Lefranc.IMGT/3Dstructure-DB and IMGT/DomainGapAlign:a database and a tool for immunoglobulins or antibodies,T cell receptors,MHC,IgSF and MhcSF.Nucleic acids research 2009；38(suppl_1):D301-D307。
通过本领域技术人员所熟知的技术手段，例如通过VBASE2数据库根据IMGT定义分析单克隆抗体序列的CDR区的氨基酸序列。
本发明涉及的抗体26B12、26B12H1L1、26B12H4L1、26B12H2L2、26B12H3L2、26B12H2L3、26B12H3L3、26B12H1L4和26B12H4L4具有相同的CDR：
其重链可变区的3个CDR区的氨基酸序列如下：
HCDR1:GHSFTSDYA(SEQ ID NO:3)
HCDR2:ISYSDST(SEQ ID NO:4)
HCDR3:ARLDYGNYGGAMDY(SEQ ID NO:5)；
其轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1：QHVSTA(SEQ ID NO:8)
LCDR2：SAS(SEQ ID NO:9)
LCDR3：QQHYITPWT(SEQ ID NO:10)。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关 术语的定义和解释。
如本文中所使用的，当提及TIGIT(NCBI GenBank ID:NP_776160.2)的氨基酸序列时，其包括TIGIT蛋白的全长，或者细胞外免疫球蛋白可变区(IgV)结构域或者包含细胞外免疫球蛋白可变区(IgV)结构域的片段；还包括TIGIT的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在TIGIT蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“TIGIT蛋白”或“TIGIT”应包括所有此类序列，包括所示的序列以及其天然或人工的变体。并且，当描述TIGIT蛋白的序列片段时，其不仅包括序列片段，还包括其天然或人工变体中的相应序列片段。
如本文中所使用的，术语EC 50是指半最大效应浓度(concentration for 50％of maximal effect)，是指能引起50％最大效应的浓度。
如本文中所使用的，术语“抗体”是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(V H)和重链恒定区(C H)组成。重链恒定区由3个结构域(C H1、C H2和C H3)组成。各轻链由轻链可变区(V L)和轻链恒定区(C L)组成。轻链恒定区由一个结构域C L组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。V H和V L区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各V H和V L由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链 /轻链对的可变区(V H和V L)分别形成抗原结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda M.d.(1987and 1991))，或Chothia & Lesk J.Mol.Biol.1987；196:901-917；Chothia等人Nature1989；342:878-883或者IMGT编号系统定义，见Ehrenmann,Francois,Quentin Kaas,and Marie-Paule Lefranc."IMGT/3Dstructure-DB and IMGT/DomainGapAlign:a database and a tool for immunoglobulins or antibodies,T cell receptors,MHC,IgSF and MhcSF."Nucleic acids research 2009；38(suppl_1):D301-D307.的定义。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。在一些情况下，抗原结合片段包括Fab、Fab'、F(ab') 2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
如本文中所使用的，术语“Fd片段”意指由V H和C H1结构域组成的抗体片段；术语“Fv片段”意指由抗体的单臂的V L和V H结构域组成的抗体片段；术语“dAb片段”意指由V H结构域组成的抗体片段(Ward等人,Nature 341:544-546(1989))；术语“Fab片段”意指由V L、V H、C L和C H1结构域组成的抗体片段；术语“F(ab') 2片段”意指包含通过铰链 区上的二硫桥连接的两个Fab片段的抗体片段。
在一些情况下，抗体的抗原结合片段是单链抗体(例如，scFv)，其中V L和V H结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见，例如,Bird等人,Science 242:423-426(1988)和Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988))。此类scFv分子可具有一般结构：NH 2-V L-接头-V H-COOH或NH 2-V H-接头-V L-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS) 4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
在一些情况下，抗体的抗原结合片段是双抗体，即，双价抗体，其中V H和V L结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger P.等人,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等人,Structure 2:1121-1123(1994))。
在另一些情况下，抗体的抗原结合片段是“双特异性抗体”，指由第一抗体(片段)和第二抗体(片段)或抗体类似物通过偶联臂所形成的偶联物，偶联的方式包括但不限于化学反应、基因融合和酶促反应。抗体的抗原结合片段可以是“多特异性抗体”包括例如：三特异性抗体和四特异性抗体，前者是具有三种不同抗原结合特异性的抗体，而后者是具有四种不同抗原结合特异性的抗体。例如，经设计的锚蛋白重复蛋白(DARPin)，与IgG抗体，scFv-Fc抗体片段相连或其组合，如CN104341529A。抗IL-17a的fynomer与抗IL-6R抗体结合，如WO2015141862A1。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的单克隆抗体26B12H1L1、26B12H4L1、26B12H2L2、26B12H3L2、26B12H2L3、26B12H3L3、26B12H1L4和26B12H4L4)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(
Figure PCTCN2022107522-appb-000001
G,Milstein C.Continuous cultures of fused cells secreting antibody of predefined specificity[J].nature,1975；256(5517):495)，但也可采用重组DNA技术获得(如参见U.S.Patent 4,816,567)。
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.,Nature 1986；321:522-525；Reichmann et al.,Nature 1988；332:323-329；Presta,Curr.Op.Struct.Biol.,1992；2:593-596；和Clark M.Antibody humanization:a case of the‘Emperor’s new clothes’？[J].Immunol.Today,2000；21(8):397-402。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或 成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草杆菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，GS细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M 或10 -10M或更小的亲和力(K D)结合该抗原。
如本文中所使用的，术语“K D”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的解离平衡常数(K D)结合抗原(例如，TIGIT蛋白)。可以使用本领域技术人员知悉的方法测定K D，例如使用Fortebio分子相互作用仪测定。
如本文中所使用的，术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用；术语“多克隆抗体”和“多抗”具有相同的含义且可互换使用；术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。
如本文中所使用的，术语“杂交瘤”和“杂交瘤细胞株”可互换使用，并且当提及术语“杂交瘤”和“杂交瘤细胞株”时，其还包括杂交瘤的亚 克隆和后代细胞。
术语“单次药物剂量单元”表示在给药方案时刻，(优选按照受试者每kg体重)待给药于受试者的包含本发明所述的抗CTLA4抗PD-1双特异性抗体、抗TIGIT抗体的单次药物剂型，如注射剂，例如置于安瓿瓶中。在本发明的具体实施方案中，给药方案例如包括根据每天两次至约每隔一天一次的给药周期来给药单次药物剂量单元，或者每3天、4天、5天、6天、10天、1周、2周、3周、4周、5周或6周给药一次。
在本发明中，如果没有特别说明，所述“第一”(例如第一蛋白功能区、第一连接片段)和“第二”(例如第二蛋白功能区、第二连接片段)是为了指代上的区分或表述上的清楚，并不具有典型的次序上的含义。
药物或治疗剂的“治疗有效量”或“治疗上有效的剂量”是当单独使用或与另一种治疗剂联合使用时保护主体免于疾病发作或促进疾病消退的药物的任何量，所述疾病消退通过疾病征状的严重程度的降低、无疾病征状阶段的频率和持续时间的增加、或由疾病折磨引起的损伤或失能的预防来证明。使用熟练的从业人员已知的多种方法可以评价治疗剂的促进疾病消退的能力，诸如在临床试验期间在人主体中，在预测对于人类的效力的动物模型系统中，或通过在体外测定法中测定所述药剂的活性。
药物的“预防有效量”是指，其为当单独地或与抗肿瘤剂联合施用给处于发生癌症的风险的主体(例如，具有恶化前病症的主体)或具有癌症复发的风险的主体时，抑制癌症的发生或复发的任何药物量。在某些实施方案中，预防有效量完全阻止癌症的发生或复发。“抑制”癌症的发生或复发是指减少癌症的发生或复发的可能性，或完全阻止癌症的发生或复发。
发明的有益效果
本发明的单克隆抗体能够很好地特异性与TIGIT结合，并且具有极强的亲和力，降低TIGIT抑制免疫细胞的作用，促进T细胞活性，逆转NK细胞耗竭，增强免疫细胞对肿瘤的杀伤作用。抗TIGIT抗体单独或联合抗CTLA4-抗PD-1双特异性抗体(和/或化疗药)可有效治疗或预防肿瘤，抗TIGIT抗体联合抗CTLA4-抗PD-1双特异性抗体具 有有效抑制肿瘤生长的药理学效果，且优于抗TIGIT抗体单药或者抗CTLA4-抗PD-1双特异性抗体单药，具有良好的应用前景和市场价值。
附图说明
图1：26B12H1L1、26B12H2L2、26B12H2L3、26B12H3L2和RG6058抗体与TIGIT-mFc的结合活性检测结果。
图2：26B12H3L3、26B12H1L4、26B12H4L1、26B12H4L4和RG6058抗体与TIGIT-mFc的结合活性检测结果。
图3：26B12H1L1、26B12H2L2、26B12H2L3、26B12H3L2和RG6058抗体与人CD155-hFc-Biotin竞争结合TIGIT-mFc的活性检测结果。
图4：26B12H3L3、26B12H1L4、26B12H4L1、26B12H4L4和RG6058抗体与人CD155-hFc-Biotin竞争结合TIGIT-mFc的活性检测结果。
图5：26B12H3L3与TIGIT-mFc亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为5nM、1.67nM、0.557nM、0.185nM、0.06nM。
图6：26B12H1L1与TIGIT-mFc亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为5nM、1.67nM、0.557nM、0.185nM、0.06nM。
图7：26B12H2L2与TIGIT-mFc亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为5nM、1.67nM、0.557nM、0.185nM、0.06nM。
图8：26B12H2L3与TIGIT-mFc亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为5nM、1.67nM、0.557nM、0.185nM、0.06nM。
图9：26B12H3L2与TIGIT-mFc亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为5nM、1.67nM、0.557nM、 0.185nM、0.06nM。
图10：26B12H4L4与TIGIT-mFc亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为5nM、1.67nM、0.557nM、0.185nM、0.06nM。
图11：26B12H1L4与TIGIT-mFc亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为5nM、1.67nM、0.557nM、0.185nM、0.06nM。
图12：26B12H4L1与TIGIT-mFc亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为5nM、1.67nM、0.557nM、0.185nM、0.06nM。
图13：RG6058与TIGIT-mFc亲和力常数检测结果图。图中从上到下的每对曲线中加入抗体的浓度分别为5nM、1.67nM、0.557nM、0.185nM、0.06nM。
图14：FACS检测人源化抗体26B12H2L2和RG6058与293T-TIGIT细胞膜表面抗原TIGIT结合活性。
图15：FACS检测人源化抗体26B12H2L2和RG6058与CD155竞争结合293T-TIGIT细胞膜表面TIGIT的活性。
图16：FACS检测人源化抗体26B12H2L2和RG6058与CD112竞争结合293T-TIGIT细胞膜表面TIGIT的活性。
图17：检测在Jurkat-TIGIT和HT1080-aCD3scFv细胞体系中加入TIGIT抗体后分泌IL-2的量。
图18：hTIGIT-BALB/c转基因小鼠CT26肿瘤模型效果。
图19：hTIGIT-BALB/c转基因小鼠CT26肿瘤模型体重变化。
图20：26B12H2L2与抗CTLA4-抗PD-1双特异性抗体CP004(hG1TM)联用对BALB/c-hPD1/hTIGIT转基因小鼠CT26肿瘤模型效果。
图21：26B12H2L2与抗CTLA4-抗PD-1双特异性抗体CP004(hG1TM)联用对BALB/c-hPD1/hTIGIT转基因小鼠CT26肿瘤模型体重变化。
涉及保藏的生物材料：
[根据细则26改正21.09.2022]　
杂交瘤细胞株LT019(TIGIT-26B12)，其于2020年10月23日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2020208，保藏地址为中国.武汉.武汉大学，邮编：430072。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或按照产品说明书进行。所用试剂或仪器未注明生产厂商者，为可以通过市场购买获得的常规产品。例如293T可以购自ATCC。
在本发明的下述实施例中，使用的BALB/c小鼠购自广东省医学实验动物中心。
在本发明的下述实施例中，所用的阳性对照抗体RG6058，其序列可参中国专利公开CN108290946中的序列34和序列36。
在本发明的下述实施例中，所用的联用抗CTLA4-抗PD-1双特异性抗体CP004(hG1TM)，生产自中山康方生物医药有限公司，其序列来源于公开专利CN112300286A中所述的抗体，参考CP004(hG1TM)重链全长序列(氨基酸序列如SEQ ID NOs:57所示，核苷酸序列如SEQ ID NO:58所示)，轻链全长序列(氨基酸序列如SEQ ID NOs:59所示，核苷酸序列如SEQ ID NO:60所示)，CP004(hG1TM)结构为IgG-scFv，其中IgG部分为抗PD1抗体，scFv部分为抗CTLA4抗体，
其中抗PD1抗体的HCDR1序列如SEQ ID NO:61所示，HCDR2序列如SEQ ID NO:62所示，HCDR3序列如SEQ ID NO:63所示，VH序列SEQ ID NO:73所示，抗PD1抗体的LCDR1序列如SEQ ID NO:70所示，LCDR2序列如SEQ ID NO:71所示，LCDR3序列如SEQ ID NO:72所示，VL序列SEQ ID NO:76所示，
抗CTLA4抗体的HCDR1序列如SEQ ID NO:64所示，HCDR2序列如SEQ ID NO:65所示，HCDR3序列如SEQ ID NO:66所示，VH序列SEQ ID NO:74所示，抗CTLA4抗体的LCDR1序列如SEQ ID NO:67所示，LCDR2序列如SEQ ID NO:68所示，LCDR3序列如SEQ ID NO:69所示，VL序列SEQ ID NO:75所示，
实施例1：抗TIGIT抗体26B12的制备
1.杂交瘤细胞株LT019的制备
制备抗TIGIT抗体所用的抗原为人TIGIT-mFc(TIGIT为GenbankID:NP_776160.2，mFc的序列如SEQ ID NO:77所示)。取免疫后的小鼠的脾细胞与小鼠骨髓瘤细胞融合，制成杂交瘤细胞。以人TIGIT-mFc作为抗原，对杂交瘤细胞进行间接ELISA法筛选，获得能够分泌与TIGIT特异性结合的抗体的杂交瘤细胞。对筛选得到的杂交瘤细胞，经过有限稀释法得到稳定的杂交瘤细胞株。将以上杂交瘤细胞株分别命名为杂交瘤细胞株LT019，其分泌的单克隆抗体分别命名为26B12。
[根据细则26改正21.09.2022]　
杂交瘤细胞株LT019(又称TIGIT-26B12)，其于2020年10月23日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2020208，保藏地址为中国.武汉.武汉大学，邮编：430072。
2.抗TIGIT抗体26B12的制备
用CD培养基(Chemical Defined Medium，含1％青链霉素)对上面制得的LT019细胞株在5％CO 2，37℃条件下进行培养。7天后收集细胞培养上清，通过高速离心、微孔滤膜抽真空过滤，并使用HiTrap protein A HP柱进行纯化，制得抗体26B12。
实施例2：抗TIGIT的抗体26B12的序列分析
按照培养细胞细菌总RNA提取试剂盒(Tiangen，货号DP430)的方法，从实施例1中培养的LT019细胞株中提取mRNA。
按照Invitrogen
Figure PCTCN2022107522-appb-000002
III First-Strand Synthesis System for  RT-PCR试剂盒说明书合成cDNA，并进行PCR扩增。
PCR扩增产物直接进行TA克隆，具体操作参考pEASY-T1Cloning Kit(Transgen CT101)试剂盒说明书进行。
将TA克隆的产物直接进行测序，测序结果如下：
重链可变区的核酸序列如SEQ ID NO:2所示，片段长363bp。
其编码的氨基酸序列为SEQ ID NO:1所示，长度为121个氨基酸。
其中重链HCDR1的序列如SEQ ID NO:3所示，HCDR2的序列如SEQ ID NO:4所示，HCDR3的序列如SEQ ID NO:5所示。
轻链可变区的核酸序列如SEQ ID NO:7所示，长度为321bp。
其编码的氨基酸序列为SEQ ID NO:6所示，长度为107个氨基酸。
其中轻链LCDR1的序列如SEQ ID NO:8所示，LCDR2的序列如SEQ ID NO:9所示，LCDR3的序列如SEQ ID NO:10所示。
实施例3：抗人TIGIT的人源化抗体的轻链和重链设计和制备
1.抗人TIGIT的人源化抗体26B12H1L1、26B12H4L1、26B12H2L2、26B12H3L2、26B12H2L3、26B12H3L3、26B12H1L4和26B12H4L4的轻链和重链设计
根据人TIGIT蛋白的三维晶体结构以及实施例2获得的抗体26B12的序列，通过计算机模拟抗体模型，随后根据模型设计突变，得到抗体26B12H1L1、26B12H4L1、26B12H2L2、26B12H3L2、26B12H2L3、26B12H3L3、26B12H1L4和26B12H4L4的可变区序列(抗体恒定区序列，来自NCBI的数据库，重链恒定区均采用Ig gamma-1 chain C region，ACCESSION:P01857；轻链恒定区为Ig kappa chain C region，ACCESSION:P01834)。
设计的可变区序列如下面的表A所示。
Figure PCTCN2022107522-appb-000003
Figure PCTCN2022107522-appb-000004
人源化单克隆抗体26B12H1L4的重链可变区和轻链可变区序列
以上的8个抗体26B12H1L1、26B12H4L1、26B12H2L2、26B12H3L2、26B12H2L3、26B12H3L3、26B12H1L4和26B12H4L4，重链可变区的核酸序列的长度均为363bp，其编码的氨基酸序列的长度均为121aa；轻链可变区的核酸序列的长度均为321bp，其编码的氨基酸序列的长度均为107aa。
并且上述8个抗体具有相同的HCDR1-HCDR3和LCDR1-LCDR3，如下：
HCDR1的序列如SEQ ID NO:3所示，HCDR2的序列如SEQ ID NO:4所示，HCDR3的序列如SEQ ID NO:5所示；
LCDR1的序列如SEQ ID NO:8所示，LCDR2的序列如SEQ ID NO:9所示，LCDR3的序列如SEQ ID NO:10所示。
2.人源化抗体26B12H1L1、26B12H4L1、26B12H2L2、26B12H3L2、26B12H2L3、26B12H3L3、26B12H1L4和26B12H4L4的制备
重链恒定区均采用Ig gamma-1chain C region，ACCESSION:P01857；轻链恒定区均采用Ig kappa chain C region，ACCESSION:P01834。
将26B12H1L1重链cDNA和轻链的cDNA、26B12H4L1重链cDNA 和轻链的cDNA、26B12H2L2重链cDNA和轻链的cDNA、26B12H3L2重链cDNA和轻链的cDNA、26B12H2L3重链cDNA和轻链的cDNA、26B12H3L3重链cDNA和轻链的cDNA、26B12H1L4重链cDNA和轻链的cDNA、26B12H2L4重链cDNA和轻链的cDNA以及26B12H4L4重链cDNA和轻链的cDNA，分别克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-26B12H1、pUC57simple-26B12L1；pUC57simple-26B12H4、pUC57simple-26B12L1；pUC57simple-26B12H2、pUC57simple-26B12L2；pUC57simple-26B12H3、pUC57simple-26B12L2；pUC57simple-26B12H2、pUC57simple-26B12L3；pUC57simple-26B12H3、pUC57simple-26B12L3；pUC57simple-26B12H1、pUC57simple-26B12L4；pUC57simple-26B12H2、pUC57simple-26B12L4；和pUC57simple-26B12H4、pUC57simple-26B12L4。参照《分子克隆实验指南(第二版)》介绍的标准技术，EcoRI&HindIII酶切合成的重、轻链全长基因，通过限制酶(EcoRI&HindIII)的酶切亚克隆到表达载体pcDNA3.1中获得表达质粒pcDNA3.1-26B12H1、pcDNA3.1-26B12L1、pcDNA3.1-26B12H4、pcDNA3.1-126B12H2、pcDNA3.1-26B12L2、pcDNA3.1-26B12H3、pcDNA3.1-26B12L3和pcDNA3.1-26B12L4，并进一步对重组表达质粒的重/轻链基因进行测序分析。随后将含有相应的轻、重链重组质粒设计基因组合(pcDNA3.1-26B12H1/pcDNA3.1-26B12L1、pcDNA3.1-26B12H4/pcDNA3.1-26B12L1、pcDNA3.1-26B12H2/pcDNA3.1-26B12L2、pcDNA3.1-26B12H3/pcDNA3.1-26B12L2、pcDNA3.1-26B12H2/pcDNA3.1-26B12L3、pcDNA3.1-26B12H3/pcDNA3.1-26B12L3、pcDNA3.1-26B12H1/pcDNA3.1-26B12L4和 pcDNA3.1-26B12H4/pcDNA3.1-26B12L4)分别共转染293F细胞后收集培养液进行纯化。测序验证正确后，制备去内毒素级别的表达质粒并将质粒瞬时转染HEK293细胞进行抗体表达，培养7天后收集细胞培养液，采用Protein A柱进行亲和纯化获得人源化抗体。
实施例4：ELISA方法测定抗体与抗原TIGIT-mFc的结合活性
实验步骤：将羊抗鼠IgG Fc(购自Jackson公司，批号：132560)，2μg/mL包被酶标板后，4℃孵育16小时。孵育结束后使用PBST洗包被了羊抗鼠IgG Fc的酶标板1次，后使用1％BSA的PBST溶液作为酶标板封闭液，封闭2小时。酶标板结束封闭后用PBST洗板3次。再加入抗原人TIGIT-mFc 1μg/mL，置于37℃条件下孵育30分钟后用PBST洗板3次。在酶标板孔内加入PBST溶液梯度稀释的抗体，抗体稀释梯度详见表1和表2。将加入了待测抗体的酶标板置于37℃条件下孵育30分钟，孵育完成后用PBST洗板3次。洗板后加入1:5000比例稀释的HRP标记的羊抗人IgG Fc(购自Jackson公司，批号：128332)二抗工作液，置于37℃条件下孵育30分钟。孵育完成后使用PBST洗板4次，后加入TMB(Neogen，308177)避光显色4min，加入终止液终止显色反应。立即把酶标板放入酶标仪中，选择450nm光波长读取酶标板各孔的OD数值。用SoftMax Pro 6.2.1软件对数据进行分析处理。
抗体与抗原TIGIT-mFc结合的结果如图1、图2所示。各剂量的OD值见表1和表2。以抗体浓度为横坐标，吸光度值为纵坐标进行曲线拟合，计算抗体与抗原的结合EC 50，结果如表1、表2和图1、图2所示。
表1：26B12H1L1、26B12H2L2、26B12H2L3、26B12H3L2和RG6058与TIGIT-mFc的结合活性检测结果
Figure PCTCN2022107522-appb-000005
Figure PCTCN2022107522-appb-000006
表2：26B12H3L3、26B12H1L4、26B12H4L1、26B12H4L4和RG6058与TIGIT-mFc的结合活性检测结果
Figure PCTCN2022107522-appb-000007
结果显示，抗体26B12H1L1、26B12H4L1、26B12H2L2、26B12H3L2、26B12H2L3、26B12H3L3、26B12H1L4和26B12H4L4均能有效地与人TIGIT-mFc结合，结合效率呈剂量依赖关系，结合活性与同靶点阳性药RG6058相当，表明26B12H1L1、26B12H4L1、26B12H2L2、26B12H3L2、26B12H2L3、26B12H3L3、26B12H1L4和26B12H4L4具有有效结合TIGIT的功能。
实施例5:竞争ELISA方法分别测定抗体与CD155-hFc-Biotin竞争结 合TIGIT-mFc的活性
实验步骤：将TIGIT-mFc，2μg/mL包被酶标板后，4℃孵育过夜。孵育结束后使用PBST漂洗包被了抗原的酶标板1次，后使用1％BSA的PBST溶液作为酶标板封闭液，封闭2小时。酶标板结束封闭后用PBST洗板3次。在酶标板内加入PBST溶液梯度稀释的抗体，抗体浓度详见表3和表4，室温孵育10分钟后加入等体积的2μg/mL(终浓度为1μg/mL)的CD155-hFc-Biotin(中山康方生物医药有限公司生产，批号：20170210，其中CD155的GenBank编号为NP_006496.4，hFc的序列如SEQ ID NO:78所示)，和抗体混合均匀后将酶标板置于37℃条件下孵育30分钟，孵育完成后用PBST洗板3次。洗板后加入1:4000比例稀释的SA-HRP工作液，置于37℃条件下孵育30分钟。孵育完成后使用PBST洗板4次后加入TMB(Neogen，308177)避光显色5min，加入终止液终止显色反应。立即把酶标板放入酶标仪中，选择450nm光波长读取酶标板各孔的OD数值。用SoftMax Pro 6.2.1软件对数据进行分析处理。
抗体与CD155-hFc-Biotin竞争结合TIGIT-mFc的活性的结果见表3和表4。以抗体浓度为横坐标，吸光度值为纵坐标进行曲线拟合，计算抗体与CD155-hFc-Biotin竞争结合TIGIT-mFc的EC 50，结果如下表3和表4、图3和图4所示。
表3：26B12H1L1、26B12H2L2、26B12H2L3、26B12H3L2和 RG6058与CD155-hFc-Biotin竞争结合TIGIT-mFc的活性检测结果
Figure PCTCN2022107522-appb-000008
表4：26B12H3L3、26B12H1L4、26B12H4L1、26B12H4L4和RG6058与CD155-hFc-Biotin竞争结合TIGIT-mFc的活性检测结果
Figure PCTCN2022107522-appb-000009
Figure PCTCN2022107522-appb-000010
结果表明，在相同实验条件下，26B12H1L1、26B12H4L1、26B12H2L2、26B12H3L2、26B12H2L3、26B12H3L3、26B12H1L4和26B12H4L4可分别与CD155-hFc-Biotin竞争结合抗原TIGIT-mFc，活性与同靶点阳性药RG6058相当，提示26B12H1L1、26B12H4L1、26B12H2L2、26B12H3L2、26B12H2L3、26B12H3L3、26B12H1L4和26B12H4L4具有有效与CD155-hFc-Biotin竞争结合TIGIT-mFc的功能。
实施例6：使用Fortebio分子相互作用仪测定人源化抗体26B12H3L3、 26B12H1L1、26B12H2L2、26B12H2L3、26B12H3L2、26B12H4L4、 26B12H1L4、26B12H4L1和RG6058与抗原TIGIT-mFc结合的动力学 参数
样品稀释缓冲液为PBS，0.02％Tween-20，0.1％BSA，pH7.4。将TIGIT-mFc以3μg/mL的浓度固定于AMC传感器上，时间为50s，传感器在缓冲液中平衡60s，固定在传感器上的TIGIT-mFc与抗体结合，浓度为0.06-5nM(三倍稀释)，时间120s，蛋白在缓冲液中解离，时间300s。传感器采用10mM甘氨酸，pH＝1.7溶液再生。检测温度为37度，检测频率为0.3Hz，样品板震动速率为1000rpm。数据以1:1模型拟合分析，得到亲和力常数。
人源化抗体(作为对照抗体)与TIGIT的亲和力常数测定结果见表5，检测结果如图5至图13所示。
表5：人源化抗体与抗原TIGIT-mFc亲和力常数检测结果
Figure PCTCN2022107522-appb-000011
K D为亲和力常数；K D＝kdis/kon。
结果显示，人源化抗体26B12H3L3、26B12H1L1、26B12H2L2、26B12H2L3、26B12H3L2、26B12H4L4、26B12H1L4、26B12H4L1和RG6058与TIGIT-mFc的亲和力常数依次为9.64E-11M、1.64E-11M、8.40E-12M、4.85E-11M、5.40E-11M、3.69E-11M、4.63E-11M、8.57E-12M和3.16E-11M。
结果表明，TIGIT各抗体与TIGIT-mFc结合的亲和力从强到弱依次为：26B12H2L2、26B12H4L1、26B12H1L1、RG6058、26B12H4L4、26B12H1L4、26B12H2L3、26B12H3L2、26B12H3L3。其中人源化抗体26B12H2L2、26B12H4L1、26B12H1L1的亲和力比阳性药物RG6058强，而26B12H4L4的亲和力与阳性药物RG6058亲和力相当。
实施例7：FACS检测人源化抗体26B12H2L2和RG6058与293T-TIGIT细胞膜表面抗原TIGIT的结合活性
实验方法：
TIGIT载体plenti6.3-TIGITFL-BSD(TIGIT为GenbankID: NP_776160.2，委托金斯瑞基因优化合成人TIGIT全长cDNA序列，命名为TIGITFL，并克隆到pUC57simple(金斯瑞公司提供)载体中，获得pUC57simple-TIGITFL质粒。以BamHI&XhoI双酶切合成的pUC57simple-TIGITFL质粒，回收TIGITFL目的基因片段并通过限制性位点BamHI&XhoI亚克隆到plenti6.3表达载体，载体pLenti6.3购自Invitrogen公司)转染293T细胞，经筛选获得稳定表达TIGIT的细胞株293T-TIGIT细胞。
收集293T-TIGIT细胞(DMEM+10％FBS)，离心5min后去上清，重悬，计数及活率(P7，95.79％)，稀释细胞，透明尖底96孔板每个孔中加入30w细胞，每管加200μL 1％PBSA，离心5min，去上清。按实验设计每孔对应加入100μL抗体(终浓度为300nM，100nM，33.3nM，11.1nM，3.7nM，1.23nM，0.41nM，0.041nM，0.0041nM)，并设计空白对照及同型对照，冰上孵育60min。每管加200μL1％PBSA，离心5min，去上清，洗两遍。每个样品加FITC羊抗人IgG抗体(购自Jackson公司，货号：109-095-098)(用PBSA 500倍稀释)，冰上避光孵育40min；每管加入200μL PBSA，离心5min，去上清。加入200μL PBSA重悬细胞，转移到流式管中，流式细胞仪检测各浓度下细胞的平均荧光强度。
表6：FACS检测人源化抗体26B12H2L2和RG6058与293T-TIGIT细胞膜表面抗原TIGIT的结合活性
Figure PCTCN2022107522-appb-000012
实验结果如表6和图14所示，阳性对照抗体RG6058与细胞膜表面抗原TIGIT结合的EC 50为1.257nM，而人源化抗体26B12H2L2与细胞膜表面抗原TIGIT结合的EC 50为0.917nM。
实验结果表明，人源化抗体26B12H2L2结合细胞膜表面抗原 TIGIT的能力比阳性对照抗体RG6058强。
实施例8：FACS检测人源化抗体26B12H2L2和RG6058与CD155或 者CD112竞争结合293T-TIGIT细胞膜表面抗原TIGIT的活性
实验方法：收集293T-TIGIT细胞，离心5min后去上清，重悬，计数及活率(94.95％)，稀释细胞，透明尖底96孔板每个孔中加入30w细胞，每管加200μL 1％PBSA，离心5min，去上清。按实验设计每孔对应加入100μL抗体(终浓度为300nM，100nM，33.3nM，11.1nM，3.7nM，1.23nM，0.123nM，0.0123nM)，并设计空白对照及同型对照，冰上孵育30min。每个样品加CD155(终浓度为10nM)中山康方生物医药有限公司生产，批号：20190726，其中CD155的GenBank编号为NP_006496.4)或CD112(终浓度为30nM)中山康方生物医药有限公司生产，批号：20190726，其中CD112的GenBank编号为NP_001036189.1)，冰上避光孵育60min，再每管加200μL 1％PBSA，离心5min，去上清，洗两遍。每个样品加APC羊抗鼠IgG(购自Biolegend公司，批号405308)(最小交叉反应活性minimal x-reactivity)抗体(PBSA 300倍稀释)，冰上避光孵育40min；每管加入200μL PBSA，离心5min，去上清。加入200μL PBSA重悬细胞，转移到流式管中，流式细胞仪检测各浓度下细胞的平均荧光强度。
实验结果分别如表7和图15、以及表8和图16所示。
表7：FACS检测人源化抗体26B12H2L2和RG6058与CD155竞争结合293T-TIGIT细胞膜表面抗原TIGIT的活性
Figure PCTCN2022107522-appb-000013
表8：FACS检测人源化抗体26B12H2L2和RG6058与CD112竞争结合293T-TIGIT细胞膜表面抗原TIGIT的活性
Figure PCTCN2022107522-appb-000014
结果显示：阳性对照抗体RG6058竞争CD155结合TIGIT的EC 50为1.212nM，而人源化抗体26B12H2L2竞争CD155结合TIGIT的EC 50为1.049nM；阳性对照抗体RG6058竞争CD112结合TIGIT的EC 50为1.224nM，而人源化抗体26B12H2L2竞争CD112结合TIGIT的EC 50为1.140nM。
结果表明，人源化抗体26B12H2L2竞争CD155或者CD112结合细胞膜表面抗原TIGIT的能力比阳性对照抗体RG6058强。
实施例9：Jurkat-TIGIT和HT1080-aCD3scFv细胞体系中加入TIGIT 抗体的混合淋巴反应
实验方法:
TIGIT载体plenti6.3/V5-TIGITFL-BSD(载体pLenti6.3购自Invitrogen公司)转染Jurkat细胞，经筛选获得稳定表达TIGIT的细胞株Jurkat-TIGIT细胞；anti-CD3抗体载体pCDH-aCD3scFv-puro(委托金斯瑞基因优化合成anti-CD3scFv的cDNA序列，并克隆到pUC57simple(金斯瑞公司提供)载体中，获得pUC57simple-anti-CD3scFv质粒。以XbaI&BamHI双酶切合成的pUC57simple-anti-CD3scFv质粒，回收anti-CD3scFv目的基因片段并通过限制性位点XbaI&BamHI亚克隆到pCDH-CMV-MCS-EF1-Puro(购自优宝生物)表达载体)(anti-CD3scFv序列来源于参考文献：Eukaryotic expression of anti-CD3single chain Fv antibody gene and the characterization of its bioactivities JOURNAL Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 20(5),552-555(2004)，PUBMED 15367345)；转染HT-1080细胞，经筛选获得稳定表达anti-CD3scFv于细胞膜上的 细胞株HT1080-aCD3scFv细胞。
收集对数生长期Jurkat-TIGIT和HT1080-aCD3scFv细胞，在96孔板中，Jurkat-TIGIT每孔加入5W细胞和HT1080-aCD3scFV每孔加入1W细胞。加入稀释的抗体(终浓度为10nM，50nM，250nM)并加入抗人CD28抗体(购自R&D公司，货号：MAB342-500)(3μg/mL)，放入培养箱中培养48h；收集培养液上清，用IL-2ELISA检测试剂盒检测IL-2含量。
实验结果如图17所示。
结果显示，人源化抗体26B12H2L2和阳性对照抗体RG6058均可促进体系中IL-2的分泌，而且在各浓度下(10nM，50nM，250nM)人源化抗体26B12H2L2与RG6058促IL-2分泌水平相当。
结果表明，人源化抗体26B12H2L2与阳性对照抗体RG6058诱使细胞分泌IL-2的能力相当。
实施例10：26B12H2L2对在hTigit-BALB/c转基因小鼠接种CT26小 鼠移植瘤的治疗作用
采用在hTigit-BALB/c转基因小鼠(小鼠购自江苏集萃药康生物科技有限公司，采购的转基因小鼠，该小鼠正常的TIGIT基因被人TIGIT基因替代)背部接种50万/只CT26细胞(小鼠结肠癌细胞株，购自ATCC)，实验具体步骤为2500万/ml的CT26细胞以每只接种200μl接种小鼠的方法建立小鼠肿瘤模型。实验小鼠每组8只，设同型对照组(给药剂量为20mg/kg，给药方式为腹腔注射(i.p.)，每周两次)，以及实验组(给药剂量为20mg/kg，给药方式为腹腔注射(i.p.)，每周两次)。具体方案如表9所示。
表9：小鼠CT26肿瘤模型的建模和抗体的给药方案
Figure PCTCN2022107522-appb-000015
Figure PCTCN2022107522-appb-000016
实验结果如图18所示。
结果显示，26B12H2L2在hTIGIT-BALB/c转基因小鼠CT26肿瘤模型上，肿瘤体积有显著的减少。
结果表明，26B12H2L2在hTIGIT-BALB/c转基因小鼠CT26肿瘤模型上有着很强的药效；具有用于治疗和/或预防肿瘤特别是结肠癌的潜力。
同时，如图19所示，26B12H2L2对作为CT26肿瘤模型的hTIGIT-BALB/c转基因小鼠的体重没有影响，表明26B12H2L2抗体对小鼠没有产生毒副作用。
实施例11：抗TIGIT抗体与抗CTLA4-抗PD-1双特异性抗体联用有效治疗
为检测抗TIGIT抗体与抗CTLA4-抗PD-1双特异性抗体CP004(hG1TM)联用的体内抑瘤活性，首先用CT26细胞(人结肠癌细胞，购自江苏集萃药康生物科技股份有限公司)，接种于5－7周龄的雌性BALB/c-hPD1/hTIGIT小鼠(购买自江苏集萃药康生物科技股份有限公司)皮下，当平均肿瘤体积达到80-120mm 3时，小鼠根据肿瘤体积随机分成4组，每组6只。分组当天定义为D0天，并于分组当天D0天开始给药。联合给药组给药方式为：药物单独配制，先后分别给药(无给药顺序和间隔时间特定要求，给完一种药物再给予另外一种药物)。造模与具体给药方式见表10。给药后测量各组肿瘤的长宽，计算肿瘤体积。
表10：抗TIGIT抗体与抗CTLA4-抗PD-1双特异性抗体联用治疗CT26移植瘤BALB/c-hPD1/hTIGIT小鼠模型的给药方案
Figure PCTCN2022107522-appb-000017
Figure PCTCN2022107522-appb-000018
结果图20所示。结果表明：相比同型对照抗体hIgG，CP004(hG1TM)，26B12H2L2均能有效抑制小鼠肿瘤的生长，且CP004(hG1TM)+26B12H2L2组对此模型展现联合抗肿瘤药效，其联合对肿瘤的抑制优于受试药单用组。
此外，如图21所示，荷瘤鼠对受试药CP004(hG1TM)和26B12H2L2单用和联用的耐受性均良好，各组对荷瘤小鼠体重无影响。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
Figure PCTCN2022107522-appb-000019
Figure PCTCN2022107522-appb-000020
Figure PCTCN2022107522-appb-000021
Figure PCTCN2022107522-appb-000022
Figure PCTCN2022107522-appb-000023
Figure PCTCN2022107522-appb-000024
Figure PCTCN2022107522-appb-000025
Figure PCTCN2022107522-appb-000026
Figure PCTCN2022107522-appb-000027
Figure PCTCN2022107522-appb-000028
Figure PCTCN2022107522-appb-000029
Figure PCTCN2022107522-appb-000030

Claims (8)

  1. 药物组合物或试剂盒，其包含抗TIGIT抗体或其抗原结合片段和抗CTLA4-抗PD-1双特异性抗体或其抗原结合片段，可选地，所述药物组合物还包括药学上可接受的载体和/或赋形剂，
    其中所述抗TIGIT抗体包含SEQ ID NO:1所示的重链可变区包含的HCDR1-HCDR3和SEQ ID NO:6所示的轻链可变区包含的LCDR1-LCDR3(优选地，按照IMGT编号系统，所述抗体的重链可变区包含氨基酸序列分别如SEQ ID NOs:3-5所示的HCDR1-HCDR3；并且所述抗体的轻链可变区包含氨基酸序列分别如SEQ ID NOs:8-10所示的LCDR1-LCDR3)，
    所述抗CTLA4-抗PD-1双特异性抗体包括：
    靶向PD-1的第一蛋白功能区，和
    靶向CTLA4的第二蛋白功能区；
    其中，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为单链抗体；或者，所述第一蛋白功能区为单链抗体，所述第二蛋白功能区为免疫球蛋白；
    其中，
    所述的免疫球蛋白，其包含SEQ ID NO:27所示的重链可变区中的HCDR1-HCDR3(优选分别如SEQ ID NOs:29－31所示的HCDR1－HCDR3)，和SEQ ID NO:28所示的轻链可变区中的LCDR1-LCDR3(优选分别如SEQ ID NOs:32－34所示的LCDR1－LCDR3)；和所述的单链抗体，其包含SEQ ID NO:35所示的重链可变区中的HCDR1-HCDR3(优选分别如SEQ ID NOs:37－39所示的HCDR1－HCDR3)和SEQ ID NO:36所示的轻链可变区中的LCDR1-LCDR3(优选分别如SEQ ID NOs:40－42所示的LCDR1－LCDR3)；
    或者，
    所述的免疫球蛋白，其包含SEQ ID NO:35所示的重链可变区中的HCDR1-HCDR3(优选分别如SEQ ID NOs:37－39所示的HCDR1－HCDR3)和SEQ ID NO:36所示的轻链可变区中的LCDR1-LCDR3(优选分别如SEQ  ID NOs:40－42所示的LCDR1－LCDR3)；和所述的单链抗体，其包含SEQ ID NO:27所示的重链可变区中的HCDR1-HCDR3(优选分别如SEQ ID NOs:29－31所示的HCDR1－HCDR3)，和SEQ ID NO:28所示的轻链可变区中的LCDR1-LCDR3(优选分别如SEQ ID NOs:32－34所示的LCDR1－LCDR3)。
  2. 权利要求1的药物组合物或试剂盒，其中所述抗TIGIT抗体的重链可变区的氨基酸序列选自SEQ ID NO:1、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15和SEQ ID NO:17；并且所述抗TIGIT抗体的轻链可变区的氨基酸序列选自SEQ ID NO:6、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23和SEQ ID NO:25；
    优选所述的抗TIGIT抗体或其抗原结合片段，其中，
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:6所示；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:11所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:19所示；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:17所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:19所示；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:13所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:21所示；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:13所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:23所示；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:15所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:21所示；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:15所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:23所示；
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:11所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:25所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:17所示，并且所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:25所示；
    优选地，所述的抗TIGIT抗体或其抗原结合片段，其中，所述的抗体包 括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体；
    优选地，所述的抗TIGIT抗体或其抗原结合片段，其中，所述抗体的重链恒定区为Ig gamma-1 chain C region(例如NCBI ACCESSION:P01857)；轻链恒定区为Ig kappa chain C region(例如NCBI ACCESSION:P01834)；
    优选地，所述的抗TIGIT抗体或其抗原结合片段，其中，所述抗TIGIT抗体或其抗原结合片段选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体、人源化抗体、嵌合抗体或双抗体；
    优选地，所述的抗TIGIT抗体或其抗原结合片段，其中，所述抗体以小于4E-10或小于4E-11的K D结合TIGIT-mFc；优选地，所述K D通过Fortebio分子相互作用仪测得；
    优选地，所述的抗TIGIT抗体或其抗原结合片段，其中，所述抗体以小于1.5nM、小于1.2nM或小于1nM的EC 50结合TIGIT-mFc；优选地，所述EC 50通过流式细胞仪测得；
    优选地，所述的抗TIGIT抗体为单克隆抗体、人源化抗体、嵌合抗体、多特异性抗体(例如双特异性抗体)；
    优选地，所述抗原结合片段选自Fab、Fab'、F(ab') 2、Fd、Fv、dAb、Fab/c、互补决定区片段、单链抗体(例如，scFv)、人源化抗体、嵌合抗体或双特异性抗体；
    优选地，所述的抗TIGIT抗体或其抗原结合片段，其中所述抗体是由杂交瘤细胞株LT019产生的抗体，所述杂交瘤细胞株LT019保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2020208。
  3. 权利要求1的药物组合物或试剂盒，所述的抗CTLA4-抗PD-1双特异性抗体，其中，
    所述免疫球蛋白的重链可变区的氨基酸序列选自SEQ ID NO:27，SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列；并且所述免疫球蛋白的轻链可变区的氨基酸序列选自SEQ ID NO:28，SEQ ID  NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列；和，所述单链抗体的重链可变区的氨基酸序列选自SEQ ID NO:35、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47，SEQ ID NO:48或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列；并且所述单链抗体的轻链可变区的氨基酸序列选自SEQ ID NO:36、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51，SEQ ID NO:52或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列；
    或者，
    所述免疫球蛋白的重链可变区的氨基酸序列选自SEQ ID NO:35、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47，SEQ ID NO:48或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列；并且所述免疫球蛋白的轻链可变区的氨基酸序列选自SEQ ID NO:36、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51，SEQ ID NO:52或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列；和，所述单链抗体的重链可变区的氨基酸序列选自SEQ ID NO:27，SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至 少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列；并且所述单链抗体的轻链可变区的氨基酸序列选自SEQ ID NO:28，SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列；
    优选地，所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述双特异性抗体选自如下的(1)－(20)中的任一项：
    (1)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:27或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:28或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:35或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:36或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源 性的序列所示；
    (2)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:27或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:28或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:45或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:49或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
    (3)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:27或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:28或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至 少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:46或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:50或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
    (4)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:35或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:36或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源 性的序列所示；
    (5)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:45或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:49或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
    (6)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至 少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:46或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:50或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
    (7)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:35或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:36或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:27或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:28或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源 性的序列所示；
    (8)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:35或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:36或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
    (9)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:45或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:49或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至 少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:27或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:28或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
    (10)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:45或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:49或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源 性的序列所示；
    (11)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:46或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:50或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:27或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:28或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
    (12)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:46或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:50或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至 少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
    (13)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:27或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:28或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:47或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:51或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源 性的序列所示；
    (14)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:27或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:28或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:48或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:52或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
    (15)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至 少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:47或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:51或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
    (16)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:48或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:52或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源 性的序列所示；
    (17)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:47或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:51或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:27或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:28或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
    (18)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:48或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:52或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至 少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:27或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:28或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
    (19)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:47或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少98％，99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:51或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％ 同源性的序列所示；
    以及，
    (20)所述免疫球蛋白的重链可变区的氨基酸序列如SEQ ID NO:48或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述免疫球蛋白的轻链可变区的氨基酸序列如SEQ ID NO:52或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；和，所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:43或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:44或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
    优选地，所述的抗CTLA4-抗PD-1双特异性抗体，其中，
    所述免疫球蛋白的重链的氨基酸序列如SEQ ID NO:53或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且其轻链的氨基酸序列如SEQ ID NO:54或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少 86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
    优选地，所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述第一蛋白功能区与所述第二蛋白功能区直接连接或者通过连接片段连接；和/或所述单链抗体的重链可变区与所述单链抗体的轻链可变区直接连接或者通过连接片段连接；
    优选地，所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述连接片段为(GGGGS)n，n为正整数；优选地，n为1、2、3、4、5或6；
    优选地，所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区独立地为1个、2个或者2个以上；
    优选地，所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述单链抗体(优选重链可变区)连接在免疫球蛋白的重链的C末端；
    优选地，所述的抗CTLA4-抗PD-1双特异性抗体，其中，
    所述免疫球蛋白为人IgG1亚型；
    其中，按照EU编号系统，所述免疫球蛋白的重链恒定区具有如下突变组合之一：
    L234A和L235A；或者
    L234A和G237A；或者
    L235A和G237A；或者
    L234A、L235A、G237A；
    优选地，所述的抗CTLA4-抗PD-1双特异性抗体，其中，所述双特异性抗体包括：
    靶向PD-1的第一蛋白功能区，和
    靶向CTLA4的第二蛋白功能区；
    所述第一蛋白功能区为1个，所述第二蛋白功能区为2个；
    其中，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为单链抗体；
    所述免疫球蛋白的重链的氨基酸序列如SEQ ID NO:53或与其具有至 少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且其轻链的氨基酸序列如SEQ ID NO:54或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
    所述单链抗体的重链可变区的氨基酸序列如SEQ ID NO:48或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示，并且所述单链抗体的轻链可变区的氨基酸序列如SEQ ID NO:52或与其具有至少60％，至少65％，至少70％，至少75％，至少80％，至少81％，至少82％，至少83％，至少84％，至少85％，至少86％，至少87％，至少88％，至少89％，至少90％，至少91％，92％，93％，94％，至少95％，至少96％，至少97％，至少98％或至少99％同源性的序列所示；
    所述单链抗体连接在免疫球蛋白的重链的C末端；
    所述第一蛋白功能区与所述第二蛋白功能区通过第一连接片段连接；并且所述单链抗体的重链可变区与所述单链抗体的轻链可变区通过第二连接片段连接；所述第一连接片段和所述第二连接片段相同或不同；
    优选地，所述第一连接片段和所述第二连接片段的氨基酸序列独立地选自SEQ ID NO:55和SEQ ID NO:56；
    优选地，所述第一连接片段和所述第二连接片段的氨基酸序列均如SEQ ID NO:56所示；
    优选地，所述的抗CTLA4-抗PD-1双特异性抗体的重链氨基酸序列如SEQ ID NOs:57所示，轻链氨基酸序列如SEQ ID NOs:59所示，双特异性抗体结构为IgG-scFv，其中IgG部分为抗PD1抗体，scFv部分为抗CTLA4 抗体，
    其中抗PD1抗体的HCDR1序列如SEQ ID NO:61所示，HCDR2序列如SEQ ID NO:62所示，HCDR3序列如SEQ ID NO:63所示，VH序列SEQ ID NO:73所示，抗PD1抗体的LCDR1序列如SEQ ID NO:70所示，LCDR2序列如SEQ ID NO:71所示，LCDR3序列如SEQ ID NO:72所示，VL序列SEQ ID NO:76所示，
    抗CTLA4抗体的HCDR1序列如SEQ ID NO:64所示，HCDR2序列如SEQ ID NO:65所示，HCDR3序列如SEQ ID NO:66所示，VH序列SEQ ID NO:74所示，抗CTLA4抗体的LCDR1序列如SEQ ID NO:67所示，LCDR2序列如SEQ ID NO:68所示，LCDR3序列如SEQ ID NO:69所示，VL序列SEQ ID NO:75所示。
  4. 权利要求1-3任一项所述的药物组合物，其中按照抗体的质量计算，抗TIGIT抗体或其抗原结合片段与抗CTLA4-抗PD-1抗体或其抗原结合片段的质量比为(1：5)-(5：1)，例如：1：5、1：4、1：3、1：2、1：1、2：1、3：1、4：1或5：1。
  5. 试剂盒，其包含独立包装的第一产品和第二产品，其中，
    所述第一产品包含权利要求1-4任一项中定义的所述抗TIGIT抗体或其抗原结合片段；
    所述第二产品包含权利要求1-4任一项中定义的所述抗CTLA4-抗PD-1双特异性抗体或其抗原结合片段；
    优选地，所述试剂盒还包含独立包装的第三产品，所述第三产品包含一种或多种化疗药物，
    优选地，所述第一产品和所述第二产品还独立地包含一种或多种药学上可接受的辅料；
    优选地，所述组合产品还包含产品说明书；
    优选地，所述的试剂盒，其中，按照抗体的质量计算，抗TIGIT抗体或其抗原结合片段与抗CTLA4-抗PD-1双特异性抗体或其抗原结合片段的质量比为(1：5)-(5：1)，例如：1：5、1：4、1：3、1：2、1：1、2：1、3：1、4：1或5：1。
  6. 治疗和/或预防肿瘤的方法，包括给予有需求的受试者以有效量的权利要求1-4任一项中定义的所述抗TIGIT抗体或其抗原结合片段；和/或权利要求1-4任一项中定义的所述抗CTLA4-抗PD-1双特异性抗体或其抗原结合片段；
    优选地，进一步联合施用一种或多种药物(如化疗剂或生长抑制剂，靶向治疗剂，抗体-药物缀合物，抗代谢药物，抗生素，抗激素剂，植物类抗癌药物和/或激素类药物，优选地，所述药物选自以下药物的一种或多种：阿霉素类，他莫昔芬，甲地孕酮，门冬酰胺酶，铂类药物(如顺铂、卡铂或奥沙利铂)，氟尿嘧啶类抗肿瘤药，环磷酰胺，培美曲塞，紫杉醇，长春碱类，阿霉素类，戈舍瑞林，烷化剂，蒽环类，抗雄激素剂，芳香酶抑制剂，蛋白激酶抑制剂(例如酪氨酸激酶抑制剂)，脂质激酶抑制剂，反义寡核苷酸，核酶，拓扑异构酶抑制剂，细胞毒剂，抗肿瘤抗生素、蛋白酶体抑制剂、抗微管剂、EGFR拮抗剂、类视色素、组蛋白脱乙酰酶抑制剂，B-raf抑制剂，MEK抑制剂，K-ras抑制剂，c-Met抑制剂，Alk抑制剂，磷脂酰肌醇3-激酶抑制剂，Akt抑制剂，mTOR抑制剂，双磷脂酰肌醇3-激酶/mTOR抑制剂，美登新碱，单甲基auristatin E，加利车霉素，esperamicin和放射性同位素螯合剂；
    优选地，所述抗TIGIT抗体、抗CTLA4-抗PD-1双特异性抗体和肿瘤化疗药物同时或顺序施用；更优选地，所述抗TIGIT抗体、抗CTLA4-抗PD-1双特异性抗体为在手术治疗之前或之后，和/或在放射治疗之前或之后施用；
    优选地，所述抗TIGIT抗体，所述抗CTLA4-抗PD-1双特异性抗体和/或所述化疗药为适于静脉注射或静脉滴注的形式，优选液体的形式；
    优选地，所述肿瘤选自如下的一种或多种：
    乳腺癌、卵巢癌、结直肠癌、宫颈瘤、多发性骨髓瘤、非霍奇金淋巴瘤、B淋巴细胞瘤、浆细胞癌，头和颈癌、脑癌、咽喉癌、鼻咽癌、食管癌、食管鳞癌、甲状腺癌、间皮瘤、腺癌(如胰腺癌)、肺癌(如小细胞肺癌、小细胞肺癌)、乳腺癌、肝癌(如肝细胞癌、肝胆癌)、胃癌、胃肠道癌、肠癌(如结肠癌、结肠直肠癌)、胆道癌(如胆管癌)、肾癌、输卵管癌、子宫内膜癌、宫颈癌、膀胱癌、尿路上皮癌、前列腺 癌、睾丸癌、皮肤癌、黑色素瘤、骨髓瘤(如多发性骨髓瘤)、非霍奇金淋巴瘤、B淋巴细胞瘤、浆细胞癌、白血病、淋巴瘤、骨癌、骨肉瘤、软骨肉瘤、高度微卫星不稳定型(MSI-H)或错配修复缺陷型(dMMR)实体瘤；
    优选地，权利要求1-4任一项中定义的所述抗TIGIT抗体和/或所述抗CTLA4抗PD-1双特异性抗体的单次给药剂量为每千克体重0.1-100mg，优选1-10mg；或者，本发明任一项所述抗TIGIT抗体和/或本发明任一项所述抗CTLA4抗PD-1双特异性抗体的单次给药剂量为每位受试者10-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg或200mg，
    优选地，每天两次至约每隔一天一次给药，或每3天、4天、5天、6天、10天、1周、2周、3周、4周、5周或6周给药一次；
    优选地，给药方式为静脉滴注或静脉注射。
  7. 单位制剂，优选用于治疗肿瘤，其中，所述单位制剂包含：1～10000mg(优选10-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg或200mg)的权利要求1-4任一项中定义的所述抗TIGIT抗体和1～10000mg(优选1-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg、200mg或100mg)的权利要求1-4任一项中定义的抗CTLA4-抗PD-1双特异性抗体，和任选地一种或多种权利要求6中定义的所述化疗药(如铂类药物和/或氟尿嘧啶类抗肿瘤药)；其中，所述抗TIGIT抗体、所述抗CTLA4-抗PD-1双特异性抗体和化疗药分别单独包装。
  8. 单次药物剂量单元，优选用于治疗肿瘤，其包含0.1-10000mg(优选1-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg、200mg或100mg)的权利要求1-4任一项中定义的所述抗TIGIT抗体和0.1-10000mg(优选1-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg、200mg或100mg)的权利要求1-4任一项中定义的抗CTLA4-抗PD-1双特异性抗体。
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