WO2022063100A1 - 抗tigit抗体及双抗体和它们的应用 - Google Patents

抗tigit抗体及双抗体和它们的应用 Download PDF

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WO2022063100A1
WO2022063100A1 PCT/CN2021/119496 CN2021119496W WO2022063100A1 WO 2022063100 A1 WO2022063100 A1 WO 2022063100A1 CN 2021119496 W CN2021119496 W CN 2021119496W WO 2022063100 A1 WO2022063100 A1 WO 2022063100A1
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amino acid
acid sequence
antibody
variable region
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French (fr)
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金亮
肖扬
赵立文
罗成
高飞
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南京圣和药业股份有限公司
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Priority to US18/027,493 priority Critical patent/US20240262915A1/en
Priority to EP21871470.7A priority patent/EP4219553A1/en
Publication of WO2022063100A1 publication Critical patent/WO2022063100A1/zh

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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—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 B7 molecules, e.g. CD80, CD86
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    • 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
    • A—HUMAN NECESSITIES
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    • 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
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • 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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    • C07K2317/62—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
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the technical field of antibody drugs, in particular to an anti-TIGIT antibody or an antigen-binding fragment thereof, an anti-TIGIT/anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising an anti-TIGIT antibody or an antigen-binding fragment thereof or an anti-TIGIT/anti-PD - Pharmaceutical compositions of L1 antibodies or antigen-binding fragments thereof and their uses.
  • TIGIT is a member of the poliovirus receptor-binding protein family, belonging to the immunoglobulin superfamily subtype, consisting of an extracellular immunoglobulin domain (IgV), a transmembrane structure, and an intracellular tyrosine-based inhibition Motif immunoreceptor (ITIM) and immunoglobulin tyrosine tail motif (ITT).
  • IgV extracellular immunoglobulin domain
  • ITIM immunoglobulin tyrosine-based inhibition Motif immunoreceptor
  • ITT immunoglobulin tyrosine tail motif
  • TIGIT is only expressed in lymphocytes, and is highly expressed in effector CD4+ T cells and regulatory T cells, CD4+ vesicular helper T cells, effector CD8+ T cells and NK cells.
  • TIGIT competes with CD226 and CD96 for binding to PVR (CD155), and competes with CD226 and CD112R for binding to CD122. Coordinates the immune system through network signaling, regulating immune activation and immune suppression. Studies have shown that TIGIT plays an important role in the regulation of tumor immunosuppression.
  • the binding of TIGIT to its ligands PVR, PVR2 and PVR3 (potential ligands) inhibits immune responses through three different modes of action.
  • the first mode of action is to trigger the rapid phosphorylation of PVR to transmit signals to antigen-presenting cells or tumor cells through the signaling of TIGIT.
  • TIGIT itself transmits its signals to lymphocytes through the intracellular ITIM/ITT domain.
  • the mode of action that affects the immune response is to suppress the activity of T cells by expressing and secreting inflammatory factors such as IL10 and TGF- ⁇ after the antigen-presenting cells receive the TIGIT signal; the third mode of action is to inhibit the balance of CD226 by competing with CD226 to bind PVR activation of cells.
  • Anti-TIGIT antibody can block its signaling pathway, relieve tumor immunosuppression, increase the cytotoxicity of effector T cells and NK cells, and can establish long-term anti-tumor immune memory to achieve the effect of tumor treatment.
  • PD-L1 is a 40KD type I transmembrane protein that plays an important role in suppressing the immune system.
  • PD-L1 negatively regulates the T cell receptor signaling pathway by forming an immune complex with PD-1 and B7-1, which in turn causes the blocking of T cell activation and inhibits the antitumor activity of T cells.
  • Receptor mainly involved in the activation of T cells, B cells, monocytes and myeloid cells, CD80 (B7-1) is a T cell costimulatory molecule. This immunomodulatory effect of PD-L1 occurs mainly in some chronic diseases, such as allogeneic transplantation, autoimmune diseases and cancer.
  • PD-L1 is highly expressed on the surface of many tumor cells and tumor-infiltrating immune cells, including most solid tumors and hematological lymphomas, such as myeloma, prostate cancer, breast cancer, colon cancer, lung cancer, gastric cancer, melanoma, etc. Therefore, PD-L1 can serve as an important tumor therapeutic target.
  • monoclonal antibodies that exert anti-tumor effects by blocking PD-L1 have been widely used in clinical practice and have shown strong anti-tumor activity in a variety of tumors.
  • Anti-TIGIT/anti-PD-L1 bispecific antibodies can activate the immune response by blocking the PD-1/PD-L1 pathway and TIGIT/CD155 pathway, and recruit tumor cells to the periphery of lymphocytes, further enhancing anti-tumor activity. Therefore, the development of anti-TIGIT/anti-PD-L1 bispecific antibodies is particularly necessary.
  • an anti-TIGIT antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and/or a light chain variable region
  • the heavy chain variable region comprises the complementarity determining region of the heavy chain variable region 1 (H1CDR1), heavy chain variable region complementarity determining region 2 (H1CDR2) and/or heavy chain variable region complementarity determining region 3 (H1CDR3)
  • the light chain variable region comprising the complementarity of the light chain variable region Determining region 1 (L1CDR1), complementarity determining region 2 (L1CDR2) of the light chain variable region and/or complementarity determining region 3 (L1CDR3) of the light chain variable region.
  • the invention provides an anti-TIGIT antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises H1CDR1, H1CDR2 and H1CDR3 selected from the group consisting of:
  • the light chain variable region comprises L1CDR1, L1CDR2 and L1CDR3 selected from the group consisting of:
  • the present invention provides an anti-TIGIT antibody or antigen-binding fragment thereof, which has the H1CDR1, H1CDR2 and H1CDR3 respectively as SEQ ID NO: 25, 26 and 27 or with SEQ ID NO: 25,
  • the amino acid sequences shown in 26 and 27 have the heavy chain variable region of the amino acid sequence of at least 85% sequence identity
  • the L1CDR1, L1CDR2 and L1CDR3 are respectively SEQ ID NO: 31, 32 and 33 or with SEQ ID NO:
  • the amino acid sequences shown in 31, 32 and 33 have light chain variable regions of amino acid sequences of at least 85% sequence identity.
  • the present invention provides an anti-TIGIT antibody or an antigen-binding fragment thereof, which has the H1CDR1, H1CDR2 and H1CDR3 respectively as SEQ ID NO: 28, 29 and 30 or with SEQ ID NO: 28,
  • the amino acid sequences shown in 29 and 30 have the heavy chain variable region of the amino acid sequence of at least 85% sequence identity
  • the L1CDR1, L1CDR2 and L1CDR3 are respectively SEQ ID NO: 34, 35 and 36 or with SEQ ID NO:
  • the amino acid sequences shown in 34, 35 and 36 have light chain variable regions of amino acid sequences of at least 85% sequence identity.
  • the anti-TIGIT antibody or antigen-binding fragment thereof according to the invention is a monoclonal antibody or antigen-binding fragment thereof.
  • the anti-TIGIT antibody or antigen-binding fragment thereof according to the invention is a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.
  • the invention provides an anti-TIGIT antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein
  • amino acid sequence of the heavy chain variable region is selected from:
  • (b1) such as SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO: 88, the amino acid sequence shown in SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92;
  • amino acid sequence of the light chain variable region is selected from:
  • the present invention provides an anti-TIGIT antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO:75, which is substituted, deleted or An amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 75 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 75, and the amino acid sequence of the light chain variable region is SEQ ID NO:77, the amino acid sequence of SEQ ID NO:77 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO:77 or having at least 85% sequence identity with SEQ ID NO:77 amino acid sequence.
  • the present invention provides an anti-TIGIT antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 76, SEQ ID NO: 76 is substituted, deleted or An amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 76 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 76, and the amino acid sequence of the light chain variable region is SEQ ID NO:78, the amino acid sequence of SEQ ID NO:78 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO:78 or having at least 85% sequence identity with SEQ ID NO:78 amino acid sequence.
  • the present invention provides an anti-TIGIT antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO:75, which is substituted, deleted or An amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO:75 or having at least 85% sequence identity with SEQ ID NO:75 and said H1CDR1, H1CDR2 and H1CDR3 as SEQ ID NO:25, 26 and the amino acid sequence shown in 27, and the amino acid sequence of the light chain variable region is SEQ ID NO: 77, SEQ ID NO: 77 is obtained by substitution, deletion or addition of one or more amino acids and is the same as SEQ ID NO: 77 A functionally identical amino acid sequence or amino acid sequence having at least 85% sequence identity to SEQ ID NO:77 and said L1CDR1, L1CDR2 and L1CDR3 are as shown in SEQ ID NO:31, 32 and 33.
  • the present invention provides an anti-TIGIT antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 76, SEQ ID NO: 76 is substituted, deleted or An amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO:76 or having at least 85% sequence identity with SEQ ID NO:76 and said H1CDR1, H1CDR2 and H1CDR3 as SEQ ID NO:28, 29 and the amino acid sequence shown in 30, and the amino acid sequence of the light chain variable region is SEQ ID NO: 78, SEQ ID NO: 78 is obtained by substitution, deletion or addition of one or more amino acids and is the same as SEQ ID NO: 78 A functionally identical amino acid sequence or amino acid sequence having at least 85% sequence identity to SEQ ID NO:78 and said L1CDR1, L1CDR2 and L1CDR3 are as set forth in SEQ ID NO:34, 35 and 36.
  • the anti-TIGIT antibody according to the invention is a murine antibody further comprising the heavy chain constant regions of murine IgGl, IgG2a, IgG2b, IgG2c, IgG3 or variants thereof, and murine kappa The light chain constant region of the chain or a variant thereof.
  • the anti-TIGIT murine antibody according to the present invention further contains the heavy chain constant region of murine IgG1, IgG2a, IgG2b, IgG2c or variants thereof, and the light weight of murine kappa chain or variants thereof chain constant region.
  • the invention provides an anti-TIGIT humanized antibody or antigen-binding fragment thereof, wherein:
  • amino acid sequence of the heavy chain variable region is selected from:
  • amino acid sequence of the light chain variable region is selected from:
  • the present invention provides an anti-TIGIT humanized antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO:98, and SEQ ID NO:98 is substituted, deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 98 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 98, and the amino acid sequence of the light chain variable region is SEQ ID NO:100, the amino acid sequence of SEQ ID NO:100 obtained by substitution, deletion or addition of one or more amino acids and is functionally identical to SEQ ID NO:100 or has at least 85% sequence identity with SEQ ID NO:100 Sexual amino acid sequence.
  • the present invention provides an anti-TIGIT humanized antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO:99, and SEQ ID NO:99 is substituted, deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 99 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 99, and the amino acid sequence of the light chain variable region is SEQ ID NO:101, the amino acid sequence obtained by substitution, deletion or addition of one or more amino acids of SEQ ID NO:101 and is functionally identical to SEQ ID NO:101 or has at least 85% sequence identity with SEQ ID NO:101 Sexual amino acid sequence.
  • the present invention provides an anti-TIGIT humanized antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO:98, and SEQ ID NO:98 is substituted, deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 98 or having at least 85% sequence identity with SEQ ID NO: 98 and said H1CDR1, H1CDR2 and H1CDR3 as SEQ ID NO:25,
  • the amino acid sequences shown in 26 and 27, and the amino acid sequence of the light chain variable region is SEQ ID NO: 100
  • SEQ ID NO: 100 is obtained by substitution, deletion or addition of one or more amino acids and is the same as SEQ ID NO. : 100 functionally identical amino acid sequences or amino acid sequences having at least 85% sequence identity with SEQ ID NO: 100 and said L1CDR1, L1CDR2 and L1CDR3 are as shown in SEQ ID NOs: 31, 32 and 33.
  • the present invention provides an anti-TIGIT humanized antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO:99, and SEQ ID NO:99 is substituted, deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO:99 or having at least 85% sequence identity with SEQ ID NO:99 and said H1CDR1, H1CDR2 and H1CDR3 as SEQ ID NO:28,
  • the amino acid sequences shown in 29 and 30, and the amino acid sequence of the light chain variable region is SEQ ID NO: 101, SEQ ID NO: 101 is obtained by substitution, deletion or addition of one or more amino acids and is the same as SEQ ID NO. :101 functionally identical amino acid sequence or amino acid sequence having at least 85% sequence identity with SEQ ID NO:101 and said L1CDR1, L1CDR2 and L1CDR3 are as shown in SEQ ID NO:34, 35 and 36.
  • the murine anti-TIGIT antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine ⁇ , ⁇ chain or a variant thereof, and/or further comprise a murine constant region Heavy chain constant regions of IgGl, IgG2a, IgG2b, IgG2c, IgG3 or variants thereof.
  • the antibody light chain of the anti-TIGIT chimeric antibody or its antigen-binding fragment further comprises a light chain constant region of a murine ⁇ , ⁇ chain or a mutant sequence thereof.
  • the antibody heavy chain of the anti-TIGIT chimeric antibody or its antigen-binding fragment further comprises the heavy G chain constant region of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or its mutant sequence, preferably human IgG1, IgG2a, IgG2b , IgG2c heavy chain constant region, or IgG4 constant region with amino acid mutation that significantly reduces ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity.
  • the anti-TIGIT humanized antibody or antigen-binding fragment thereof of the present invention further comprises the heavy chain constant region of human IgG1, IgG2a, IgG2b, IgG2c, IgG3 or variants thereof, and human kappa, The light chain constant region of the lambda chain or variant thereof.
  • the anti-TIGIT humanized antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region of human IgG1, IgG2a, IgG2b, IgG2c or a variant thereof, and a human kappa chain or its The light chain constant region of the variant.
  • the present invention provides an anti-TIGIT antibody or antigen-binding fragment thereof, wherein the antigen-binding fragment is a Fab, Fv, sFv, or F(ab)2.
  • Another aspect of the present invention provides an isolated nucleic acid encoding an anti-TIGIT antibody or antigen-binding fragment thereof according to the present invention.
  • the isolated nucleic acid according to the present invention comprises an amino acid sequence encoding a heavy chain variable region such as SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:98 or SEQ ID NO:99 and the nucleotide sequence encoding the light chain variable region amino acid sequence such as SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:100 or SEQ ID NO:101.
  • the isolated nucleic acid according to the present invention comprises a nucleotide sequence encoding a heavy chain variable region of SEQ ID NO:75; and a nucleotide sequence encoding a light chain variable region of SEQ ID NO:77 acid sequence.
  • the isolated nucleic acid according to the present invention comprises a nucleotide sequence encoding a heavy chain variable region of SEQ ID NO:76; and a nucleotide sequence encoding a light chain variable region of SEQ ID NO:78 acid sequence.
  • the isolated nucleic acid according to the present invention comprises a nucleotide sequence encoding a heavy chain variable region of SEQ ID NO:98; and a nucleotide sequence encoding a light chain variable region of SEQ ID NO:100 acid sequence.
  • the isolated nucleic acid according to the present invention comprises a nucleotide sequence encoding a heavy chain variable region of SEQ ID NO:99; and a nucleotide sequence encoding a light chain variable region of SEQ ID NO:101 acid sequence.
  • Another aspect of the present invention provides an expression vector expressing the anti-TIGIT antibody or antigen-binding fragment thereof of the present invention.
  • the expression vector according to the present invention comprises the isolated nucleic acid molecule of the present invention.
  • Another aspect of the present invention provides a host cell transformed with the expression vector as described above.
  • the host cell according to the present invention is selected from prokaryotic cells and eukaryotic cells.
  • the host cell is a bacterium, preferably E. coli.
  • the host cell is a mammalian cell.
  • Another aspect of the present invention provides a method of making an anti-TIGIT antibody or antigen-binding fragment thereof of the present invention, comprising the steps of expressing the antibody in the host cell and isolating the antibody from the host cell.
  • compositions comprising the anti-TIGIT humanized antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.
  • the present invention provides pharmaceutical compositions comprising the anti-TIGIT humanized antibody or antigen-binding fragment thereof of the present invention, and other active components, such as other antibodies, targeted drugs, and the like.
  • the pharmaceutically acceptable carrier is selected from the group consisting of antioxidants, polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, sugar alcohols, ions, and surfactants.
  • the pharmaceutically acceptable carrier is a buffered aqueous solution.
  • the pharmaceutically acceptable carrier is in the form of a liposome.
  • the anti-TIGIT humanized antibody or antigen-binding fragment thereof of the present invention can be mixed with a pharmaceutically acceptable carrier, diluent or excipient to prepare a pharmaceutical preparation suitable for oral or parenteral administration.
  • Methods of administration include, but are not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, intracerebral, intraocular, intratracheal, subcutaneous, intranasal routes.
  • the formulations may be administered by any route, such as by infusion or bolus injection, by route of absorption through the epithelium or mucocutaneous (eg, oral mucosa or rectum, etc.). Administration can be systemic or local.
  • the formulations can be prepared by methods known in the art and include carriers, diluents or excipients conventionally used in the art of pharmaceutical formulations.
  • Another aspect of the present invention provides a method of inhibiting TIGIT activity, the method comprising administering to an individual in need thereof an anti-TIGIT antibody or antigen-binding fragment thereof of the present invention or a pharmaceutical composition of the present invention.
  • Another aspect of the present invention provides a method for detecting or measuring human TIGIT, the method comprising the step of using an anti-TIGIT antibody or antigen-binding fragment thereof of the present invention.
  • Another aspect of the present invention provides a reagent for detecting or measuring human TIGIT, the reagent comprising the anti-TIGIT antibody or antigen-binding fragment thereof of the present invention.
  • Another aspect of the present invention provides a method of treating a disease associated with TIGIT, the method comprising administering to a subject a pharmaceutically effective amount of a TIGIT antibody or antigen-binding fragment thereof of the present invention, or a pharmaceutical composition comprising the above, or The above isolated nucleic acid molecule.
  • Another aspect of the present invention provides the use of the anti-TIGIT antibody or antigen-binding fragment thereof of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicine for TIGIT-related diseases.
  • the medicament for a disease associated with TIGIT is used to treat a T-cell dysfunctional disorder, such as a tumor, an immune disease, or an infectious disorder.
  • the tumor is non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma tumor, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, adrenocortical carcinoma, Liver hepatocellular carcinoma, pancreatic duct adenocarcinoma, pheochromocytoma, ganglion cell tumor, endometrial carcinoma and ovarian serous cystadenocarcinoma, etc.
  • the immune disease is arthritis, inflammatory bowel disease, psoriasis.
  • the infectious disease is a chronic viral infection.
  • the present invention provides an anti-TIGIT/anti-PD-L1 antibody, comprising an anti-TIGIT antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-TIGIT antibody or an antigen-binding fragment thereof comprises a first A heavy chain variable region and/or a first light chain variable region, wherein the first heavy chain variable region comprises the complementarity determining region 1 (H1CDR1) of the first heavy chain variable region, the first heavy chain variable region The complementarity determining region 2 (H1CDR2) of the first heavy chain variable region and/or the complementarity determining region 3 (H1CDR3) of the first heavy chain variable region comprising the complementarity determining region 1 of the first light chain variable region ( L1CDR1), the first light chain variable region complementarity determining region 2 (L1CDR2) and/or the first light chain variable region complementarity determining region 3 (L1CDR3); and the anti-PD-L1 antibody or antigen
  • the present invention provides an anti-TIGIT/anti-PD-L1 antibody comprising an anti-TIGIT antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-TIGIT antibody or its antigen-binding fragment
  • the antigen-binding fragment comprises a first heavy chain variable region and a first light chain variable region, wherein:
  • the first heavy chain variable region comprises H1CDR1, H1CDR2 and H1CDR3 selected from the group consisting of:
  • the first light chain variable region comprises L1CDR1, L1CDR2 and L1CDR3 selected from the group consisting of:
  • the anti-TIGIT antibody or antigen-binding fragment thereof has:
  • the H1CDR1, H1CDR2 and H1CDR3 are respectively SEQ ID NO: 25, 26 and 27 or the first heavy chain of the amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 25, 26 and 27.
  • the variable region, and the L1CDR1, L1CDR2 and L1CDR3 are respectively SEQ ID NO: 31, 32 and 33 or the first amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 31, 32 and 33 a light chain variable region; or
  • the H1CDR1, H1CDR2 and H1CDR3 are respectively SEQ ID NO: 28, 29 and 30 or the first heavy chain of the amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 28, 29 and 30.
  • the variable region, and the L1CDR1, L1CDR2 and L1CDR3 are respectively SEQ ID NO: 34, 35 and 36 or the first amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 34, 35 and 36 A light chain variable region.
  • the anti-TIGIT antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:
  • amino acid sequence of the first heavy chain variable region is selected from:
  • (b1) such as SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO: 88, the amino acid sequence shown in SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92;
  • amino acid sequence of the first light chain variable region is selected from:
  • the anti-TIGIT antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:
  • the amino acid sequence of the variable region of the first heavy chain is SEQ ID NO: 75, the amino acid sequence obtained by substitution, deletion or addition of one or more amino acids in SEQ ID NO: 75 and having the same function as SEQ ID NO: 75 or An amino acid sequence having at least 85% sequence identity with SEQ ID NO:75 and the H1CDR1, H1CDR2 and H1CDR3 amino acid sequences shown in SEQ ID NOs:25, 26 and 27, and the first light chain variable region
  • the amino acid sequence of SEQ ID NO: 77, SEQ ID NO: 77 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 77
  • the amino acid sequence of the variable region of the first heavy chain is SEQ ID NO: 76, the amino acid sequence of SEQ ID NO: 76 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO: 76 or An amino acid sequence having at least 85% sequence identity with SEQ ID NO: 76 and the H1CDR1, H1CDR2 and H1CDR3 amino acid sequences shown in SEQ ID NOs: 28, 29 and 30, and the first light chain variable region
  • the amino acid sequence of SEQ ID NO: 78, SEQ ID NO: 78 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 78.
  • the anti-TIGIT antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof comprising a first heavy chain variable region and a second A light chain variable region, wherein:
  • amino acid sequence of the first heavy chain variable region is selected from:
  • amino acid sequence of the first light chain variable region is selected from:
  • the anti-TIGIT antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof comprising a first heavy chain variable region and a second A light chain variable region, wherein
  • the amino acid sequence of the first heavy chain variable region is SEQ ID NO: 98, the amino acid sequence of SEQ ID NO: 98 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO: 98 or An amino acid sequence having at least 85% sequence identity with SEQ ID NO: 98, and the amino acid sequence of the first light chain variable region is SEQ ID NO: 100, which is substituted, deleted, or added with one or more An amino acid sequence obtained by multiple amino acids and functionally identical to SEQ ID NO: 100 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 100; or
  • the amino acid sequence of the first heavy chain variable region is SEQ ID NO: 99, the amino acid sequence obtained by substitution, deletion or addition of one or more amino acids in SEQ ID NO: 99 and having the same function as SEQ ID NO: 99 or An amino acid sequence having at least 85% sequence identity with SEQ ID NO: 99, and the amino acid sequence of the first light chain variable region is SEQ ID NO: 101, which is substituted, deleted, or added with one or more An amino acid sequence obtained by multiple amino acids that is functionally identical to SEQ ID NO: 101 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 101.
  • the present invention provides an anti-TIGIT/anti-PD-L1 antibody comprising an anti-TIGIT antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-TIGIT antibody or an antigen-binding fragment thereof as defined in the above embodiments; and the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and/or a second light chain variable region, wherein the second The heavy chain variable region comprises the complementarity determining region 1 (H2CDR1) of the second heavy chain variable region, the complementarity determining region 2 (H2CDR2) of the second heavy chain variable region and/or the complementarity determining region of the second heavy chain variable region Region 3 (H2CDR3), the second light chain variable region comprising complementarity determining region 1 (L2CDR1) of the second light chain variable region, complementarity determining region 2 (L2CDR2) of the second light chain variable region and/or Comp
  • the present invention provides an anti-TIGIT/anti-PD-L1 antibody comprising an anti-TIGIT antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti- The TIGIT antibody or antigen-binding fragment thereof is as defined in the above embodiments, and the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein:
  • the second heavy chain variable region comprises H2CDR1, H2CDR2 and H2CDR3 selected from the group consisting of:
  • (A5) an amino acid sequence having at least 85% sequence identity to the amino acid sequence shown in (A1), (A2), (A3) or (A4);
  • the second light chain variable region comprises L2CDR1, L2CDR2 and L2CDR3 selected from the group consisting of:
  • the present invention provides an anti-TIGIT/anti-PD-L1 antibody comprising an anti-TIGIT antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein:
  • the anti-TIGIT antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:
  • the first heavy chain variable region comprises H1CDR1, H1CDR2 and H1CDR3 selected from the group consisting of:
  • the first light chain variable region comprises L1CDR1, L1CDR2 and L1CDR3 selected from the group consisting of:
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein:
  • the second heavy chain variable region comprises H2CDR1, H2CDR2 and H2CDR3 selected from the group consisting of:
  • (A5) an amino acid sequence having at least 85% sequence identity to the amino acid sequence shown in (A1), (A2), (A3) or (A4);
  • the second light chain variable region comprises L2CDR1, L2CDR2 and L2CDR3 selected from the group consisting of:
  • the present invention provides an anti-TIGIT/anti-PD-L1 antibody comprising an anti-TIGIT antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-TIGIT antibody or an antigen-binding fragment thereof comprising said H1CDR1, H1CDR2 and H1CDR3 respectively as SEQ ID NOs: 28, 29 and 30 or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 28, 29 and 30
  • the first heavy chain variable region, and the L1CDR1, L1CDR2 and L1CDR3 are respectively SEQ ID NO: 34, 35 and 36 or have at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 34, 35 and 36 and the anti-PD-L1 antibody or antigen-binding fragment thereof comprising the H2CDR1, H2CDR2 and H2CDR3 are respectively SEQ ID NO: 1, 2 and 3 or with SEQ ID NO
  • the present invention provides an anti-TIGIT/anti-PD-L1 antibody comprising an anti-TIGIT antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-TIGIT antibody or an antigen-binding fragment thereof comprising said H1CDR1, H1CDR2 and H1CDR3 respectively as SEQ ID NOs: 28, 29 and 30 or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 28, 29 and 30
  • the first heavy chain variable region, and the L1CDR1, L1CDR2 and L1CDR3 are respectively SEQ ID NO: 34, 35 and 36 or have at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 34, 35 and 36 and the anti-PD-L1 antibody or antigen-binding fragment thereof comprising the H2CDR1, H2CDR2 and H2CDR3 are respectively SEQ ID NO: 7, 8 and 9 or with SEQ ID NO
  • the present invention provides an anti-TIGIT/anti-PD-L1 antibody comprising an anti-TIGIT antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-TIGIT antibody or an antigen-binding fragment thereof comprising said H1CDR1, H1CDR2 and H1CDR3 respectively as SEQ ID NOs: 28, 29 and 30 or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 28, 29 and 30
  • the first heavy chain variable region, and the L1CDR1, L1CDR2 and L1CDR3 are respectively SEQ ID NO: 34, 35 and 36 or have at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 34, 35 and 36 and the anti-PD-L1 antibody or antigen-binding fragment thereof comprising the H2CDR1, H2CDR2 and H2CDR3 are respectively SEQ ID NO: 4, 5 and 6 or with SEQ ID NO
  • the present invention provides an anti-TIGIT/anti-PD-L1 antibody comprising an anti-TIGIT antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-TIGIT antibody or an antigen-binding fragment thereof comprising said H1CDR1, H1CDR2 and H1CDR3 respectively as SEQ ID NOs: 28, 29 and 30 or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 28, 29 and 30
  • the first heavy chain variable region, and the L1CDR1, L1CDR2 and L1CDR3 are respectively SEQ ID NO: 34, 35 and 36 or have at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 34, 35 and 36 and the anti-PD-L1 antibody or antigen-binding fragment thereof comprising the H2CDR1, H2CDR2 and H2CDR3 are respectively SEQ ID NO: 10, 11 and 12 or with SEQ ID NO
  • the anti-TIGIT antibody or its antigen-binding fragment and the anti-PD-L1 antibody or its antigen-binding fragment are each independently a murine antibody, Chimeric, humanized or fully human antibodies.
  • the present invention provides an anti-TIGIT/anti-PD-L1 antibody, wherein the anti-TIGIT antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, in:
  • amino acid sequence of the first heavy chain variable region is selected from:
  • (b2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (b1) and having the same or similar function to the amino acid sequence shown in (b1);
  • amino acid sequence of the first light chain variable region is selected from:
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein:
  • amino acid sequence of the second heavy chain variable region is selected from:
  • amino acid sequence of the second light chain variable region is selected from:
  • the present invention provides an anti-TIGIT/anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO:98, and SEQ ID NO:98 is substituted, An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 98 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 98, and the first light chain variable region.
  • the present invention provides an anti-TIGIT/anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO:98, and SEQ ID NO:98 is substituted, An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO:98 or having at least 85% sequence identity with SEQ ID NO:98 and said H1CDR1, H1CDR2 and H1CDR3 as SEQ ID NO:25
  • the amino acid sequences shown in , 26 and 27, and the amino acid sequence of the first light chain variable region is SEQ ID NO: 100, SEQ ID NO: 100 is obtained by substitution, deletion or addition of one or more amino acids and with
  • the amino acid sequence of SEQ ID NO:100 is functionally identical or has at least 85% sequence identity to SEQ ID NO:100 and the L1CDR1, L1CDR2 and L1CDR3 amino acid sequences are set forth in SEQ ID NO:31, 32 and 33.
  • the anti-TIGIT/anti-PD-L1 antibody of the present invention wherein the anti-TIGIT antibody or anti-PD-L1 antibody can be a murine antibody, which further contains murine IgG1, IgG2a, IgG2b , the heavy chain constant region of IgG2c, IgG3 or a variant thereof, and the light chain constant region of a murine kappa chain or a variant thereof.
  • the anti-TIGIT/anti-PD-L1 antibody according to the present invention wherein the anti-TIGIT murine antibody further contains the heavy chain constant region of murine IgG1 or IgG2 or a variant thereof, and a murine The light chain constant region of a kappa chain or a variant thereof.
  • the invention provides an anti-TIGIT/anti-PD-L1 antibody, wherein the anti-TIGIT antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:
  • amino acid sequence of the first heavy chain variable region is selected from:
  • amino acid sequence of the first light chain variable region is selected from:
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein:
  • amino acid sequence of the second heavy chain variable region is selected from:
  • (C2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (C1) and having the same or similar function to the amino acid sequence shown in (C1);
  • (C3) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (C1);
  • amino acid sequence of the second light chain variable region is selected from:
  • (C5) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (C4) and having the same or similar function to the amino acid sequence shown in (C4);
  • the present invention provides an anti-TIGIT/anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO:99, SEQ ID NO:99 is substituted, deleted or An amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO:99 or having at least 85% sequence identity with SEQ ID NO:99 and said H1CDR1, H1CDR2 and H1CDR3 as SEQ ID NO:28, 29 and the amino acid sequence shown in 30, and the amino acid sequence of the first light chain variable region is SEQ ID NO: 101, SEQ ID NO: 101 is obtained by substitution, deletion or addition of one or more amino acids and is the same as SEQ ID NO: 101
  • the amino acid sequence of NO: 101 is functionally identical or has at least 85% sequence identity to SEQ ID NO: 101 and the L1CDR1, L1CDR2 and L1CDR3 amino acid sequences are as shown in SEQ ID NOs: 34, 35 and 36; and The amino acid sequence of the amino acid sequence
  • the present invention provides an anti-TIGIT/anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO:99, SEQ ID NO:99 is substituted, deleted or An amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO:99 or having at least 85% sequence identity with SEQ ID NO:99 and said H1CDR1, H1CDR2 and H1CDR3 as SEQ ID NO:28, 29 and the amino acid sequence shown in 30, and the amino acid sequence of the first light chain variable region is SEQ ID NO: 101, SEQ ID NO: 101 is obtained by substitution, deletion or addition of one or more amino acids and is the same as SEQ ID NO: 101
  • the amino acid sequence of NO: 101 is functionally identical or has at least 85% sequence identity to SEQ ID NO: 101 and the L1CDR1, L1CDR2 and L1CDR3 amino acid sequences are as shown in SEQ ID NOs: 34, 35 and 36; and The amino acid sequence of the amino acid sequence
  • the present invention provides an anti-TIGIT/anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO:99, SEQ ID NO:99 is substituted, deleted or An amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO:99 or having at least 85% sequence identity with SEQ ID NO:99 and said H1CDR1, H1CDR2 and H1CDR3 as SEQ ID NO:28, 29 and the amino acid sequence shown in 30, and the amino acid sequence of the first light chain variable region is SEQ ID NO: 101, SEQ ID NO: 101 is obtained by substitution, deletion or addition of one or more amino acids and is the same as SEQ ID NO: 101
  • the amino acid sequence of NO: 101 is functionally identical or has at least 85% sequence identity to SEQ ID NO: 101 and the L1CDR1, L1CDR2 and L1CDR3 amino acid sequences are as shown in SEQ ID NOs: 34, 35 and 36; and The amino acid sequence of the amino acid sequence
  • the present invention provides an anti-TIGIT/anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO:99, SEQ ID NO:99 is substituted, deleted or An amino acid sequence obtained by adding one or more amino acids that is functionally identical to SEQ ID NO:99 or has at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity to SEQ ID NO:99 and Said H1CDR1, H1CDR2 and H1CDR3 are the amino acid sequences shown in SEQ ID NOs: 28, 29 and 30, and the amino acid sequence of the first light chain variable region is SEQ ID NO: 101, and SEQ ID NO: 101 is substituted An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 101 or having at least 85%, or at least 90%, or at least 95%, or at least 98% sequence with SEQ ID NO: 101 identity and the amino acid sequences of the L1CDR1, L1
  • the present invention provides an anti-TIGIT/anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO:99, SEQ ID NO:99 is substituted, deleted or An amino acid sequence obtained by adding one or more amino acids that is functionally identical to SEQ ID NO:99 or has at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity to SEQ ID NO:99 and Described H1CDR1, H1CDR2 and H1CDR3 are the aminoacid sequence shown in SEQ ID NO:28, 29 and 30, and the aminoacid sequence of described first light chain variable region is SEQ ID NO:101, SEQ ID NO:101 is substituted An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 101 or having at least 85%, or at least 90%, or at least 95%, or at least 98% sequence with SEQ ID NO: 101 identity and the amino acid sequences of the L1CDR1, L
  • the present invention provides an anti-TIGIT/anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO:99, SEQ ID NO:99 is substituted, deleted or An amino acid sequence obtained by adding one or more amino acids that is functionally identical to SEQ ID NO:99 or has at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity to SEQ ID NO:99 and Said H1CDR1, H1CDR2 and H1CDR3 are the amino acid sequences shown in SEQ ID NOs: 28, 29 and 30, and the amino acid sequence of the first light chain variable region is SEQ ID NO: 101, and SEQ ID NO: 101 is substituted An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 101 or having at least 85%, or at least 90%, or at least 95%, or at least 98% sequence with SEQ ID NO: 101 identity and the amino acid sequences of the L1CDR1, L1
  • the present invention provides an anti-TIGIT/anti-PD-L1 humanized antibody, wherein the heavy chain comprises the heavy chain constant region of a human IgG1, IgG2, IgG3, IgG4 or a variant thereof, wherein The light chain comprises the light chain constant region of a human kappa, lambda chain or a variant thereof.
  • the murine anti-TIGIT/anti-PD-L1 antibody may further comprise a light chain constant region of a murine ⁇ , ⁇ chain or a variant thereof, and/or further comprise a murine constant region Heavy chain constant regions of IgGl, IgG2a, IgG2b, IgG2c, IgG3 or variants thereof.
  • the antibody light chain of the anti-TIGIT antibody or its antigen-binding fragment further comprises murine ⁇ , ⁇ chains or mutated sequences thereof. light chain constant region.
  • the antibody heavy chain of the anti-TIGIT antibody or its antigen-binding fragment further comprises a heavy chain constant region of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or a mutant sequence thereof, preferably a human IgG1, IgG2, IgG4 heavy chain constant Area.
  • the anti-TIGIT humanized antibody or antigen-binding fragment thereof further comprises human IgG1, IgG2a, IgG2b, IgG2c, IgG3 or a variant thereof The constant region of the heavy chain of the human body, and the constant region of the light chain of the human kappa, lambda chain or its variants.
  • the anti-TIGIT humanized antibody or antigen-binding fragment thereof of the present invention further comprises the heavy chain constant region of human IgG1, IgG2, IgG4 or a variant thereof, and a human kappa chain or variant thereof light chain constant region.
  • the antibody heavy chain of the anti-PD-L1 antibody or its antigen-binding fragment further comprises murine IgG1, IgG2a, IgG2b, IgG2c , the heavy chain constant region of IgG3 or its mutant sequence, preferably comprising the heavy chain constant region of human IgG or its mutant sequence;
  • the antibody light chain of the anti-PD-L1 antibody or its antigen-binding fragment further comprises murine ⁇ , ⁇ light chain constant region of the chain or a mutated sequence thereof.
  • the anti-PD-L1 humanized antibody or antigen-binding fragment thereof further comprises human IgG1, IgG2, IgG3 or IgG4 or a variant thereof The constant region of the heavy chain of the human body, and the constant region of the light chain of the human kappa, lambda chain or its variants.
  • the anti-TIGIT humanized antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region of human IgG4 or a variant thereof, and a light chain constant region of a human kappa chain or a variant thereof Area.
  • the present invention provides anti-TIGIT/anti-PD-L1 antibodies, wherein the anti-TIGIT antibodies or antigen-binding fragments thereof and anti-PD-L1 antibodies or antigen-binding fragments thereof are Fab, Fv, sFv or F, respectively (ab) 2 .
  • the anti-TIGIT/anti-PD-L1 antibody provided by the present invention is scF(ab) 2 .
  • the anti-TIGIT/anti-PD-L1 antibody in the above embodiment of the invention is an anti-TIGIT/anti-PD-L1 bispecific antibody.
  • the bispecific antibody is a human antibody or a humanized antibody.
  • one of the binding specificities is for TIGIT and the other binding specificity is for any other antigen.
  • one of the binding specificities is for TIGIT and the other binding specificity is for PD-L1.
  • the bispecific antibody binds two different epitopes of TIGIT.
  • the bispecific antibodies can also be used to localize cytotoxic agents to TIGIT-expressing cells.
  • bispecific antibodies of the invention can be prepared as full-length antibodies or antibody fragments (eg, F(ab') 2 bispecific antibodies).
  • bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, Nature 305:537 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) may produce a mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of this correct molecule is usually carried out by an affinity chromatography step, which is rather cumbersome and yields low product. Similar methods are disclosed in WO93/08829 and Traunecker et al., EMBOJ. 10:3655 (1991).
  • antibody variable regions with the desired binding specificities are fused to immunoglobulin constant region sequences.
  • the fusion is to an immunoglobulin heavy chain constant region comprising at least a portion of the hinge, CH2 and CH3 regions.
  • the first heavy chain constant region (CH1 ) comprising the site necessary for binding to the light chain is present in at least a portion of the fusion.
  • the DNA encoding the immunoglobulin heavy chain fusion fragment and, if desired, the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host organism.
  • the bispecific antibody consists of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair in the other arm (provides a second binding specificity) composition. Since the presence of the immunoglobulin light chain in only one half of the bispecific molecule provides a convenient separation route, this asymmetric structure was found to facilitate separation of the desired bispecific material from the undesired immunoglobulin chain composition . This method is disclosed in WO 94/04690. For further information on the production of bispecific antibodies see, e.g., Suresh et al., Methods in Enzymology 121:210 (1986).
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture.
  • the interface comprises at least a portion of the CH3 domain of the antibody constant region.
  • one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (eg, tyrosine or tryptophan).
  • larger side chains eg, tyrosine or tryptophan.
  • a compensatory "cavity" of the same or similar size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers compared to other undesired end products such as homodimers.
  • Bispecific antibodies include cross-linked or "heteroconjugate" antibodies.
  • one heteroconjugated antibody can be conjugated to avidin and the other heteroconjugated antibody can be conjugated to biotin.
  • Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable crosslinking agents are well known in the art and are disclosed in US Pat. No. 4,676,980, along with a number of crosslinking techniques.
  • Bispecific antibodies of the present invention can be produced from antibody fragments.
  • bispecific antibodies can be prepared using chemical ligation techniques.
  • Brennetal., Science 229:81 (1985) describes a method for proteolytic cleavage of intact antibodies to generate F(ab') 2 fragments. These fragments are decomposed in the presence of the dithiol complexing agent sodium arsenite (to stabilize adjacent dithiols and prevent the formation of intermolecular disulfide bonds).
  • the resulting Fab' fragments were then converted into thionitrobenzoate (TNB) derivatives.
  • TAB thionitrobenzoate
  • One of the Fab'-TNB derivatives was then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody.
  • Fab'-SH fragments can be recovered directly from E. coli and these fragments can be chemically coupled to form bispecific antibodies.
  • Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the generation of fully humanized bispecific antibody F(ab') 2 molecules.
  • Each Fab' fragment is secreted separately from E. coli and subjected to targeted chemical coupling in vitro to form bispecific antibodies.
  • bispecific antibody fragments of the invention can be produced and isolated directly from recombinant cell culture.
  • bispecific antibodies can be generated using leucine zippers (Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)).
  • Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by genetic fusion.
  • Antibody homodimers break down at the hinge region to form monomers, which are then reoxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers.
  • Diabody technology provides additional mechanisms for making bispecific antibody fragments.
  • the bispecific antibody fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL) joined by a linker that is too short to allow pairing between the two domains on the same chain .
  • VH and VL domains on one fragment are forced to pair with the complementary VL and VH domains on the other fragment, thereby forming two antigen binding sites.
  • bispecific antibody fragments can be constructed by using single-chain Fv (sFv) dimers.
  • the present invention encompasses multivalent antibodies with more than two valences, eg, trispecific antibodies can be prepared.
  • Multivalent antibodies can be internalized (and/or dissimilated) more rapidly than bivalent antibodies by cells expressing the antigen to which the antibody binds.
  • Antibodies of the invention may be multivalent antibodies having three or more antigen binding sites (eg, tetravalent antibodies) that can be readily produced by recombinant expression of nucleic acids encoding antibody polypeptide chains.
  • Multivalent antibodies may contain a dimerization domain and three or more antigen binding sites. In some embodiments, the dimerization domain comprises (or consists of) an Fc region or a hinge region.
  • the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region.
  • the multivalent antibody comprises (or consists of) three to about eight antigen binding sites.
  • the multivalent antibody comprises four antigen binding sites.
  • Multivalent antibodies comprise at least one polypeptide chain (eg, two polypeptide chains), wherein the polypeptide chains comprise two or more variable regions.
  • the multivalent antibodies of the present invention may further comprise at least two (eg, four) light chain variable region polypeptides.
  • the multivalent antibodies of the invention may comprise, for example, from about two to about eight light chain variable region polypeptides.
  • the light chain variable region polypeptides of the invention comprise a light chain variable region, and optionally further comprise a CL domain.
  • one of the TIGIT targeting moiety and the PD-L1 targeting moiety may be a full-length antibody, and the other may be a heavy chain CDR, light Antigen-binding fragments (eg, scFvs) of chain CDRs or combinations thereof.
  • a full-length antibody targeting one of the TIGIT and PD-L1 proteins and an antigen-binding fragment targeting the other protein can be linked (eg, covalently) directly or chemically via a linking peptide.
  • Antigen-binding fragments can be directly or by linking peptides to the N-terminus of a full-length antibody (eg, the N-terminus of a light or heavy chain of a full-length antibody), the C-terminus of a full-length antibody (eg, of a full-length antibody the C-terminus of the heavy chain (or Fc or CH3 domains) or both.
  • the bispecific antibody may comprise a full-length anti-TIGIT antibody, an antigen-binding fragment (eg, scFab, scFv) of an anti-PD-L1 antibody, and a linker peptide therebetween.
  • the bispecific antibody may comprise a full-length anti-TIGIT antibody, an antigen-binding fragment of an anti-PD-L1 antibody (eg, scFab, scFv), and linking peptides therebetween.
  • the scFv comprised in the bispecific antibody may comprise a heavy chain variable region and a light chain variable region in any order.
  • the scFv contained in a bispecific antibody may comprise a heavy chain variable region and a light chain variable region and optionally a linking peptide between them in the N-terminal to C-terminal direction, or alternatively
  • the scFv comprised in the bispecific antibody may comprise, in the direction from the N-terminus to the C-terminus, a light chain variable region and a heavy chain variable region and optionally a linking peptide therebetween.
  • the linker peptide may include, for example, Gly, Asn, and/or Ser residues, and may also include neutral amino acids, such as Thr and/or Ala.
  • Amino acid sequences suitable for linking peptides may be those known in the relevant art.
  • the length of the linker peptide can be determined differently within such limits that the function of the fusion protein is not affected.
  • the linker peptide can be formed by including a total of about 1 to about 100, about 2 to about 50, or about 5 to about 25 one or more selected from the group consisting of Gly, Asn, Ser, Thr, and Ala .
  • the linker peptide can be represented as (GmSl)n (m, 1, and n are independently integers from about 1 to about 10, particularly from about 2 to about 5).
  • the PD-L1 targeting moiety and the TIGIT targeting moiety can both be full-length antibodies or antigen-binding fragments comprising heavy chain CDRs, light chain CDRs, or a combination thereof.
  • the bispecific antibody may be in the form of a heterodimer comprising a first arm and a second arm, the first arm comprising a pair of first heavy chains targeting one of TIGIT and PD-L1 and a first light chain, the second arm includes a pair of a second heavy chain and a second light chain that target the other.
  • full-length antibodies may be in the form of full-length immunoglobulins (eg, IgG, IgM, IgA, IgE, or IgD, eg, human IgG, human IgM, human IgA, human IgE, or human IgD), and antigen-binding fragments It can be selected from the group consisting of Fab, Fab', F(ab') 2 , Fd, Fv, scFv, scFab, single chain antibody, sdFv, and the like.
  • immunoglobulins eg, IgG, IgM, IgA, IgE, or IgD
  • antigen-binding fragments It can be selected from the group consisting of Fab, Fab', F(ab') 2 , Fd, Fv, scFv, scFab, single chain antibody, sdFv, and the like.
  • the full-length antibody can be in the form of a full-length human IgG (human IgGl, human IgG2, human IgG3, or human IgG4), and the antigen-binding fragment can be an scFv.
  • the antibodies described herein can contain flexible linker sequences, or can be modified to add functional moieties (eg, PEG, drugs, toxins, or labels).
  • the structure of the anti-TIGIT antibody or antigen-binding fragment thereof is (VL-CL)-linking peptide-(VH)- IgG4CH
  • the structure of the anti-PD-L1 antibody or its antigen-binding fragment is (VL-CL)-linking peptide-(VH)-IgG4CH.
  • the structure of the anti-TIGIT antibody or its antigen-binding fragment is (VL-VH)-linking peptide-IgG4FC, so The structure of the anti-PD-L1 antibody or its antigen-binding fragment is (VL-CL)-linking peptide-(VH)-IgG4CH.
  • the linking peptide is in the form of (GGGGS)n, wherein n is 1-12, preferably 3-10, more preferably 6-8, eg, 6, 7, 8 GGGGS repeats.
  • the IgG4FC in the (VL-CL)-linked peptide-(VH)-IgG4CH targeting the TIGIT moiety is an IgG4CH segment containing S228P, L235E mutations, and the (VL-CL) targeting the PD-L1 moiety
  • the IgG4FC in the -CL)-linking peptide-(VH)-IgG4CH is the IgG4CH segment containing S228P and L235E mutations.
  • the IgG4FC in the (VL-CL)-connecting peptide-(VH)-IgG4CH targeting the TIGIT moiety is an IgG4CH segment containing S228P, S354C, T366W mutations to form a "Hole" structure
  • targeting The IgG4FC in the (VL-CL)-linking peptide-(VH)-IgG4CH of the PD-L1 part is an IgG4CH segment containing S228P, Y349C, T366S, L368A, Y407V mutations to form a "Knob" structure.
  • the IgG4FC in the (VL-CL)-linking peptide-(VH)-IgG4CH targeting the TIGIT moiety is an IgG4CH segment containing S228P, L235E, S354C, T366W mutated to form a "Hole" structure
  • the IgG4FC in the (VL-CL)-linking peptide-(VH)-IgG4CH targeting the PD-L1 moiety is an IgG4CH segment containing S228P, L235E, Y349C, T366S, L368A, Y407V mutations to form a "Knob" structure.
  • the anti-TIGIT/anti-PD-L1 bispecific antibody according to the invention consists of two peptide chains, wherein:
  • peptide chain 1 The structure of peptide chain 1 is:
  • IgG4CH/Ks represents the IgG4 constant region (S228P, Y349C, T366S, L368A, Y407V mutations), and IgG4CH/Hs represents the IgG4 constant region (S228P, S354C, T366W mutations).
  • the anti-TIGIT/anti-PD-L1 bispecific antibody according to the present invention consists of two peptide chains, wherein:
  • peptide chain 1 The structure of peptide chain 1 is:
  • IgG4CH/PE represents IgG4 constant region (S228P, L235E mutation).
  • the anti-TIGIT/anti-PD-L1 bispecific antibody according to the present invention consists of two peptide chains, wherein the amino acid sequence of peptide chain 1 is SEQ ID NO: 102, and the amino acid sequence of peptide chain 2 is SEQ ID NO: 102.
  • the amino acid sequence is SEQ ID NO:103.
  • the anti-TIGIT/anti-PD-L1 bispecific antibody according to the present invention is composed of two peptide chains, wherein the amino acid sequence of peptide chain 1 is SEQ ID NO: 104, and the amino acid sequence of peptide chain 2 is SEQ ID NO: 104.
  • the amino acid sequence of SEQ ID NO: 105 is composed of two peptide chains, wherein the amino acid sequence of peptide chain 1 is SEQ ID NO: 104, and the amino acid sequence of peptide chain 2 is SEQ ID NO: 104.
  • the amino acid sequence of SEQ ID NO: 105 is composed of two peptide chains, wherein the amino acid sequence of peptid
  • the anti-TIGIT/anti-PD-L1 bispecific antibody according to the present invention is composed of two peptide chains, wherein the amino acid sequence of peptide chain 1 is SEQ ID NO: 102, and the amino acid sequence of peptide chain 2 is SEQ ID NO: 102.
  • the anti-TIGIT/anti-PD-L1 bispecific antibody according to the present invention is composed of two peptide chains, wherein the amino acid sequence of peptide chain 1 is SEQ ID NO: 107, and the amino acid sequence of peptide chain 2 is SEQ ID NO: 107.
  • the anti-TIGIT/anti-PD-L1 bispecific antibody according to the present invention is composed of two peptide chains, wherein the amino acid sequence of peptide chain 1 is SEQ ID NO: 102, and the amino acid sequence of peptide chain 2 is SEQ ID NO: 102.
  • the anti-TIGIT/anti-PD-L1 bispecific antibody according to the present invention consists of two peptide chains, wherein the amino acid sequence of peptide chain 1 is SEQ ID NO: 110, and the amino acid sequence of peptide chain 2 is SEQ ID NO: 110.
  • the amino acid sequence of SEQ ID NO: 111 Another aspect of the invention provides isolated nucleic acids.
  • an isolated nucleic acid according to the present invention encodes an anti-TIGIT/anti-PD-L1 antibody of the present invention. In some embodiments, an isolated nucleic acid according to the present invention encodes an anti-TIGIT antibody or antigen-binding fragment thereof of the present invention. In other embodiments, the isolated nucleic acid according to the present invention encodes an anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention.
  • the isolated nucleic acid according to the present invention comprises a variable region encoding a first heavy chain such as SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:98 or SEQ ID NO:99 and the nucleotide sequence encoding the first light chain variable region such as SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:100 or SEQ ID NO:101.
  • the isolated nucleic acid according to the present invention comprises a nucleotide sequence encoding a second heavy chain variable region such as SEQ ID NO:50, SEQ ID NO:52 or SEQ ID NO:61 and a nucleotide sequence encoding a second light chain variable region such as SEQ ID NO:54, SEQ ID NO:56 or SEQ ID NO:64.
  • the isolated nucleic acid according to the present invention comprises a nucleotide sequence encoding a first heavy chain variable region such as SEQ ID NO:99 and a first light chain variable region encoding such as SEQ ID Nucleotide sequence of NO:101.
  • the isolated nucleic acid according to the present invention comprises a nucleotide sequence encoding the second heavy chain variable region SEQ ID NO:50 and encoding the second light chain variable region SEQ ID NO: 54 nucleotide sequence.
  • an isolated nucleic acid combination according to the present invention comprises a nucleotide sequence encoding a first heavy chain variable region such as SEQ ID NO:99 encoding a first light chain variable region such as SEQ ID The nucleotide sequence of NO:101, the nucleotide sequence encoding the second heavy chain variable region such as SEQ ID NO:50, and the nucleotide sequence encoding the second light chain variable region such as SEQ ID NO:54.
  • an isolated nucleic acid combination comprises a nucleotide sequence encoding a first heavy chain variable region such as SEQ ID NO:99 encoding a first light chain variable region such as SEQ ID NO: 99
  • the nucleotide sequence of ID NO:101, the nucleotide sequence encoding the second heavy chain variable region such as SEQ ID NO:52, and the nucleotide sequence encoding the second light chain variable region such as SEQ ID NO:56 is a nucleotide sequence encoding a first heavy chain variable region such as SEQ ID NO:99 encoding a first light chain variable region such as SEQ ID NO: 99
  • the nucleotide sequence of ID NO:101, the nucleotide sequence encoding the second heavy chain variable region such as SEQ ID NO:52, and the nucleotide sequence encoding the second light chain variable region such as SEQ ID NO:56 is a nucleotide sequence encoding a first heavy chain variable region such as SEQ ID NO:99
  • an isolated nucleic acid combination comprises a nucleotide sequence encoding a first heavy chain variable region such as SEQ ID NO:99 encoding a first light chain variable region such as SEQ ID NO: 99
  • the nucleotide sequence of ID NO:101, the nucleotide sequence encoding the second heavy chain variable region such as SEQ ID NO:61, and the nucleotide sequence encoding the second light chain variable region such as SEQ ID NO:64 is a nucleotide sequence encoding a first heavy chain variable region such as SEQ ID NO:99 encoding a first light chain variable region such as SEQ ID NO: 99
  • the expression vector of the invention expresses an anti-TIGIT/anti-PD-L1 bispecific antibody of the invention. In some embodiments, the expression vector of the present invention expresses an anti-TIGIT antibody or antigen-binding fragment thereof of the present invention. In other embodiments, the expression vector of the present invention expresses an anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention. In some embodiments, according to the expression vector of the present invention, the vector expressing the anti-TIGIT antibody or antigen-binding fragment thereof of the present invention and the vector expressing the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention are the same expression vector.
  • the expression vector according to the present invention comprises the isolated nucleic acid molecule of the present invention.
  • Another aspect of the present invention provides a host cell transformed with the expression vector as described above.
  • the host cell according to the present invention is selected from prokaryotic cells and eukaryotic cells.
  • the host cell is a bacterium, preferably E. coli.
  • the host cell is a mammalian cell.
  • Another aspect of the present invention provides a method for preparing an anti-TIGIT/anti-PD-L1 bispecific antibody of the present invention, comprising the steps of expressing the antibody in the host cell and isolating the antibody from the host cell.
  • Another aspect of the present invention provides a pharmaceutical composition comprising the anti-TIGIT/anti-PD-L1 bispecific antibody of the present invention and a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition comprising the anti-TIGIT/anti-PD-L1 bispecific antibody of the present invention and other active components, such as other antibodies, targeted drugs, and the like.
  • the pharmaceutically acceptable carrier is selected from the group consisting of antioxidants, polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, sugar alcohols, ions, and surfactants.
  • the pharmaceutically acceptable carrier is a buffered aqueous solution.
  • the pharmaceutically acceptable carrier is in the form of a liposome.
  • a chimeric antigen receptor (CAR) fusion protein comprising an anti-TIGIT antibody or an antigen-binding fragment thereof and/or an anti-PD-L1 antibody or an antigen-binding fragment thereof of the present invention.
  • the chimeric antigen receptor fusion protein comprises an anti-TIGIT antibody or antigen-binding fragment thereof of the invention, which is a single-chain variable fragment (scFv) directed against the VH and VL of the TIGIT antigen.
  • the chimeric antigen receptor fusion protein comprises an anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention, which is a single-chain variable fragment directed against the VH and VL of the PD-L1 antigen (scFv).
  • the chimeric antigen receptor fusion protein comprises a first single-chain variable fragment (scFv) directed against the VH and VL of the TIGIT antigen and a VH and VL directed against the PD-L1 antigen.
  • a second single-chain variable fragment (scFv) directed against the VH and VL of the TIGIT antigen and a VH and VL directed against the PD-L1 antigen.
  • the first scFv for VH and VL of the TIGIT antigen has H1CDR1, H1CDR2 and H1CDR3 of the first heavy chain variable region and L1CDR1, L1CDR2 and L1CDR3 of the first light chain variable region as described in the above embodiments.
  • the second scFv for VH and VL of the PD-L1 antigen has the H2CDR1, H2CDR2 and H2CDR3 of the second heavy chain variable region and the L2CDR1, L2CDR2 and L2CDR1, L2CDR2 and H2CDR3 of the second light chain variable region described in the above embodiment. L2CDR3.
  • the anti-TIGIT/anti-PD-L1 bispecific antibody of the present invention can be mixed with a pharmaceutically acceptable carrier, diluent or excipient to prepare a pharmaceutical preparation suitable for oral or parenteral administration.
  • Methods of administration include, but are not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, intracerebral, intraocular, intratracheal, subcutaneous, intranasal routes.
  • the formulations may be administered by any route, such as by infusion or bolus injection, by route of absorption through the epithelium or mucocutaneous (eg, oral mucosa or rectum, etc.). Administration can be systemic or local.
  • the formulations can be prepared by methods known in the art and include carriers, diluents or excipients conventionally used in the art of pharmaceutical formulations.
  • Another aspect of the invention provides a method of treating and/or preventing a disease associated with TIGIT, PD-L1 or both, the method comprising administering to an individual in need thereof an anti-TIGIT/anti-PD-L1 bispecific of the invention Antibodies or pharmaceutical compositions of the present invention.
  • Another aspect of the present invention provides an anti-TIGIT/anti-PD-L1 bispecific antibody of the present invention or a pharmaceutical composition of the present invention in the manufacture of a medicament for the treatment and/or prevention of diseases associated with TIGIT, PD-L1 or both Applications.
  • the disease associated with TIGIT, PD-L1, or both comprises hematological tumors, lymphomas, breast cancer, lung cancer, gastric cancer, bowel cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer cancer, pancreatic cancer, glioma and/or melanoma.
  • the tumor can be any tumor that expresses PD-L1 protein, such as bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, lung cancer (eg, small cell lung cancer, non-small cell lung cancer) etc.), breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, thyroid cancer, etc.
  • the tumor can be a primary or metastatic tumor.
  • the present invention provides the use of the above-mentioned anti-TIGIT/anti-PD-L1 bispecific antibody or the pharmaceutical composition of the present invention in the preparation of anti-tumor drugs, for example, the tumor is selected from hematological tumors, lymphoma, Breast cancer, lung cancer, stomach cancer, bowel cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma, melanoma, myeloma, prostate cancer.
  • the anti-TIGIT/anti-PD-L1 bispecific antibody provided by the invention has significant anti-tumor effect, can significantly inhibit tumor growth, can be used in the preparation of drugs for treating various tumor diseases, and has broad market prospects.
  • the term "at least 80% sequence identity” means at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.
  • the term “at least 85% sequence identity” means at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.
  • sequence identity of the present invention may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% %. Sequence comparison and percent identity determination between two sequences can be performed by the BLASTN/BLASTP algorithm on the National Center For Biotechnology Institute website.
  • the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface.
  • the antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy and light chain are referred to as "complementarity determining regions" or "CDRs.”
  • CDRs complementarity determining regions
  • the “antibody” of the present invention refers to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen.
  • Antibodies can be whole antibodies and any antigen-binding fragments or single chains thereof.
  • An “antibody” of the present invention includes any protein or peptide containing at least a portion of an Ig molecule that has the biological activity of binding an antigen.
  • Examples of “antibodies” of the invention include, but are not limited to, heavy or light chain CDRs or ligand binding portions thereof, heavy or light chain variable regions, heavy or light chain constant regions, framework regions, or any portion thereof.
  • the "antigen-binding fragments" of the present invention include Fab fragments, Fab' fragments, F(ab')2 fragments with antigen-binding activity, and Fv fragments and scFv fragments that bind to human TIGIT or PD-L1.
  • An Fv fragment is the smallest antibody fragment that contains the first heavy chain variable region and the first light chain variable region of an antibody, but no constant region, and has all antigen-binding sites.
  • Fv antibodies also contain a multi-linker peptide between the VH and VL domains and are capable of forming the structure required for antigen binding.
  • scFv single-chain antibody or single-chain Fv
  • the anti-TIGIT or anti-PD-L1 antibody of the present invention may be a single-chain variable fragment (scFv), which is derived from the single-chain polypeptide of the antibody and retains the ability to bind antigen.
  • scFvs include antibody polypeptides formed by recombinant DNA techniques in which the Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer sequence.
  • H chain immunoglobulin heavy chain
  • L chain light chain
  • Antibodies in the context of the present invention refer to immunoglobulin molecules or immunologically active portions thereof, ie molecules comprising an antigen-binding site that specifically binds to (immunoreacts with) an antigen.
  • "Specifically binds” means that an antibody reacts with one or more epitopes of an antigen but does not react with other polypeptides or binds other polypeptides with very low affinity (Kd>10<" 6 >).
  • Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAbs (domain antibodies), single chain, Fab, Fab' and F(ab')2 fragments, Fv, scFv, and Fab expression libraries.
  • Monoclonal antibodies are antibodies obtained from a single clonal cell line, which is not limited to eukaryotic, prokaryotic or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombinant techniques such as hybridoma technology, recombinant technology, phage display technology, and synthetic technology such as CDR grafting or other existing techniques.
  • the "murine antibody” in the present invention is a monoclonal antibody to human TIGIT prepared according to the knowledge and skills in the art. In preparation, test subjects are injected with TIGIT antigen, and hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • the "chimeric antibody” of the present invention is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, and can reduce the immune response induced by the murine antibody.
  • To establish a chimeric antibody first establish a hybridoma that secretes a mouse-specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as needed, and then clone the variable region of the mouse.
  • the gene is linked with the human constant region gene to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the "humanized antibody” of the present invention is also referred to as a CDR-grafted antibody, and is an antibody produced by grafting mouse CDR sequences into a human antibody variable region framework (FR).
  • FR human antibody variable region framework
  • Such variable region framework sequences can be obtained from public DNA databases or published references, eg from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr or from J. Immunoglobulins, 2001 ISBN012441351.
  • Bispecific antibodies as used in the present invention refer to monoclonal antibodies that have binding specificities for at least two different antigens.
  • linking peptides may be those comprising any amino acid from 1 to 10, especially 2 to 50, and may comprise any kind of amino acid without any limitation.
  • Figure 1 is a schematic diagram of the bispecific anti-TIGIT/anti-PD-L1 antibody structure of ScFab structure.
  • Figure 2 is a schematic diagram of the bispecific anti-TIGIT/anti-PD-L1 antibody structure of the ScFv structure.
  • mice and SD strains of experimental rats were immunized with mFc-labeled PD-L1 antigen protein (purchased from Beijing Biopsis Biotechnology Co., Ltd., China) and adjuvant.
  • mFc-labeled PD-L1 antigen protein purchased from Beijing Biopsis Biotechnology Co., Ltd., China
  • the immune adjuvant was Freund's Adjuvant, Complete (SIGMA, F5881-10ML) for the first time, and Freund's Adjuvant, Incomplete (SIGMA, F5506-10ML) was used later.
  • the PD-L1 antigen protein samples with different labels were added dropwise to the adjuvant solution, vortexed while adding dropwise to mix thoroughly, and the dosage of adjuvant was carried out with reference to the instructions. Mice or rats were immunized after mixing well to form a water-in-oil milk.
  • the immunization schedule is shown in Table 1:
  • the fused cell suspension was transferred to 15 mL of RPMI 1640 complete medium containing 20% FBS, and placed at room temperature for 20 min. Confluent cells were resuspended in RPMI 1640 medium containing 1 ⁇ HAT, 1 ⁇ BIOMYC3, 20% FBS.
  • the cell supernatant was taken, and the hybridoma parent clones with binding and blocking ability were screened by ELISA and expanded and cultured. tumor-positive cell lines.
  • the positive cell lines were subcloned by the limiting dilution method, and after one week of culture, the binding activity of the subclone supernatant to PD-L1 molecules and the activity of blocking the interaction of PD-L1/PD-1 were detected by ELISA to obtain PL-7,
  • the four strains PL-15, PL-16 and PL-18 are preferably double-positive cell strains.
  • the subcloned positive hybridoma cells were expanded and cultured, and an appropriate amount of cells were taken to extract total RNA according to the instructions of the RNeasy Plus Mini Kit (Qiagen, 74134), and reverse transcribed using Prime Script 1st strand cDNA Synthesis Kit (Takara, 6110A).
  • the kit synthesizes the first strand of cDNA.
  • Design specific primers according to the variable region of the mouse antibody subtype (the 5' end contains the homology arm sequence for homologous recombination with the eukaryotic expression vector), and use the cDNA as the template to carry out PCR amplification of the variable region gene of the antibody , to obtain the gene fragments of the variable region of the light chain and heavy chain of the mouse antibody, respectively, named SHS009PL-7, SHS009PL-15, SHS009PL-16, SHS009PL-18; design primers (references: 1. Anke Krebber, Susanne Bornhauser , Jorg Burmester et al.
  • the purified mouse antibody light chain and heavy chain variable region gene fragments were co-transformed into E. coli DH5 ⁇ competent cells with linearized eukaryotic expression plasmids containing human antibody light chain or heavy chain constant region gene fragments, respectively.
  • the chimeric antibodies with correct sequencing were labeled as PL-7CHI, PL-15CHI, PL-16CHI, and PL-18CHI, respectively. The sequencing results of the chimeric antibodies are shown in Table 3.
  • PL-7CHI, PL-15CHI, PL-16CHI, PL-18CHI light and heavy chain variable region amino acid sequences respectively identical to murine antibodies SHS009PL-7, SHS009PL-15, SHS009PL-16, SHS009PL-18 light and heavy chain variable region amino acids The sequence is the same.
  • the chimeric antibody light chain plasmid and heavy chain plasmid were extracted and transfected into HEK 293F cells, and a large number of antibodies were obtained by expression and purification. Purity detection, activity analysis and affinity detection were carried out.
  • PL-7CHI and PL-16CHI were selected for humanized antibody transformation according to the results of the activity analysis and affinity KD value of the chimeric antibody.
  • PL-7CHI and PL-16CHI antibody heavy chain variable region FR regions are respectively associated with human antibody germline genes M99683
  • PL-7CHI humanized heavy chains were obtained (PL-7CHI humanized heavy chain variable region sequences: VH1-0 (SEQ ID NO: 48), VH1-1 (SEQ ID NO: 49), VH1- 2 (SEQ ID NO:50), VH1-3 (SEQ ID NO:51) or VH1-4 (SEQ ID NO:52)), 4 humanized light chains (PL-7CHI humanized light chain variable Region sequence: VL1-0 (SEQ ID NO:53), VL1-1 (SEQ ID NO:54), VL1-2 (SEQ ID NO:55) or VL1-3 (SEQ ID NO:56)); PL- 16CHI humanized heavy chain 5 (PL-16CHI humanized heavy chain variable region sequence: VH1-0 (SEQ ID NO: 57), VH1-1 (SEQ ID NO: 58), VH1-2 (SEQ ID NO: 58) NO:59), VH1-3 (SEQ ID NO:60) or VH1-4 (SEQ ID NO:61)), 3 humanized light chains (PL-16CHI humanized light chain variable region sequence: VH1-0
  • the above-designed humanized antibody light chain and heavy chain variable region amino acid sequences are synthesized to correspond to nucleotide coding sequences, and oligonucleotide fragments containing complementary sequences between adjacent fragments are generated.
  • the oligonucleotide fragments are annealed and connected, and then specific primers (the 5' end contains the homology arm sequence for homologous recombination with the eukaryotic expression vector) are used to amplify the complete light chain and heavy chain variable regions Nucleotide fragment; the purified light chain variable region nucleotide fragment and the linearized eukaryotic expression plasmid containing the IgG4 light chain constant region were co-transformed into E.
  • E. coli DH5 ⁇ competent cells and the purified heavy chain variable region was transformed into E. coli DH5 ⁇ competent cells.
  • the nucleotide fragment of the region and the eukaryotic expression plasmid containing the S228P/L235E mutated IgG4 heavy chain constant region were co-transformed into E. coli DH5 ⁇ competent cells, and the competent cells of the transformed plasmid were evenly coated on the surface of an agar plate containing the corresponding antibiotics. , after overnight incubation in a constant temperature incubator at 37°C, several single colonies were picked for DNA sequencing.
  • the positive clones with correct sequencing were subjected to plasmid extraction to obtain humanized antibody light chain and heavy chain expression plasmids, and a nucleic acid quantitative analyzer was used to detect the concentration and purity of the plasmids.
  • the plasmid was transfected into HEK293F cells, expressed and purified to obtain a large number of antibodies, and the purity detection, activity analysis and affinity detection were carried out. See Table 4 for the sequence.
  • the tagged TIGIT protein extracellular region gene fragments TIGIT-hFc and TIGIT-mFc were synthesized and cloned into the eukaryotic expression plasmid pHR to obtain the expression plasmid pHR-TIGIT- hFc, pHR-TIGIT-mFc.
  • the amino acid sequence of the extracellular region of human TIGIT protein is fused with his amino acid sequence, and the amino acid sequence design is shown in SEQ ID NO: 67. After codon optimization of the amino acid sequence, a complete expression plasmid pcDNA3.1-TIGIT-his was synthesized.
  • the tagged CD155 protein extracellular region gene fragments CD155-hFc and CD155-mFc were synthesized and cloned into the eukaryotic expression plasmid pHR to obtain the expression plasmid pHR-CD155- hFc, pHR-CD155-mFc.
  • the amino acid sequence of the extracellular region of the human CD155 protein is fused with his amino acid sequence, and the amino acid sequence design is shown in SEQ ID NO: 70.
  • the complete expression plasmid pcDNA3.1(+)-CD155-his was synthesized after codon optimization of the amino acid sequence.
  • the humanized antibody 22G2 (herein referred to as 22G2) disclosed in patent application US 2016/0176963A1 was used as a positive control antibody. 22G2 was prepared with reference to the method disclosed in US 2016/0176963A1. The amino acid sequence of 22G2 is shown below:
  • 22G2 heavy chain variable region amino acid sequence SEQ ID NO: 71;
  • the amino acid sequence corresponding to the 22G2 antibody was artificially optimized for codons, and the heavy chain gene fragment was cloned into the eukaryotic expression plasmid pHR containing the constant region of the IgG4 light chain to obtain the heavy chain eukaryotic expression plasmid pHR-22G2-hG4 of 22G2 , and its light chain expression plasmid is pHR-22G2-hk.
  • a gene fragment encoding the full-length TIGIT protein was synthesized, and the amino acid sequence design was shown in SEQ ID NO: 73, and then cloned into the eukaryotic expression plasmid pTargeT to obtain its expression plasmid pTargeT-TIGIT.
  • the eukaryotic expression plasmid pTargeT-TIGIT was transfected into CHO-K1 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) by electroporation at a voltage of 160V and a square pulse of 15msec, in a 37°C, 5% CO2 incubator cultivated in. After 24h, pressurized culture medium containing 500 ⁇ g/ml G418 was used. After 16 days, the positive rate of the pool was detected by FACS, and the cells after electroporation of the plasmid were plated (1x10 6 cells/ml, 100ul/well), and the cells were incubated with PE mouse anti-human TIGIT antibody (BD, 556046).
  • a cytometer (BD, FACSJazz) was used to read the mean value at a wavelength of 585 nm, and GraphPad was used to generate the data for analysis.
  • the positive cell line was subcloned, and the cloned CHO-K1 cell line was selected, which expressed TIGIT molecule at a high level and named it hTIGIT-CHO-K1.
  • the full-length gene fragment encoding CD155 protein was synthesized, and the amino acid sequence design was shown in SEQ ID NO: 74, and then cloned into the eukaryotic expression plasmid pTargeT to obtain its expression plasmid pTargeT-CD155.
  • the eukaryotic expression plasmid pTargeT-CD155 was transfected into CHO-K1 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) by electroporation under the square pulse of 160V and 15msec, in a 37°C, 5% CO2 incubator cultivated in. After 24h, pressurized culture medium containing 500 ⁇ g/ml G418 was used. After 16 days, the positive rate of the pool was detected by FACS, and the electroporated cells were plated (1x10 6 cells/ml cell density, 100ul/well), and the cells were incubated with PE mouse anti-human CD155 antibody (BD, 556046).
  • a cytometer (BD, FACSJazz) was used to read the mean value at a wavelength of 585 nm, and GraphPad was used to generate the data for analysis.
  • the positive cell line was subcloned, and the cloned CHO-K1 cell line was selected, which expressed CD155 molecule at a high level and named it hCD155-CHO-K1.
  • the mixed solution of PEI and FreeStyle 293 expression medium was added to the plasmid, mixed well, then added to the cell culture, and cultured in a humidified CO 2 incubator at 37° C., 8% CO 2 .
  • Cells were fed on days 1 and 3 post-transfection with 2.5 ml of glutamine (200 mM stock concentration) and 5 ml of glucose (180 g/L stock concentration) per flask. When the cell viability dropped to 65%-75%, the cell supernatant was collected. The cell culture was centrifuged at 1500 rpm for 5 min to collect the supernatant, and then centrifuged at 8000 rpm for 20 min to collect the supernatant.
  • Loading Load the cell expression supernatant with a retention time of 5min;
  • TIGIT-His (Sino Biological, 10917-H08H), TIGIT-hFc (R&D, 7898-TG), TIGIT-mFc (Acro Biosystems, Cat. No. TIT-H5253)
  • the adjuvant co-immunization method was used to immunize the experimental mice of C57 and SJL strains with 50 ⁇ g antigen for the first immunization and 25 ⁇ g antigen for the later immunization. 100 ⁇ g of antigen was used for the first time, and 50 ⁇ g of antigen was used for immunization in the later stage.
  • the immune adjuvant can be Quick Antibody-Mouse5W (Beijing Boaolong Immune Technology Co., Ltd., Beijing) or Titer Max (Sigma) and CpG (synthesized by GenScript Biotechnology Co., Ltd.)/Alum (thermo) adjuvant spacer.
  • the TIGIT antigen protein samples with different labels were added dropwise to the adjuvant solution, vortexed while adding dropwise to mix thoroughly, and the dosage of adjuvant was carried out with reference to the instructions. Mice and rats were immunized after mixing to form a water-in-oil emulsion.
  • Cell lines expressing high levels of the TIGIT assay were also used to immunize rats to produce antibodies. Treat the hTIGIT-CHO-K1 positive single cells obtained in Example 1 under culture with trypsinization, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cell pellet with PBS, take samples and count with a cell counter, and centrifuge the remaining samples at 1000 rpm for 5 min , discard the supernatant, resuspend the cell pellet with PBS, and add the appropriate amount of PBS to obtain a cell suspension of 1 x 10 8 cells/ml. Each mouse in the experimental group was immunized with 1 ⁇ 10 7 cells.
  • the immunization program is shown in Table 6 and Table 7:
  • spleen cells The boosted mice/rat were sacrificed and immersed in 75% alcohol. The spleen was dissected out, ground with a grinding rod, and filtered through a cell mesh to prepare a single-cell suspension. The spleen cell suspension was centrifuged at 2000 rpm for 5 min, and the supernatant was discarded. Add 2 mL of erythrocyte lysis solution, lyse the erythrocytes for 2 min at room temperature, add PBS to 20 mL, centrifuge at 1500 rpm for 7 min, discard the supernatant, resuspend and count the viable cells.
  • the Sp2/0 cells in the culture flask were collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the viable cells were counted after resuspension.
  • Cells were resuspended in 20 mL of electroporation buffer and centrifuged at 1500 rpm for 7 min. Discard the supernatant and repeat once. Resuspend the cells with an appropriate amount of electroporation buffer to ensure the cell concentration is about 2 ⁇ 10 7 cells/mL.
  • the cell suspension was added to a 9mL electroporation fusion tank for fusion. After fusion, the cell suspension was transferred to 15 mL of RPMI 1640 complete medium containing 20% FBS, and placed at room temperature for 20 min. Confluent cells were resuspended in RPMI 1640 medium containing 1 ⁇ HAT, 1 ⁇ BIOMYC-3, 20% FBS. Add 100 ⁇ l/well of the cell suspension to several 96-well cell culture plates to ensure that the amount of cells in each well is about 4 ⁇ 10 4 cells/well, and culture in a 37°C cell incubator. After 5 days, 100 ⁇ L/well of RPMI 1640 complete medium (containing 20% FBS, 1 ⁇ HAT, 1 ⁇ BIOMYC-3) was supplemented.
  • TIGIT-his was used to screen antibodies against TIGIT but not hFc and mFc.
  • the hybridoma supernatants were then assayed for their ability to block the TIGIT-CD155 interaction by ELISA.
  • Coat CD155-hFc on the ELISA plate add the mixture of recombinant human protein TIGIT-mFc and hybridoma supernatant for 2h, add HRP-labeled anti mouse IgG Fc-specific antibody (Jackson Immuno Research) and incubate for 1h, use enzyme The absorbance value at 450nm was detected by a standard analyzer.
  • the hybridoma parent clones with binding ability and blocking ability obtained by screening were expanded and cultured, and the retest of ELISA binding activity was carried out; the hybridoma supernatant that could bind TIGIT on the surface of hTIGIT-CHO-K1 cells was screened by FACS; The hybridoma supernatant that can bind to the cyno-TIGIT-his protein was screened by ELISA; the hybridoma supernatant with positive crossover after three experiments was used as a candidate positive clone.
  • the positive cell lines were subcloned by the limiting dilution method. After one week of culture, the binding activity of the subclone supernatant to TIGIT molecules and the activity of blocking the interaction of TIGIT-CD155 were detected by ELISA. Five double-positive cell lines were obtained, which were marked as SHS006-17, SHS006-23.
  • the monoclonal antibody parent clones SHS006-17 and SHS006-23 were determined and expanded.
  • the culture conditions are 1640 medium containing 10% fetal bovine serum, 1 ⁇ NAEE, 1 ⁇ sodium pyruvate, and 1% penicillin-streptomycin double antibody.
  • the cell confluence is greater than 80%, the cells are subcultured and expanded.
  • the culture reaches about 50ml, collect the supernatant and purify the antibody.
  • the obtained antibody was confirmed to be of good purity by SDS-PAGE gel electrophoresis.
  • the subcloned positive hybridoma cells were expanded and cultured, and an appropriate amount of cells were taken to extract total RNA according to the instructions of the RNeasy Plus Mini Kit (Qiagen, 74134), and reverse transcribed using Prime Script 1st strand cDNA Synthesis Kit (Takara, 6110A).
  • the kit synthesizes the first strand of cDNA.
  • Design specific primers according to the variable region of the rat antibody subtype (the 5' end contains the homology arm sequence for homologous recombination with the eukaryotic expression vector), and use cDNA as the template to carry out PCR amplification of the variable region gene of the antibody , so as to obtain the gene fragments of the variable region of the light chain and heavy chain of the rat antibody respectively; design primers (reference: 1. Anke Krebber, Susanne Bornhauser, Jorg Burmester etal. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. Journal of Immunological Methods, 1997, 201: 35–55; 2.
  • the purified mouse anti-light chain and heavy chain variable region gene fragments were co-transformed into E. coli DH5 ⁇ with the linearized eukaryotic expression plasmids containing human antibody light chain or heavy chain constant regions, respectively.
  • the mixed solution was evenly coated on the surface of the agar plate containing the corresponding antibiotics, and after overnight incubation in a constant temperature incubator at 37°C, a number of single colonies were picked for DNA sequencing; the correctly sequenced chimeric antibodies were marked as SHS006-17CHI , SHS006-23CHI.
  • the positive clones with correct sequencing were inoculated into 2 ⁇ YT liquid medium containing the corresponding antibiotics, shaken and cultured at 37°C for more than 12 hours, and then the cells were collected for plasmid extraction to obtain chimeric antibody light chain and heavy chain expression plasmids. Use a nucleic acid quantitative analyzer to detect the concentration and purity of the plasmid.
  • the chimeric antibody was transfected into HEK293E cells, expressed and purified to obtain a large number of antibodies, and the purity detection, activity analysis and affinity detection were carried out.
  • SHS006-17CHI and SHS006-23CHI were selected for humanized antibody transformation according to the results of the activity analysis and affinity KD value of the chimeric antibody.
  • the humanization transformation of antibodies is firstly performed by aligning with the mouse antibody sequences in the Immunity Gene Database (IMGT) to confirm the mouse strains of the variable regions of the SHS006-17CHI and SHS006-23CHI antibodies.
  • IMGT Immunity Gene Database
  • SHS006-17CHI, SHS006-23CHI antibody heavy chain variable region sequence FR region is most similar to mouse antibody germline gene IGHV2-26*01 and IGHV3-7*01 respectively; FR sequence of antibody light chain variable region They are most similar to mouse antibodies IGKV2-28*01 and IGKV1-39*01, respectively.
  • SHS006-17CHI/SHS006-23CHI antibody framework region sequences FR1-FR3 as templates search for a fully human framework with similar 3D structure but lower immunogenicity in the human framework region library to replace FR1-FR3 of SHS006-17CHI/SHS006-23CHI Sequence, 3D modeling of the full-length heavy chain/light chain sequence and structural alignment analysis with the original antibody heavy chain/light chain sequence, comprehensive consideration of antigenicity and 3D structural similarity, and the structure of the antibody will be displayed in the structural simulation.
  • the amino acid sites that play a key role in stabilization are backmutated to murine amino acid residues.
  • the above-designed humanized antibody light chain and heavy chain variable region amino acid sequences are reverse transcribed into corresponding nucleotide sequences, and oligonucleotide fragments containing complementary sequences between adjacent fragments are generated.
  • the oligonucleotide fragments are annealed and connected by PCR, and then the complete light chain and heavy chain are amplified by specific primers (the 5' end contains the homology arm sequence for homologous recombination with the eukaryotic expression vector).
  • Variable region nucleotide fragment; the purified light chain variable region nucleotide fragment and the linearized eukaryotic expression plasmid containing the IgG4 light chain constant region were co-transformed into E.
  • variable region nucleotide fragments and the eukaryotic expression plasmid containing the S228P/L235E mutated IgG4 heavy chain constant region were co-transformed into Escherichia coli DH5 ⁇ competent cells, and the competent cells of the transformed plasmid were uniformly coated on agar containing the corresponding antibiotics. The surface of the plate was cultured overnight in a constant temperature incubator at 37°C, and several single colonies were picked for DNA sequencing.
  • the positive clones with correct sequencing were inoculated into 2 ⁇ YT liquid medium containing the corresponding antibiotics, shaken at 37°C for more than 12 hours, and then the cells were collected for plasmid extraction to obtain humanized antibody light chain and heavy chain expression plasmids , use a nucleic acid quantitative analyzer to detect the concentration and purity of the plasmid.
  • the plasmid was transfected into HEK293E cells, expressed and purified to obtain a large number of antibodies, and the purity detection, activity analysis and affinity detection were carried out.
  • the luciferase reporter gene system (purchased from BPS Bioscience, USA) was used to measure the cell function of the anti-TIGIT antibody of the present invention, and the antibody 22G2 was used as a control.
  • 1Target cell preparation Plate CD155/TCR Activator-CHO cells at a density of 2.5x 10 4 cells/ml (detection well and NTC), 100ul/well, and place the cell culture plate in a 37°C, 5% CO2 incubator Incubate overnight;
  • the experimental results showed that the EC50 value of the anti-TIGIT antibody of the present invention induced luciferase expression was significantly lower than that of the control antibody 22G2. This indicates that the anti-TIGIT antibody of the present invention can more effectively activate T cells by blocking the binding of TIGIT-CD155, and has a significantly better function than 22G2 in activating T cells to kill tumors.
  • Human TIGIT-His protein (1 ⁇ g/well, Sino Biological, 10917-H08H) was coated on a 96-well microtiter plate and incubated at 37°C for 2 h. After washing three times with 1 ⁇ PBST, the cells were blocked with 5% skim milk overnight at 4°C.
  • the anti-TIGIT antibody provided by the present invention was used as the primary antibody, starting from 2 ⁇ g/mL, 5-fold gradient dilution was added to the ELISA plate, a total of 8 concentrations, the concentrations were 2000ng/mL, 400ng/mL, 80ng/mL, 16ng/mL, 3.2ng/mL, 0.64ng/mL, 0.128ng/mL, 0ng/mL, incubated at 37°C for 1.5h; washed with 1 ⁇ PBST for 5 times, the secondary antibody was Anti-Human IgG HRP (Jackson, 109-035-003, 1:10000), incubated at 37°C for 40 min. After washing 5 times with 1 ⁇ PBST, the color developing solution TMB was added, and the OD450 value was read by a microplate reader (thermo, Multiskan FC) after termination. EC50s were generated using GraphPad and the results are shown in Table 12.
  • the experimental results show that the anti-TIGIT antibodies SHS006-17CHI, SHS006-T-Hu17-31, SHS006-23CHI and SHS006-T-Hu23-53 of the present invention all have good binding ability to human TIGIT.
  • Antibody binding activity was analyzed by FACS.
  • hTIGIT-CHO-K1 cells were plated with 1 ⁇ 10 5 cells per well, and cultured overnight at 37°C under 5% CO 2 ; washed twice with Cell stanining buffer the next day; TIGIT antibody was used as primary antibody from 2 ⁇ g/mL, and was added to the cell plate by gradient dilution, with a total of 8 concentrations, the concentrations were 2000ng/mL, 400ng/mL, 80ng/mL, 40ng/mL, 20ng/mL, 4ng/mL, 0.8 ng/mL, 0.16ng/mL, the positive control antibody was 22G2, incubated at 37°C for 1 h; washed twice with Cell stanining buffer; the secondary antibody was PE anti-human IgG Fc (Biolegend, 409304, 1.2ul/well), avoid at 4°C Incubate in light for 30 min; wash twice with Cell stanining buffer and then use flow cytometer (ACE
  • the experimental results show that the humanized anti-TIGIT antibodies SHS006-T-Hu23-53 and SHS006-T-Hu17-31 of the present invention can bind to cell surface TIGIT, and the binding ability is better than that of the antibody 22G2.
  • the binding affinity of the humanized anti-TIGIT antibodies prepared in Examples 1 and 2 to the antigen TIGIT-his was measured by Fortebio Octet.
  • the humanized anti-TIGIT antibody was diluted with SD buffer (PBST+0.02%Tween20+0.1%BSA) to a concentration of 5 ⁇ g/ml
  • the antigen TIGIT-His was diluted with SD buffer 4-fold concentration gradient to make its concentration 1 ⁇ g /ml, 0.25 ⁇ g/ml, 0.0625 ⁇ g/ml, 0 ⁇ g/ml
  • the SA sensor was used to immobilize the antigen, and the affinity was determined according to the operating procedures of Fortebio Octet RED96.
  • Table 14 The specific parameters and experimental results are shown in Table 14.
  • the experimental results show that the humanized anti-TIGIT antibodies SHS006-T-Hu17-31 and SHS006-T-Hu23-53 of the present invention have high affinity with human TIGIT protein.
  • Example 11 ELISA detection to block the binding activity of TIGIT and CD155
  • the blocking activity of the antibodies was analyzed by ELISA.
  • Human CD155-hFc protein (1 ⁇ g/well) was coated on a 96-well microtiter plate and incubated at 37°C for 2h. After washing three times with 1 ⁇ PBST, the cells were blocked with 5% skim milk overnight at 4°C.
  • the anti-TIGIT antibody provided by the present invention is mixed with TIGIT-mFc as the primary antibody, wherein the anti-TIGIT antibody starts from 5 ⁇ g/mL, and is added to the ELISA plate by gradient dilution, with a total of 8 concentrations, and the concentrations are respectively 5000ng/mL, 1667ng/mL, 556ng/mL, 278ng/mL, 139ng/mL, 46ng/mL, 15ng/mL, 0ng/mL, positive control antibody is 22G2, TIGIT-mFc concentration is constant at 1 ⁇ g/ml, 37°C Incubate for 2h; after washing 5 times with 1 ⁇ PBST, the secondary antibody was incubated with Goat anti mouse IgG-HRP antibody (abcam, Ab6789, 1:10000) at 37°C for 40min.
  • Goat anti mouse IgG-HRP antibody abcam, Ab6789, 1:10000
  • FACS was used to detect the ability of the anti-TIGIT antibody provided by the present invention to block the binding of TIGIT to CD155 on the cell surface.
  • the hCD155-CHO-K1 positive cell line was used as CD155 provider, and the binding ability of TIGIT-mFc to CD155-CHO-K1 was observed in the presence of serially diluted anti-TIGIT antibody.
  • the secondary antibody used PE Goat anti mouse IgG (Biolegend, 405307, 1.2ul/well) to monitor changes in TIGIT-mFc. 22G2 served as a positive control for blocking TIGIT binding to cell surface CD155.
  • Flow cytometer (ACEABIO, Novocyte) read the mean value at a wavelength of 585 nm, and used GraphPad to generate IC50. The results are shown in Table 16.
  • the anti-PD-L1 antibody used in the present invention is a mouse monoclonal antibody PL-7 (the heavy chain variable region sequence is SEQ ID NO: 37, the light chain variable region sequence is SEQ ID NO: 37, and the The region sequence is SEQ ID NO: 41), PL-16 (the heavy chain variable region sequence is SEQ ID NO: 39, and the light chain variable region sequence is SEQ ID NO: 43), which are humanized and screened to obtain human Monoclonal antibody HuPL7-21 (heavy chain variable region sequence SEQ ID NO:50, light chain variable region sequence SEQ ID NO:54), HuPL7-43 (heavy chain variable region sequence SEQ ID NO:52, light chain variable region sequence SEQ ID NO:56), HuPL16-42 (heavy chain variable region sequence SEQ ID NO:61, light chain variable region sequence SEQ ID NO:64).
  • the anti-TIGIT antibody used in the present invention is the mouse monoclonal antibody T-23 (the heavy chain variable region sequence is SEQ ID NO: 76, the light chain variable region sequence is SEQ ID NO: 76), which is obtained by immunizing mice with human TIGIT-his protein. ID NO: 78), screened to obtain humanized monoclonal antibody HuT23-53 after humanization (the heavy chain variable region sequence is SEQ ID NO: 99, and the light chain variable region sequence is SEQ ID NO: 101) .
  • the present invention utilizes the variable region sequences of the above-mentioned anti-PD-L1 and anti-TIGIT humanized antibodies to construct an anti-TIGIT/anti-PD-L1 bispecific antibody involving two structures.
  • the two structures are an asymmetric structure composed of ScFab as an element (such as the bispecific antibody HuPL721-T2353-ScFab in this example) and a symmetric structure composed of ScFv as an element (such as the bispecific antibody in this example). HuPL721-T2353-ScFv).
  • the bispecific antibody of the ScFab structure is shown in Figure 1. Specifically, the bispecific antibody is formed by heterodimerization of two anti-PD-L1 and anti-TIGIT antibody single chains; it is different from natural IgG.
  • G glycine
  • S serine
  • the bispecific antibody of the ScFv structure is shown in Figure 2.
  • the ScFv heavy chain of the TIGIT antibody is connected to the C-terminal of the complete anti-PD-L1 antibody heavy chain through a flexible connecting peptide, and the connecting peptide contains glycine (G) and A serine (S) residue comprising a GGGGS repeat, preferably a repeat of 8 GGGGS.
  • G glycine
  • S serine residue
  • cysteine mutations can be introduced at the VH44 position of the heavy chain variable region and the VL100 position of the light chain variable region at the same time, so that they form a VH44-VL100 disulfide bond , to increase the stability of the antibody structure.
  • IgG4CH/Ks represents IgG4 constant region (S228P, Y349C, T366S, L368A, Y407V mutation)
  • IgG4CH/Hs represents IgG4 constant region (S228P, S354C, T366W mutation)
  • IgG4CH/PE represents IgG4 constant region (S228P, L235E mutation) .
  • variable region sequences of anti-PD-L1 antibody HuPL7-21 and anti-TIGIT antibody HuT23-53 were used to construct the expression vector ScFab-T2353-Knob-PHR of bispecific antibody HuPL721-T2353-ScFab ( The sequence is shown in SEQ ID NO: 102) and HuPL721-scfab-hole-PHR (the sequence is shown in SEQ ID NO: 103);
  • variable region sequences of the anti-PD-L1 antibody HuPL7-21 and the anti-TIGIT antibody HuT23-53 were used to construct the expression vector PL721-T2353-scFv-HC-PHR of the bispecific antibody HuPL721-T2353-ScFv (the sequence is shown in SEQ ID NO: 104) and HuPL721-VL1-PHR (the sequence is shown in SEQ ID NO: 105);
  • variable region sequences of anti-PD-L1 antibody HuPL7-43 and anti-TIGIT antibody HuT23-53 were used to construct the expression vector ScFab-T2353-Knob-PHR of bispecific antibody HuPL743-T2353-ScFab ( The sequence is shown in SEQ ID NO: 102) and HuPL743-scfab-hole-PHR (the sequence is shown in SEQ ID NO: 106);
  • variable region sequences of anti-PD-L1 antibody HuPL7-43 and anti-TIGIT antibody HuT23-53 were used to construct the expression vector PL743-T2353-scFv-HC-PHR of bispecific antibody HuPL743-T2353-ScFv (the sequence is shown in SEQ ID NO: 107) and HuPL743-VL3-PHR (the sequence is shown in SEQ ID NO: 108);
  • variable region sequences of the anti-PD-L1 antibody HuPL16-42 and the anti-TIGIT antibody HuT23-53 were used to construct the expression vector ScFab-T2353-Knob-PHR of the bispecific antibody HuPL1642-T2353-ScFab ( The sequence is shown in SEQ ID NO: 102) and HuPL1642-scfab-hole-PHR (the sequence is shown in SEQ ID NO: 109);
  • variable region sequences of anti-PD-L1 antibody HuPL16-42 and anti-TIGIT antibody HuT23-53 were used to construct the expression vector PL1642-T2353-scFv-HC-PHR of bispecific antibody HuPL1642-T2353-ScFv (the sequence is shown in SEQ ID NO: 110) and HuPL1642-VL2-PHR (the sequence is shown in SEQ ID NO: 111);
  • HEK293E cells Inoculate HEK293E cells at a density of 1.0 ⁇ 10 6 cells/ml in a 1L cell culture flask, add fresh pre-warmed FreeStyle 293 expression medium to make the total volume after inoculation reach 250 mL, set at 37°C, 8% CO 2 , and humidify Incubate overnight in a CO 2 incubator.
  • FreeStyle 293 expression medium Take 8mL of FreeStyle 293 expression medium, add 500ul of 1mg/ml PEI solution, mix well, add 250 ⁇ g of the expression vector to be transfected into 8.0ml of FreeStyle 293 expression medium, and mix evenly, wherein the bispecific antibody is based on the ratio of the two vectors. for 1:1 co-transfection.
  • the mixed solution of PEI and FreeStyle 293 expression medium was added to the plasmid, mixed well, then added to the cell culture, and cultured in a humidified CO 2 incubator at 37° C., 8% CO 2 .
  • Cells were fed on days 1 and 3 post-transfection with 2.5 ml of glutamine (200 mM stock concentration) per flask, 12.5 mL of OPM-CHO PFF05 and 5 ml of glucose (stock solution) per 250 mL The concentration is 180g/L).
  • the cell viability dropped to 65%-75%, the cell supernatant was collected.
  • the cell culture was centrifuged at 1500 rpm for 5 min to collect the supernatant, and then centrifuged at 8000 rpm for 20 min to collect the supernatant.
  • AKTA (GE, AKTA pure-150) was used for purification using different affinity chromatography columns according to the properties of the protein (see Table 17 for the affinity chromatography columns adapted to the anti-TIGIT/anti-PD-L1 bispecific antibody). Proceed as follows:
  • Loading Load the cell expression supernatant with a retention time of 5min;
  • the purified antibodies HuPL721-T2353-ScFab/ScFv, HuPL743-T2353-ScFab/ScFv, HuPL1642-T2353-ScFab/ScFv were purified by SDS-PAGE and SEC-HPLC. If the purity is less than 95%, further purification is performed to obtain bispecific antibody molecules with SEC purity >95% for subsequent experiments.
  • Example 14 Determination of the binding activity of anti-TIGIT/anti-PD-L1 bispecific antibody to human PD-L1 protein
  • the binding activity of the antibody was analyzed by FACS. hPD-L1-CHO-K1 cells were plated with 1.5 ⁇ 10 5 cells per well; the anti-TIGIT/anti-PD-L1 bispecific antibody provided by the present invention was used as the primary antibody, starting from 10 ⁇ g/mL, and added in gradient dilution.
  • Cell plate a total of 8 concentrations, the concentrations were 10000ng/mL, 2000ng/mL, 400ng/mL, 80ng/mL, 16ng/mL, 3.2ng/mL, 0.64ng/mL, 0.128ng/mL, incubated at 4°C for 1.5 h; wash twice with diluent (PBS+2% FBS); use PE anti-human IgG Fc (Abcam, ab98596, 0.8ul/well) for secondary antibody, incubate at 4°C for 60 min in the dark; A cytometer (ACEABIO, Novocyte) was used to measure the mean value at a wavelength of 585 nm. During data processing, the mass concentration was converted to molar concentration and then EC50 was generated using GraphPad. The results are shown in Table 18.
  • the experimental results show that the anti-TIGIT/anti-PD-L1 bispecific antibodies HuPL721-T2353-ScFab, HuPL1642-T2353-ScFv, HuPL721-T2353-ScFv and HuPL743-T2353-ScFv provided by the present invention all have better affinity with PD-L1. binding ability.
  • Example 15 Determination of the binding activity of anti-TIGIT/anti-PD-L1 bispecific antibody to human TIGIT
  • the binding activity of the antibody was analyzed by FACS.
  • hTIGIT-CHO-K1 cells were plated with 1.5 ⁇ 10 5 cells per well; the anti-TIGIT/anti-PD-L1 bispecific antibody provided by the present invention was used as the primary antibody, starting from 2 ⁇ g/mL, and was added to the cell plate by gradient dilution , a total of 8 concentrations, the concentrations were 2000ng/mL, 400ng/mL, 80ng/mL, 40ng/mL, 20ng/mL, 4ng/mL, 0.8ng/mL, 0.16ng/mL, incubated at 4°C for 1.5h; diluted Wash twice with PBS + 2% FBS; use PE anti-human IgG Fc (Abcam, ab98596, 0.8ul/well) as secondary antibody, incubate at 4°C for 60 min in the dark; wash twice with diluent and use flow cytometer (ACEABIO, Novocyte) measured the mean value at 585 nm wavelength.
  • the experimental results show that the anti-TIGIT/anti-PD-L1 bispecific antibodies HuPL721-T2353-ScFab and HuPL721-T2353-ScFv of the present invention have better binding ability to PD-L1.
  • Example 16 Activity detection of anti-TIGIT/anti-PD-L1 bispecific antibody to block the binding of PD-L1 to PD-1
  • the binding activity of the antibody was analyzed by FACS.
  • hPD-L1-CHO-K1 cells were plated with 2.5 ⁇ 10 5 cells per well; the anti-TIGIT/anti-PD-L1 bispecific antibody provided by the present invention was diluted with a final concentration of 2 ⁇ g/ml PD
  • the -1-mFc was mixed evenly and used as the primary antibody.
  • the gradient dilution method of the antibody was: starting from 20 ⁇ g/mL, 3-fold gradient dilution was added to the cell plate, a total of 8 concentrations, the concentrations were 20000ng/mL, 6666.66ng/mL, 2222.22ng/mL, 740.74ng/mL, 246.9ng/mL, 82.3ng/mL, 27.4ng/mL, 9.14ng/mL, incubate at 4°C for 1.5h; wash twice with diluent (PBS+2%FBS); The anti-PE anti-Mouse IgG Fc (Biolegend, 405307, 0.8ul/well) was used as the antibody, and incubated at 4°C for 60 min in the dark; after washing twice with the diluent, the mean value at a wavelength of 585 nm was measured by flow cytometer (ACEABIO, Novocyte). During data processing, the mass concentration was converted to molar concentration and IC50 was generated using Graph
  • the experimental results show that the anti-TIGIT/anti-PD-L1 bispecific antibodies HuPL721-T2353-ScFab and HuPL721-T2353-ScFv of the present invention all have the ability to block the binding of PD-L1 to PD-1.
  • the blocking activity of the antibodies was analyzed by FACS.
  • the hTIGIT-CHO-K1 cells were plated with 2.5 ⁇ 10 5 cells per well; the anti-TIGIT/anti-PD-L1 bispecific antibodies provided by the present invention were serially diluted with TIGIT- The mFc was mixed evenly and used as the primary antibody.
  • the gradient dilution method of the antibody was as follows: starting from 20 ⁇ g/mL, the gradient dilution was added to the cell plate, with a total of 8 concentrations, which were 20000ng/mL, 4000ng/mL, 800ng/mL, and 400ng/mL.
  • the experimental results show that the anti-TIGIT/anti-PD-L1 bispecific antibodies HuPL721-T2353-ScFab, HuPL721-T2353-ScFv, HuPL1642-T2353-ScFab and HuPL1642-T2353-ScFv of the present invention all have good blocking effects on TIGTI and CD155 ability to combine.
  • the affinity of the humanized anti-TIGIT/anti-PD-L1 antibody prepared in Example 15 for binding to the antigens PD-L1-his and TIGIT-his was measured using Fortebio Octet, respectively.
  • the antibodies HuPL721-T2353-ScFab/ScFv, HuPL743-T2353-ScFab/ScFv, HuPL1642-T2353-ScFab/ScFv were diluted to 67 nM
  • the AHC sensor was used to immobilize the above antibodies
  • the PD-L1-his was diluted with SD buffer (0.02% Tween20+0.1%BSA solution) was serially diluted to 2 ⁇ g/ml, 0.4 ⁇ g/ml, 0.08 ⁇ g/ml
  • TIGIT-his was serially diluted with SD buffer to 0.5 ⁇ g/ml, 0.1 ⁇ g/ml, 0.02 ⁇ g/ml, press
  • the operating procedures of Fortebio Octet RED96 are used for affinity determination
  • the experimental results show that the anti-TIGIT/anti-PD-L1 bispecific antibodies HuPL1642-T2353-ScFab, HuPL1642-T2353-ScFv, HuPL721-T2353-ScFab and HuPL721-T2353-ScFv of the present invention have better affinity with human PD-L1. Protein affinity and affinity with human TIGIT protein.
  • SEB Staphylococcus aureus enterotoxin B
  • PBMC peripheral blood mononuclear cells
  • Resuscitate PBMC according to the required cell amount, add it to 8-9ml of IMDM complete medium, centrifuge at 1200rpm for 10min, discard the supernatant; resuspend with an appropriate amount of medium, count with a hemocytometer, add it to a 6-well plate, and add it at the same time.
  • the experimental results show that the antibody of the present invention has a better ability to promote the release of IFN- ⁇ .
  • Antibody HuPL16-42 and HuPL7-21 combined with antibody HuT23-53 had better effect than antibody alone.
  • Bispecific antibody HuPL721-2353 promotes IFN- ⁇ release in a dose-dependent manner.
  • HuPL721-T2353-ScFv is still better than the combination group at half the number of moles [for monoclonal antibody combination group 20 ⁇ g/ml+20 ⁇ g/ml, scFab double antibody group 40 ⁇ g/ml and scFv double antibody group 26.8 ⁇ g/ml,
  • These three groups of concentrations are the concentrations with equal numbers of TIGIT and PD-L1 antigen-binding arms (called “isoarm” concentrations); compared with the monoclonal antibody combination group of 20 ⁇ g/ml+20 ⁇ g/ml, the scFab double antibody group
  • the concentration of 20 ⁇ g/ml and 13.4 ⁇ g/ml of scFv double antibody group is equivalent to the concentration of halving the number of binding arms; in terms of moles, the total amount of the two molecules in the monoclonal antibody combination group 20 ⁇ g/ml+20 ⁇ g/ml
  • the number of moles is equal to the mole number of 40 ⁇ g/m
  • the present invention utilizes NSG mice (purchased from Beijing Biositu Gene Biotechnology Co., Ltd., China) and Raji-PD-L1 tumor cells (purchased from Immune Onco Biomedical Technology Co., Ltd., China) to establish a tumor transplantation model—Raji - PBMC-NSG model to study the anti-tumor effect of the antibody of the present invention in the Raji-hPD-L1 lymphoma subcutaneous transplantation model.
  • the anti-PD-L1 positive control antibody was Atezolizumab (Sino Biological, Cat: 68049-H001).
  • Table 25 shows the experimental design of the antitumor effect of the test drugs in the Raji-PBMC-NSG tumor model.
  • N refers to the number of mice per group; i.p.: intraperitoneal injection; BIW refers to twice weekly dosing.
  • TGI TV tumor inhibition rate
  • the antibodies HuPL7-21 and HuPL721-T2353-ScFab of the present invention have significantly better tumor-inhibiting effects in vivo than Atezolizumab.
  • B-hPD-L1/hTIGIT double-derived mice purchased from Beijing Biositu Gene Biotechnology Co., Ltd., China
  • MC38-hPD-L1 colon cancer cells purchased from Shunran Shanghai Biotechnology Co., Ltd., China
  • Pharmacological experiments were carried out in animal models.
  • the MC38-hPD-L1 colon cancer cells resuspended in PBS were subcutaneously inoculated on the right side of the B-hPD-L1/hTIGIT humanized mice at a concentration of 5 ⁇ 10 5 cells/0.1 mL and a volume of 0.1 mL/cell.
  • appropriate mice were selected according to the tumor volume and body weight of the mice, and they were equally divided into 6 experimental groups, with 8 mice in each group, and the administration started on the day of the grouping. 27.
  • PBS saline control group
  • the administration volume is calculated according to the body weight of the experimental animal at 10 ⁇ L/g;
  • BIW refers to dosing twice a week.
  • TGI TV tumor inhibition rate
  • the tumor inhibition rate of Atezolizumab at a dose of 3 mg/kg was 35.3%; the tumor inhibition rates of HuPL721-T2353-ScFab at a dose of 3.14 mg/kg and 6.27 mg/kg, respectively were 30.6% and 50.7%.
  • the experimental results showed that the anti-TIGIT/anti-PD-L1 bispecific antibody HuPL721-T2353-ScFab had a significant inhibitory effect on the growth of MC38-hPD-L1 tumor subcutaneous xenografts in a dose-dependent manner.
  • anti-TIGIT antibody, anti-PD-L1 antibody and anti-TIGIT/anti-PD-L1 bispecific antibody provided by the present invention can significantly inhibit tumor growth and have significant anti-tumor effects, suggesting that these antibodies can be used in the preparation of anti-tumor The application in medicine has good market prospect.

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Abstract

本发明涉及抗体药物技术领域，尤其涉及抗TIGIT抗体或其抗原结合片段，抗TIGIT/抗PD-L1抗体或其抗原结合片段，包含抗TIGIT抗体或其抗原结合片段或者抗TIGIT/抗PD-L1抗体或其抗原结合片段的药物组合物以及它们的应用。本发明的抗TIGIT/抗PD-L1抗体具有显著的抗肿瘤活性，可在制备抗肿瘤的药物中应用。

Description

抗TIGIT抗体及双抗体和它们的应用 技术领域
本发明涉及抗体药物技术领域，尤其涉及一种抗TIGIT抗体或其抗原结合片段，抗TIGIT/抗PD-L1抗体或其抗原结合片段，包含抗TIGIT抗体或其抗原结合片段或者抗TIGIT/抗PD-L1抗体或其抗原结合片段的药物组合物以及它们的应用。
背景技术
TIGIT是脊髓灰质炎病毒受体结合蛋白家族成员之一，属于免疫球蛋白超家族亚型，由胞外的免疫球蛋白结构域(IgV)、一个跨膜结构、胞内的基于酪氨酸抑制基序免疫受体(ITIM)和免疫球蛋白酪氨酸尾巴基序(ITT)组成。TIGIT仅表达于淋巴细胞，高表达于效应CD4+T细胞与调节T细胞、CD4+囊泡辅助T细胞、效应CD8+T细胞以及NK细胞。免疫系统中TIGIT与CD226、CD96竞争结合PVR(CD155)，与CD226、CD112R竞争结合CD122。通过网络信号传导协调免疫系统，调节免疫激活和免疫抑制。研究表明TIGIT在肿瘤免疫抑制调节中起到重要的作用。TIGIT与其配体PVR、PVR2及PVR3(潜在配体)结合后通过三种不同的作用方式抑制免疫反应。第一种作用方式是通过TIGIT的信号传导，触发PVR快速磷酸化向抗原递呈细胞或者肿瘤细胞传递信号，TIGIT自身是通过胞内ITIM/ITT结构域将其信号传递给淋巴细胞；第二种影响免疫反应的作用方式是通过抗原递呈细胞接收TIGIT信号后表达分泌IL10、TGF-β等炎症因子抑制T细胞活性；第三种作用方式是通过与CD226竞争结合PVR干扰CD226的平衡来抑制T细胞的激活。抗TIGIT抗体可阻断其信号通路，解除肿瘤免疫抑制，提高效应T细胞、NK细胞细胞毒性，并能建立长期的抗肿瘤免疫记忆，达到治疗肿瘤的效果。
考虑到TIGIT在免疫检查点调节中的重要作用，本领域仍需要开发对TIGIT具有特异性的拮抗性抗体以单独或与其它试剂组合用于免疫疗法和癌症治疗。
PD-L1是一种大小为40KD的I型跨膜蛋白，在抑制免疫系统中起重要作用。 PD-L1通过与PD-1和B7-1形成免疫复合物，负调控T细胞受体信号通路，进而引起T细胞激活受阻，抑制T细胞的抗肿瘤活性，其中PD-1作为PD-L1的受体，主要参与激活T细胞、B细胞、单核细胞和髓系细胞，CD80(B7-1)是一种T细胞共刺激分子。PD-L1的这种免疫调节作用主要发生在一些慢性疾病中，诸如异体移植、自身免疫性疾病和癌症等。PD-L1在许多肿瘤细胞和肿瘤浸润免疫细胞表面高表达，包括大多数实体瘤和血液淋巴瘤，例如骨髓瘤、前列腺癌、乳腺癌、结肠癌、肺癌、胃癌、黑色素瘤等。因此，PD-L1可以作为一个重要的肿瘤治疗靶标。目前，通过阻断PD-L1发挥抗肿瘤作用的单克隆抗体已经广泛应用于临床，并在多种肿瘤中展示出强大的抗肿瘤活性。
同一种疾病在体内往往是受不同的信号通路、不同的细胞因子以及不同的受体调节机制等控制。多个免疫检查点通路的联合阻断，可能是巧妙且必要的抗肿瘤免疫治疗策略。抗TIGIT/抗PD-L1双特异性抗体可以通过阻断PD-1/PD-L1通路和TIGIT/CD155通路激活免疫应答，并将肿瘤细胞招募至淋巴细胞周围，进一步提高抗肿瘤活性。因此，开发抗TIGIT/抗PD-L1双特异性抗体显得尤为必要。
发明内容
本发明一方面提供一种抗TIGIT抗体或其抗原结合片段，其包含重链可变区和/或轻链可变区，其中所述重链可变区包含重链可变区的互补决定区1(H1CDR1)、重链可变区的互补决定区2(H1CDR2)和/或重链可变区的互补决定区3(H1CDR3)，所述轻链可变区包含轻链可变区的互补决定区1(L1CDR1)、轻链可变区的互补决定区2(L1CDR2)和/或轻链可变区的互补决定区3(L1CDR3)。
在一些实施方案中，本发明提供一种抗TIGIT抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
(1)所述重链可变区包含选自如下组的H1CDR1、H1CDR2和H1CDR3：
(a1)如SEQ ID NO:25、26和27所示的氨基酸序列；
(a2)如SEQ ID NO:28、29和30所示的氨基酸序列；和
(a3)与(a1)或(a2)所示的氨基酸序列具有至少85％序列同一性的氨基 酸序列；和
(2)所述轻链可变区包含选自如下组的L1CDR1、L1CDR2和L1CDR3：
(a4)如SEQ ID NO:31、32和33所示的氨基酸序列；
(a5)如SEQ ID NO:34、35和36所示的氨基酸序列；和
(a6)与(a4)或(a5)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。
在一个具体的实施方案中，本发明提供一种抗TIGIT抗体或其抗原结合片段，其具有所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:25、26和27或与SEQ ID NO:25、26和27所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:31、32和33或与SEQ ID NO:31、32和33所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的轻链可变区。
在一个具体的实施方案中，本发明提供一种抗TIGIT抗体或其抗原结合片段，其具有所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:28、29和30或与SEQ ID NO:28、29和30所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:34、35和36或与SEQ ID NO:34、35和36所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的轻链可变区。
在一些具体的实施方案中，根据本发明的抗TIGIT抗体或其抗原结合片段是单克隆抗体或其抗原结合片段。
在一些具体的实施方案中，根据本发明的抗TIGIT抗体或其抗原结合片段是鼠源抗体或其抗原结合片段、嵌合抗体或其抗原结合片段或人源化抗体或其抗原结合片段。
在一些具体的实施方案中，本发明提供一种抗TIGIT抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中
(1)所述重链可变区的氨基酸序列选自：
(b1)如SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:79、SEQ ID NO:80、SEQ ID NO:81、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91、SEQ ID NO:92所示的氨基酸序列；
(b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(b4)如SEQ ID NO:77、SEQ ID NO:78、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、SEQ ID NO:97所示的氨基酸序列；
(b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗TIGIT抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:75，SEQ ID NO:75经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:75功能相同的氨基酸序列或与SEQ ID NO:75具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:77，SEQ ID NO:77经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:77功能相同的氨基酸序列或与SEQ ID NO:77具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗TIGIT抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:76，SEQ ID NO:76经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:76功能相同的氨基酸序列或与SEQ ID NO:76具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:78，SEQ ID NO:78经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:78功能相同的氨基酸序列或与SEQ ID NO:78具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗TIGIT抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:75，SEQ ID NO:75经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:75功能相同的氨基酸序列或与SEQ ID NO:75具有至少85％序列同一性且所述H1CDR1、H1CDR2和H1CDR3 如SEQ ID NO:25、26和27所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:77，SEQ ID NO:77经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:77功能相同的氨基酸序列或与SEQ ID NO:77具有至少85％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:31、32和33所示的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗TIGIT抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:76，SEQ ID NO:76经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:76功能相同的氨基酸序列或与SEQ ID NO:76具有至少85％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:28、29和30所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:78，SEQ ID NO:78经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:78功能相同的氨基酸序列或与SEQ ID NO:78具有至少85％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:34、35和36所示的氨基酸序列。
在一些具体的实施方案中，根据本发明的抗TIGIT抗体为鼠源抗体，其还含有鼠源的IgG1、IgG2a、IgG2b、IgG2c、IgG3或其变体的重链恒定区，和鼠源的κ链或其变体的轻链恒定区。
在一些优选的实施方案中，根据本发明的抗TIGIT鼠源抗体还含有鼠源的IgG1、IgG2a、IgG2b、IgG2c或其变体的重链恒定区，和鼠源κ链或其变体的轻链恒定区。
在一些具体的实施方案中，本发明提供抗TIGIT人源化抗体或其抗原结合片段，其中：
(1)所述重链可变区的氨基酸序列选自：
(c1)如SEQ ID NO:98或SEQ ID NO:99所示的氨基酸序列；
(c2)(c1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c3)与(c1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(c4)如SEQ ID NO:100或SEQ ID NO:101所示的氨基酸序列；
(c5)(c4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c6)与(c4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供抗TIGIT人源化抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:98，SEQ ID NO:98经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:98功能相同的氨基酸序列或与SEQ ID NO:98具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:100，SEQ ID NO:100经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:100功能相同的氨基酸序列或与SEQ ID NO:100具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供抗TIGIT人源化抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:99，SEQ ID NO:99经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:99功能相同的氨基酸序列或与SEQ ID NO:99具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:101，SEQ ID NO:101经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:101功能相同的氨基酸序列或与SEQ ID NO:101具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供抗TIGIT人源化抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:98，SEQ ID NO:98经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:98功能相同的氨基酸序列或与SEQ ID NO:98具有至少85％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:25、26和27所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:100，SEQ ID NO:100经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:100功能相同的氨基酸序列或与SEQ ID NO:100具有至少85％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:31、32和33所示的氨基酸序列。
在一些具体的实施方案中，本发明提供抗TIGIT人源化抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:99，SEQ ID NO:99经取代、 缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:99功能相同的氨基酸序列或与SEQ ID NO:99具有至少85％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:28、29和30所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:101，SEQ ID NO:101经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:101功能相同的氨基酸序列或与SEQ ID NO:101具有至少85％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:34、35和36所示的氨基酸序列。
在本发明一个优选的实施方案中，所述的鼠源抗TIGIT抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，和/或进一步包含鼠源IgG1、IgG2a、IgG2b、IgG2c、IgG3或其变体的重链恒定区。
在本发明一个优选的实施方案中，所述的抗TIGIT嵌合抗体或其抗原结合片段的抗体轻链进一步包含鼠源κ、λ链或其突变序列的轻链恒定区。所述的抗TIGIT嵌合抗体或其抗原结合片段的抗体重链进一步包含鼠源IgG1、IgG2a、IgG2b、IgG2c、IgG3或其突变序列的重G链恒定区，优选包含人源IgG1、IgG2a、IgG2b、IgG2c重链恒定区，或者使用氨基酸突变后显著降低ADCC(抗体依赖的细胞介导的细胞毒作用)毒性的IgG4恒定区。
在一些具体的实施方案中，本发明的抗TIGIT人源化抗体或其抗原结合片段还包含人源IgG1、IgG2a、IgG2b、IgG2c、IgG3或其变体的重链恒定区，和人源κ、λ链或其变体的轻链恒定区。在一些优选的实施方案中，本发明的抗TIGIT人源化抗体或其抗原结合片段还包含人源IgG1、IgG2a、IgG2b、IgG2c或其变体的重链恒定区，和人源κ链或其变体的轻链恒定区。
在一些实施方案中，本发明提供抗TIGIT抗体或其抗原结合片段，其中所述的抗原结合片段为Fab、Fv、sFv或F(ab)2。
本发明的另一方面提供一种分离的核酸，其编码根据本发明的抗TIGIT抗体或其抗原结合片段。
在一些具体的实施方案中，根据本发明的分离的核酸，其包含编码重链可变区氨基酸序列如SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:98或SEQ ID NO:99的核苷酸序列；和编码轻链可变区氨基酸序列如SEQ ID NO:77、SEQ ID NO:78、SEQ ID NO:100或SEQ ID NO:101的核苷酸序列。
在一个具体的实施方案中，根据本发明的分离的核酸，其包含编码重链可变区SEQ ID NO:75的核苷酸序列；和编码轻链可变区SEQ ID NO:77的核苷酸序列。
在一个具体的实施方案中，根据本发明的分离的核酸，其包含编码重链可变区SEQ ID NO:76的核苷酸序列；和编码轻链可变区SEQ ID NO:78的核苷酸序列。
在一个具体的实施方案中，根据本发明的分离的核酸，其包含编码重链可变区SEQ ID NO:98的核苷酸序列；和编码轻链可变区SEQ ID NO:100的核苷酸序列。
在一个具体的实施方案中，根据本发明的分离的核酸，其包含编码重链可变区SEQ ID NO:99的核苷酸序列；和编码轻链可变区SEQ ID NO:101的核苷酸序列。
本发明的另一方面提供一种表达载体，其表达本发明的抗TIGIT抗体或其抗原结合片段。根据本发明的表达载体其包含本发明的分离的核酸分子。
本发明的另一方面提供一种如上所述的表达载体转化的宿主细胞。
在一些实施方案中，根据本发明的宿主细胞选自原核细胞和真核细胞。在一些实施方案中，所述的宿主细胞为细菌，优选为大肠杆菌。在另一个优选的实施方案中，所述的宿主细胞为哺乳动物细胞。
本发明的另一方面提供制备本发明的抗TIGIT抗体或其抗原结合片段的方法，包括在所述宿主细胞中表达抗体以及从宿主细胞中分离所述抗体的步骤。
本发明的另一方面提供一种药物组合物，其包含本发明的抗TIGIT人源化抗体或其抗原结合片段和药学可接受的载体。在一些实施方案中，本发明提供药物组合物，其包含本发明的抗TIGIT人源化抗体或其抗原结合片段，还包含其他活性组分，如其他抗体、靶向药物等。在一些实施方案中，所述药学可接受的载体选自抗氧化剂、多肽、蛋白质、亲水性聚合物、氨基酸、糖、螯合剂、糖醇、离子和表面活性剂。在一个具体的实施方案中，所述药学可接受的载体为缓冲水溶液。在另一个具体的实施方案中，所述药学可接受的载体为脂质体的形式。
可以将本发明的抗TIGIT人源化抗体或其抗原结合片段与药学上可接受的载体、稀释剂或赋形剂混合制备成药物制剂，以适合于经口或胃肠外给药。给药 方法包括，但不限于经口、皮内、肌内、腹膜内、静脉内、脑内、眼内、气管内、皮下、鼻内途径。所述制剂可以通过任何途径施用，例如通过输注或推注，通过经上皮或皮肤粘膜(例如口腔粘膜或直肠等)吸收的途径施用。给药可以是全身的或局部的。所述制剂可通过本领域已知的方法制备，且包含药物制剂领域常规使用的载体、稀释剂或赋形剂。
本发明的另一方面提供抑制TIGIT活性的方法，所述方法包括向有此需要的个体施用本发明的抗TIGIT抗体或其抗原结合片段或本发明的药物组合物。
本发明的另一方面提供用于检测或测定人TIGIT的方法，所述方法包括使用本发明的抗TIGIT抗体或其抗原结合片段的步骤。
本发明的另一方面提供用于检测或测定人TIGIT的试剂，所述试剂包本发明的抗TIGIT抗体或其抗原结合片段。
本发明的另一方面提供一种治疗与TIGIT相关的疾病的方法，所述方法包括向受试者施用药物有效量的本发明的TIGIT抗体或其抗原结合片段，或包含上述药物组合物，或上述分离的核酸分子。本发明的另一方面提供本发明的抗TIGIT抗体或其抗原结合片段或本发明的药物组合物在制备与TIGIT相关的疾病的药物中的应用。在一些实施方案中，所述与TIGIT相关的疾病的药物用于治疗T细胞功能障碍性病症，例如肿瘤、免疫性疾病或感染性病症。在一些实施方案中，所述肿瘤为非小细胞肺癌、小细胞肺癌、肾细胞癌、结直肠癌、卵巢癌、乳腺癌、胰腺癌、胃癌、膀胱癌、食道癌、间皮瘤、黑素瘤、头和颈癌、甲状腺癌、肉瘤、前列腺癌、成胶质细胞瘤、宫颈癌、胸腺癌、白血病、淋巴瘤、骨髓瘤、蕈样肉芽肿、梅克尔细胞癌、肾上腺皮质癌、肝脏肝细胞癌、胰管腺癌、嗜铬细胞瘤、神经节细胞瘤、子宫内膜癌和卵巢浆液性囊腺癌等。在一些实施方案中，所述免疫性疾病为关节炎、炎性肠病、银屑病。在一些实施方案中，所述感染性疾病为慢性病毒感染。
本发明提供一种抗TIGIT/抗PD-L1抗体，其包括抗TIGIT抗体或其抗原结合片段和抗PD-L1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包含第一重链可变区和/或第一轻链可变区，其中所述第一重链可变区包含第一重链可变区的互补决定区1(H1CDR1)、第一重链可变区的互补决定区2(H1CDR2)和/或第一重链可变区的互补决定区3(H1CDR3)，所述第一轻链 可变区包含第一轻链可变区的互补决定区1(L1CDR1)、第一轻链可变区的互补决定区2(L1CDR2)和/或第一轻链可变区的互补决定区3(L1CDR3)；和所述抗PD-L1抗体或其抗原结合片段为特异性结合PD-L1的抗体或其抗原结合片段。
在一些实施方案中，本发明提供一种抗TIGIT/抗PD-L1抗体，其包括抗TIGIT抗体或其抗原结合片段和抗PD-L1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包含第一重链可变区和第一轻链可变区，其中：
(1)所述第一重链可变区包含选自如下组的H1CDR1、H1CDR2和H1CDR3：
(a1)如SEQ ID NO:25、26和27所示的氨基酸序列；
(a2)如SEQ ID NO:28、29和30所示的氨基酸序列；和
(a3)与(a1)或(a2)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
(2)所述第一轻链可变区包含选自如下组的L1CDR1、L1CDR2和L1CDR3：
(a4)如SEQ ID NO:31、32和33所示的氨基酸序列；
(a5)如SEQ ID NO:34、35和36所示的氨基酸序列；和
(a6)与(a4)或(a5)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。
在一些实施方案中，根据本发明的抗TIGIT/抗PD-L1抗体，所述抗TIGIT抗体或其抗原结合片段具有：
所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:25、26和27或与SEQ ID NO:25、26和27所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:31、32和33或与SEQ ID NO:31、32和33所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一轻链可变区；或
所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:28、29和30或与SEQ ID NO:28、29和30所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:34、35和36或与SEQ ID NO:34、35和36所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一轻链可变区。
在一些实施方案中，根据本发明的抗TIGIT/抗PD-L1抗体，所述抗TIGIT 抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区，其中：
(1)所述第一重链可变区的氨基酸序列选自：
(b1)如SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:79、SEQ ID NO:80、SEQ ID NO:81、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91、SEQ ID NO:92所示的氨基酸序列；
(b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述第一轻链可变区的氨基酸序列选自：
(b4)如SEQ ID NO:77、SEQ ID NO:78、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、SEQ ID NO:97所示的氨基酸序列；
(b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些实施方案中，根据本发明的抗TIGIT/抗PD-L1抗体，所述抗TIGIT抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区，其中：
所述第一重链可变区的氨基酸序列为SEQ ID NO:75，SEQ ID NO:75经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:75功能相同的氨基酸序列或与SEQ ID NO:75具有至少85％序列同一性的氨基酸序列且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:25、26和27所示的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:77，SEQ ID NO:77经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:77功能相同的氨基酸序列或与SEQ ID NO:77具有至少85％序列同一性的氨基酸序列且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:31、32和33所示的氨基酸序列；或
所述第一重链可变区的氨基酸序列为SEQ ID NO:76，SEQ ID NO:76经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:76功能相同的氨基酸序列或与SEQ ID NO:76具有至少85％序列同一性的氨基酸序列且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:28、29和30所示的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:78，SEQ ID NO:78经取代、缺失或添 加一个或多个氨基酸获得的且与SEQ ID NO:78功能相同的氨基酸序列或与SEQ ID NO:78具有至少85％序列同一性的氨基酸序列且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:34、35和36所示的氨基酸序列。在一些实施方案中，根据本发明的抗TIGIT/抗PD-L1抗体，所述抗TIGIT抗体或其抗原结合片段为人源化抗体或其抗原结合片段，其包含第一重链可变区和第一轻链可变区，其中：
(1)所述第一重链可变区的氨基酸序列选自：
(c1)如SEQ ID NO:98或SEQ ID NO:99所示的氨基酸序列；
(c2)(c1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c3)与(c1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述第一轻链可变区的氨基酸序列选自：
(c4)如SEQ ID NO:100或SEQ ID NO:101所示的氨基酸序列；
(c5)(c4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c6)与(c4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些实施方案中，根据本发明的抗TIGIT/抗PD-L1抗体，所述抗TIGIT抗体或其抗原结合片段为人源化抗体或其抗原结合片段，其包含第一重链可变区和第一轻链可变区，其中
所述第一重链可变区的氨基酸序列为SEQ ID NO:98，SEQ ID NO:98经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:98功能相同的氨基酸序列或与SEQ ID NO:98具有至少85％序列同一性的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:100，SEQ ID NO:100经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:100功能相同的氨基酸序列或与SEQ ID NO:100具有至少85％序列同一性的氨基酸序列；或
所述第一重链可变区的氨基酸序列为SEQ ID NO:99，SEQ ID NO:99经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:99功能相同的氨基酸序列或与SEQ ID NO:99具有至少85％序列同一性的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:101，SEQ ID NO:101经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:101功能相同的氨基酸序列或与SEQ ID NO:101具有至少85％序列同一性的氨基酸序列。
在一些优选的实施方案中，本发明提供一种抗TIGIT/抗PD-L1抗体，其包括抗TIGIT抗体或其抗原结合片段和抗PD-L1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如以上实施方案中所限定；和所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和/或第二轻链可变区，其中所述第二重链可变区包含第二重链可变区的互补决定区1(H2CDR1)、第二重链可变区的互补决定区2(H2CDR2)和/或第二重链可变区的互补决定区3(H2CDR3)，所述第二轻链可变区包含第二轻链可变区的互补决定区1(L2CDR1)、第二轻链可变区的互补决定区2(L2CDR2)和/或第二轻链可变区的互补决定区3(L2CDR3)。
进一步优选地，在一些实施方案中，本发明提供一种抗TIGIT/抗PD-L1抗体，其包括抗TIGIT抗体或其抗原结合片段和抗PD-L1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如以上实施方案中所限定，和所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和第二轻链可变区，其中：
(1)所述第二重链可变区包含选自如下组的H2CDR1、H2CDR2和H2CDR3：
(A1)如SEQ ID NO:1、2和3所示的氨基酸序列；
(A2)如SEQ ID NO:4、5和6所示的氨基酸序列；
(A3)如SEQ ID NO:7、8和9所示的氨基酸序列；和
(A4)如SEQ ID NO:10、11和12所示的氨基酸序列；
(A5)与(A1)、(A2)、(A3)或(A4)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
(2)所述第二轻链可变区包含选自如下组的L2CDR1、L2CDR2和L2CDR3：
(A6)如SEQ ID NO:13、14和15所示的氨基酸序列；
(A7)如SEQ ID NO:16、17和18所示的氨基酸序列；
(A8)如SEQ ID NO:19、20和21所示的氨基酸序列；
(A9)如SEQ ID NO:22、23和24所示的氨基酸序列；
(A10)与(A6)、(A7)、(A8)或(A9)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗TIGIT/抗PD-L1抗体，其包括抗TIGIT抗体或其抗原结合片段和抗PD-L1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包含第一重链可变区和第一轻链可变区，其中：
(1)所述第一重链可变区包含选自如下组的H1CDR1、H1CDR2和H1CDR3：
(a1)如SEQ ID NO:25、26和27所示的氨基酸序列；
(a2)如SEQ ID NO:28、29和30所示的氨基酸序列；和
(a3)与(a1)或(a2)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
(2)所述第一轻链可变区包含选自如下组的L1CDR1、L1CDR2和L1CDR3：
(a4)如SEQ ID NO:31、32和33所示的氨基酸序列；
(a5)如SEQ ID NO:34、35和36所示的氨基酸序列；和
(a6)与(a4)或(a5)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和第二轻链可变区，其中：
(1)所述第二重链可变区包含选自如下组的H2CDR1、H2CDR2和H2CDR3：
(A1)如SEQ ID NO:1、2和3所示的氨基酸序列；
(A2)如SEQ ID NO:4、5和6所示的氨基酸序列；
(A3)如SEQ ID NO:7、8和9所示的氨基酸序列；和
(A4)如SEQ ID NO:10、11和12所示的氨基酸序列；
(A5)与(A1)、(A2)、(A3)或(A4)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
(2)所述第二轻链可变区包含选自如下组的L2CDR1、L2CDR2和L2CDR3：
(A6)如SEQ ID NO:13、14和15所示的氨基酸序列；
(A7)如SEQ ID NO:16、17和18所示的氨基酸序列；
(A8)如SEQ ID NO:19、20和21所示的氨基酸序列；
(A9)如SEQ ID NO:22、23和24所示的氨基酸序列；
(A10)与(A6)、(A7)、(A8)或(A9)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。
在一个具体的实施方案中，本发明提供一种抗TIGIT/抗PD-L1抗体，其包括抗TIGIT抗体或其抗原结合片段和抗PD-L1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包含所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:28、29和30或与SEQ ID NO:28、29和30所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一重链可变区，和所述L1CDR1、 L1CDR2和L1CDR3分别为SEQ ID NO:34、35和36或与SEQ ID NO:34、35和36所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一轻链可变区；和所述抗PD-L1抗体或其抗原结合片段包含所述H2CDR1、H2CDR2和H2CDR3分别为SEQ ID NO:1、2和3或与SEQ ID NO:1、2和3所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二重链可变区，和所述L2CDR1、L2CDR2和L2CDR3分别为SEQ ID NO:13、14和15或与SEQ ID NO:13、14和15所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二轻链可变区。
在一个具体的实施方案中，本发明提供一种抗TIGIT/抗PD-L1抗体，其包括抗TIGIT抗体或其抗原结合片段和抗PD-L1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包含所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:28、29和30或与SEQ ID NO:28、29和30所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:34、35和36或与SEQ ID NO:34、35和36所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一轻链可变区；和所述抗PD-L1抗体或其抗原结合片段包含所述H2CDR1、H2CDR2和H2CDR3分别为SEQ ID NO:7、8和9或与SEQ ID NO:7、8和9所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二重链可变区，和所述L2CDR1、L2CDR2和L2CDR3分别为SEQ ID NO:19、20和21或与SEQ ID NO:19、20和21所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二轻链可变区。
在一个具体的实施方案中，本发明提供一种抗TIGIT/抗PD-L1抗体，其包括抗TIGIT抗体或其抗原结合片段和抗PD-L1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包含所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:28、29和30或与SEQ ID NO:28、29和30所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:34、35和36或与SEQ ID NO:34、35和36所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一轻链可变区；和所述抗PD-L1抗体或其抗原结合片段包含所述H2CDR1、H2CDR2和H2CDR3分别为SEQ ID NO:4、5和6或与SEQ ID NO:4、5和6所示的氨基酸 序列具有至少85％序列同一性的氨基酸序列的第二重链可变区，和所述L2CDR1、L2CDR2和L2CDR3分别为SEQ ID NO:16、17和18或与SEQ ID NO:16、17和18所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二轻链可变区。
在一个具体的实施方案中，本发明提供一种抗TIGIT/抗PD-L1抗体，其包括抗TIGIT抗体或其抗原结合片段和抗PD-L1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包含所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:28、29和30或与SEQ ID NO:28、29和30所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:34、35和36或与SEQ ID NO:34、35和36所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一轻链可变区；和所述抗PD-L1抗体或其抗原结合片段包含所述H2CDR1、H2CDR2和H2CDR3分别为SEQ ID NO:10、11和12或与SEQ ID NO:10、11和12所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二重链可变区，和所述L2CDR1、L2CDR2和L2CDR3分别为SEQ ID NO:22、23和24或与SEQ ID NO:22、23和24所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二轻链可变区。
在一些具体的实施方案中，根据本发明的抗TIGIT/抗PD-L1抗体，所述抗TIGIT抗体或其抗原结合片段以及抗PD-L1抗体或其抗原结合片段各自独立地为鼠源抗体、嵌合抗体、人源化抗体或完全人抗体。
在一些具体的实施方案中，本发明提供一种抗TIGIT/抗PD-L1抗体，其中所述抗TIGIT抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区，其中：
(1)所述第一重链可变区的氨基酸序列选自：
(b1)如SEQ ID NO:98或SEQ ID NO:99所示的氨基酸序列；
(b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述第一轻链可变区的氨基酸序列选自：
(b4)如SEQ ID NO:100或SEQ ID NO:101所示的氨基酸序列；
(b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和第二轻链可变区，其中：
(1)所述第二重链可变区的氨基酸序列选自：
(B1)如SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:50、SEQ ID NO:52或SEQ ID NO:61所示的氨基酸序列；
(B2)(B1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(B1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(B3)与(B1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述第二轻链可变区的氨基酸序列选自：
(B4)如SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:54、SEQ ID NO:56或SEQ ID NO:64所示的氨基酸序列；
(B5)(B4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(B4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(B6)与(B4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗TIGIT/抗PD-L1抗体，其中所述第一重链可变区的氨基酸序列为SEQ ID NO:98，SEQ ID NO:98经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:98功能相同的氨基酸序列或与SEQ ID NO:98具有至少85％序列同一性的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:100，SEQ ID NO:100经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:100功能相同的氨基酸序列或与SEQ ID NO:100具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗TIGIT/抗PD-L1抗体，其中 所述第一重链可变区的氨基酸序列为SEQ ID NO:98，SEQ ID NO:98经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:98功能相同的氨基酸序列或与SEQ ID NO:98具有至少85％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:25、26和27所示的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:100，SEQ ID NO:100经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:100功能相同的氨基酸序列或与SEQ ID NO:100具有至少85％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:31、32和33所示的氨基酸序列。
在一些具体的实施方案中，根据本发明的抗TIGIT/抗PD-L1抗体，其中所述抗TIGIT抗体或抗PD-L1抗体可以为鼠源抗体，其还含有鼠源的IgG1、IgG2a、IgG2b、IgG2c、IgG3或其变体的重链恒定区，和鼠源的κ链或其变体的轻链恒定区。
在一些优选的实施方案中，根据本发明的抗TIGIT/抗PD-L1抗体，其中所述抗TIGIT鼠源抗体还含有鼠源的IgG1或IgG2或其变体的重链恒定区，和鼠源κ链或其变体的轻链恒定区。
在一些具体的实施方案中，本发明提供抗TIGIT/抗PD-L1抗体，其中所述抗TIGIT抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区，其中：
(1)所述第一重链可变区的氨基酸序列选自：
(c1)如SEQ ID NO:99所示的氨基酸序列；
(c2)(c1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(c1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c3)与(c1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述第一轻链可变区的氨基酸序列选自：
(c4)如SEQ ID NO:101所示的氨基酸序列；
(c5)(c4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(c4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c6)与(c4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和第二轻链可变区，其中：
(1)所述第二重链可变区的氨基酸序列选自：
(C1)如SEQ ID NO:50、SEQ ID NO:52或SEQ ID NO:61所示的氨基酸序列；
(C2)(C1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(C1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(C3)与(C1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述第二轻链可变区的氨基酸序列选自：
(C4)如SEQ ID NO:54、SEQ ID NO:56或SEQ ID NO:64所示的氨基酸序列；
(C5)(C4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(C4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(C6)与(C4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供抗TIGIT/抗PD-L1抗体，其中所述第一重链可变区的氨基酸序列为SEQ ID NO:99，SEQ ID NO:99经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:99功能相同的氨基酸序列或与SEQ ID NO:99具有至少85％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:28、29和30所示的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:101，SEQ ID NO:101经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:101功能相同的氨基酸序列或与SEQ ID NO:101具有至少85％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:34、35和36所示的氨基酸序列；和所述第二重链可变区的氨基酸序列为SEQ ID NO:50所示的氨基酸序列，SEQ ID NO:50经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:50功能相同的氨基酸序列或与SEQ ID NO:50具有至少85％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:1、2和3所示的氨基酸序列，且所述第二轻链可变区的氨基酸序列为SEQ ID NO:54，SEQ ID NO:54经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:54功能相同的氨基酸序列或与SEQ ID NO:54具有至少85％序列同一性且所述 L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:13、14和15所示的氨基酸序列。
在一些具体的实施方案中，本发明提供抗TIGIT/抗PD-L1抗体，其中所述第一重链可变区的氨基酸序列为SEQ ID NO:99，SEQ ID NO:99经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:99功能相同的氨基酸序列或与SEQ ID NO:99具有至少85％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:28、29和30所示的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:101，SEQ ID NO:101经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:101功能相同的氨基酸序列或与SEQ ID NO:101具有至少85％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:34、35和36所示的氨基酸序列；和所述第二重链可变区的氨基酸序列为SEQ ID NO:52所示的氨基酸序列，SEQ ID NO:52经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:52功能相同的氨基酸序列或与SEQ ID NO:52具有至少85％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:1、2和3所示的氨基酸序列，且所述第二轻链可变区的氨基酸序列为SEQ ID NO:56，SEQ ID NO:56经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:56功能相同的氨基酸序列或与SEQ ID NO:56具有至少85％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:13、14和15所示的氨基酸序列。
在一些具体的实施方案中，本发明提供抗TIGIT/抗PD-L1抗体，其中所述第一重链可变区的氨基酸序列为SEQ ID NO:99，SEQ ID NO:99经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:99功能相同的氨基酸序列或与SEQ ID NO:99具有至少85％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:28、29和30所示的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:101，SEQ ID NO:101经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:101功能相同的氨基酸序列或与SEQ ID NO:101具有至少85％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:34、35和36所示的氨基酸序列；和所述第二重链可变区的氨基酸序列为SEQ ID NO:61所示的氨基酸序列，SEQ ID NO:61经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:61功能相同的氨基酸序列或与SEQ ID NO:61具有至少85％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:7、8 和9所示的氨基酸序列，且所述第二轻链可变区的氨基酸序列为SEQ ID NO:64，SEQ ID NO:64经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:64功能相同的氨基酸序列或与SEQ ID NO:64具有至少85％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:19、20和21所示的氨基酸序列。
在一些优选的实施方案中，本发明提供抗TIGIT/抗PD-L1抗体，其中所述第一重链可变区的氨基酸序列为SEQ ID NO:99，SEQ ID NO:99经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:99功能相同的氨基酸序列或与SEQ ID NO:99具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:28、29和30所示的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:101，SEQ ID NO:101经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:101功能相同的氨基酸序列或与SEQ ID NO:101具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:34、35和36所示的氨基酸序列；和所述第二重链可变区的氨基酸序列为SEQ ID NO:50所示的氨基酸序列，SEQ ID NO:50经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:50功能相同的氨基酸序列或与SEQ ID NO:50具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:1、2和3所示的氨基酸序列，且所述第二轻链可变区的氨基酸序列为SEQ ID NO:54，SEQ ID NO:54经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:54功能相同的氨基酸序列或与SEQ ID NO:54具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:13、14和15所示的氨基酸序列。
在一些具体的实施方案中，本发明提供抗TIGIT/抗PD-L1抗体，其中所述第一重链可变区的氨基酸序列为SEQ ID NO:99，SEQ ID NO:99经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:99功能相同的氨基酸序列或与SEQ ID NO:99具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:28、29和30所示的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:101，SEQ ID  NO:101经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:101功能相同的氨基酸序列或与SEQ ID NO:101具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:34、35和36所示的氨基酸序列；和所述第二重链可变区的氨基酸序列为SEQ ID NO:52所示的氨基酸序列，SEQ ID NO:52经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:52功能相同的氨基酸序列或与SEQ ID NO:52具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:1、2和3所示的氨基酸序列，且所述第二轻链可变区的氨基酸序列为SEQ ID NO:56，SEQ ID NO:56经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:56功能相同的氨基酸序列或与SEQ ID NO:56具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:13、14和15所示的氨基酸序列。
在一些具体的实施方案中，本发明提供抗TIGIT/抗PD-L1抗体，其中所述第一重链可变区的氨基酸序列为SEQ ID NO:99，SEQ ID NO:99经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:99功能相同的氨基酸序列或与SEQ ID NO:99具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:28、29和30所示的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:101，SEQ ID NO:101经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:101功能相同的氨基酸序列或与SEQ ID NO:101具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:34、35和36所示的氨基酸序列；和所述第二重链可变区的氨基酸序列为SEQ ID NO:61所示的氨基酸序列，SEQ ID NO:61经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:61功能相同的氨基酸序列或与SEQ ID NO:61具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:7、8和9所示的氨基酸序列，且所述第二轻链可变区的氨基酸序列为SEQ ID NO:64，SEQ ID NO:64经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:64功能相同的氨基酸序列或与SEQ ID  NO:64具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:19、20和21所示的氨基酸序列。在一些实施方案中，本发明提供一种抗TIGIT/抗PD-L1人源化抗体，其中所述重链包含人源的IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，所述轻链包含人源的κ、λ链或其变体的轻链恒定区。
在本发明一个优选的实施方案中，所述的鼠源抗TIGIT/抗PD-L1抗体，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，和/或进一步包含鼠源IgG1、IgG2a、IgG2b、IgG2c、IgG3或其变体的重链恒定区。
在本发明一个优选的实施方案中，根据本发明的抗TIGIT/抗PD-L1抗体，所述抗TIGIT抗体或其抗原结合片段的抗体轻链进一步包含鼠源κ、λ链或其突变序列的轻链恒定区。所述的抗TIGIT抗体或其抗原结合片段的抗体重链进一步包含鼠源IgG1、IgG2a、IgG2b、IgG2c、IgG3或其突变序列的重链恒定区，优选包含人源IgG1、IgG2、IgG4重链恒定区。
在一些具体的实施方案中，根据本发明的抗TIGIT/抗PD-L1抗体，所述抗TIGIT人源化抗体或其抗原结合片段还包含人源IgG1、IgG2a、IgG2b、IgG2c、IgG3或其变体的重链恒定区，和人源κ、λ链或其变体的轻链恒定区。在一些优选的实施方案中，本发明的抗TIGIT人源化抗体或其抗原结合片段还包含人源IgG1、IgG2、IgG4或其变体的重链恒定区，和人源κ链或其变体的轻链恒定区。
在本发明一个优选的实施方案中，根据本发明的抗TIGIT/抗PD-L1抗体，所述抗PD-L1抗体或其抗原结合片段的抗体重链进一步包含鼠源IgG1、IgG2a、IgG2b、IgG2c、IgG3或其突变序列的重链恒定区，优选包含人源IgG或其突变序列的重链恒定区；所述抗PD-L1抗体或其抗原结合片段的抗体轻链进一步包含鼠源κ、λ链或其突变序列的轻链恒定区。
在一些具体的实施方案中，根据本发明的抗TIGIT/抗PD-L1抗体，所述抗PD-L1人源化抗体或其抗原结合片段还包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，和人源κ、λ链或其变体的轻链恒定区。在一些优选的实施方案中，本发明的抗TIGIT人源化抗体或其抗原结合片段还包含人源IgG4或其变体的重链恒定区，和人源κ链或其变体的轻链恒定区。
在一些实施方案中，本发明提供抗TIGIT/抗PD-L1抗体，其中所述的抗TIGIT 抗体或其抗原结合片段以及抗PD-L1抗体或其抗原结合片段分别为Fab、Fv、sFv或F(ab) 2。在一个具体的实施方案中，本发明提供的抗TIGIT/抗PD-L1抗体为scF(ab) 2。
优选地，本发明以上实施方案中的抗TIGIT/抗PD-L1抗体是抗TIGIT/抗PD-L1双特异性抗体。在一些实施方案中，所述双特异性抗体是人抗体或人源化抗体。在一些实施方案中，所述结合特异性之一是针对TIGIT，而另一个结合特异性是针对任何其它抗原。在一些实施方案中，所述结合特异性之一是针对TIGIT，而另一个结合特异性是针对PD-L1。在一些实施方案中，所述双特异性抗体可结合TIGIT的两种不同表位。所述双特异性抗体还可用于将细胞毒剂定位于表达TIGIT的细胞。这些抗体拥有TIGIT结合臂和细胞毒剂结合臂，所述细胞毒剂例如肥皂草毒蛋白、抗干扰素-α、长春花生物碱类、蓖麻毒蛋白A链、甲氨蝶呤或放射性同位素半抗原。可将本发明的双特异性抗体制备成全长抗体或抗体片段(例如F(ab') 2双特异性抗体)。
制备双特异性抗体的方法是本领域已知的。传统上，双特异性抗体的重组制备基于两个免疫球蛋白重链-轻链对的共表达，其中两个重链具有不同的特异性(Millstein and Cuello,Nature 305:537(1983))。由于免疫球蛋白重链和轻链的随机分配，这些杂交瘤(quadroma))可能产生10种不同抗体分子的混合物，其中只有一种分子具有正确的双特异性结构。该正确分子的纯化通常通过亲和层析步骤进行，相当麻烦且产物产量低。类似的方法在WO93/08829及Trauneckeretal.,EMBOJ.10:3655(1991)中有公开。
根据一种不同的方法，具有期望结合特异性(抗体-抗原结合位点)的抗体可变区与免疫球蛋白恒定区序列融合。在一些实施方案中，与包含至少部分铰链、CH2和CH3区的免疫球蛋白重链恒定区进行融合。在一些实施方案中，包含与轻链结合所必需位点的第一重链恒定区(CH1)存在于该融合的至少一部分中。将编码免疫球蛋白重链融合片段以及免疫球蛋白轻链(如果需要)的DNA插入不同的表达载体中并共转染入合适的宿主生物体。在用于构建的三种多肽链比例不等时提供最佳产量的实施方案中，这为调整三种多肽片段的相互比例提供极大的灵活性。不过，在至少两种多肽链以相同比例表达产生高产量时或比例没有特别意义时，有可能将两种或所有三种多肽链的编码序列插入一个表达载体中。
在该方法的一个实施方案中，所述双特异性抗体由一个臂中具有第一结合特异性的杂合免疫球蛋白重链和另一个臂中的杂合免疫球蛋白重链-轻链对(提供第二结合特异性)组成。由于免疫球蛋白轻链仅在该双特异性分子的一半中存在提供了便利的分离途径，因此发现该不对称性结构便于将期望的双特异性物质与不想要的免疫球蛋白链组合物分开。该方法在WO 94/04690中公开。关于产生双特异性抗体的进一步信息参见例如Sureshetal.,Methods in Enzymology 121:210(1986)。
根据另一种方法，可改造一对抗体分子间的界面以使从重组细胞培养物回收的异二聚体的百分比最大化。该界面包含抗体恒定区CH3结构域的至少一部分。在该方法中，将第一抗体分子界面的一个或多个小氨基酸侧链用较大侧链(例如酪氨酸或色氨酸)替换。通过将大氨基酸侧链用较小氨基酸侧链(例如丙氨酸或苏氨酸)替换，在第二抗体分子的界面上产生与大侧链相同或相似大小的补偿性“空腔”。这提供了提高异二聚体相比于其它不想要的终产物诸如同二聚体的产量的机制。
双特异性抗体包括交联或“异源缀合”抗体。例如，一种异源缀合抗体可以与亲合素偶联，另一种异源缀合抗体可以与生物素偶联。可使用任何便利的交联方法来制备异源缀合抗体。合适的交联剂是本领域众所周知的，连同许多交联技术一起在美国专利No.4,676,980中公开。
本发明的双特异性抗体可以由抗体片段生成。例如，可使用化学连接技术来制备双特异性抗体。Brennanetal.,Science 229:81(1985)描述了通过蛋白水解切割完整抗体以生成F(ab') 2片段的方法。将这些片段在存在二硫醇络合剂亚砷酸钠的情况下(用以稳定邻近的二硫醇和防止分子间二硫键的形成)分解。然后将产生的Fab'片段转变为硫代硝基苯甲酸酯(TNB)衍生物。然后将Fab'-TNB衍生物之一通过巯基乙胺的还原重新恢复成Fab'-硫醇，并与等摩尔量的另一种Fab'-TNB衍生物混合，以形成双特异性抗体。
可以从大肠杆菌直接回收Fab'-SH片段，这些片段可化学偶联以形成双特异性抗体。Shalaby et al.,J.Exp.Med.175:217-225(1992)描述了完全人源化的双特异性抗体F(ab') 2分子的生成。每个Fab'片段由大肠杆菌单独分泌，并在体外进行定向化学偶联以形成双特异性抗体。
在一些实施方案中，本发明的双特异性抗体片段可以直接从重组细胞培养物生成和分离。例如，可以使用亮氨酸拉链生成双特异性抗体(Kostelnyetal.,J.Immunol.148(5):1547-1553(1992))。将来自Fos和Jun蛋白的亮氨酸拉链肽通过基因融合与两种不同抗体的Fab'部分连接。抗体同二聚体在铰链区分解以形成单体，然后重新氧化以形成抗体异二聚体。该方法也可用于生成抗体同二聚体。双抗体技术提供了制备双特异性抗体片段的其他机制。所述双特异性抗体片段包含通过接头相连的重链可变区(VH)和轻链可变区(VL)，所述接头太短以使得同一条链上的两个结构域之间不能配对。因此，迫使一个片段上的VH和VL结构域与另一个片段上的互补VL和VH结构域配对，由此形成两个抗原结合位点。在另一实施方案中，可以通过使用单链Fv(sFv)二聚体构建双特异性抗体片段。
本发明涵盖具有超过两价的多价抗体，例如，可制备三特异性抗体。多价抗体可以比二价抗体更快的受到表达该抗体结合的抗原的细胞的内在化(和/或异化)。本发明的抗体可以是可容易地通过编码抗体多肽链的核酸的重组表达而生成的、具有三个或更多抗原结合位点(例如四价抗体)的多价抗体。多价抗体可包含二聚化结构域和三个或更多抗原结合位点。在一些实施方案中，二聚化结构域包含(或由其组成)Fc区或铰链区。在这种情况中，抗体会包含Fc区及Fc区氨基末端的三个或更多抗原结合位点。在一些实施方案中，多价抗体包含(或由其组成)三个至大约八个抗原结合位点。在一些实施方案中，多价抗体包含四个抗原结合位点。多价抗体包含至少一条多肽链(例如两条多肽链)，其中所述多肽链包含两个或更多可变区。本发明的多价抗体可进一步包含至少两条(例如四条)轻链可变区多肽。本发明的多价抗体可包含例如约两条至约八条轻链可变区多肽。本发明的轻链可变区多肽包含轻链可变区，且任选进一步包含CL结构域。
在包含TIGIT靶向部分和PD-L1靶向部分的双特异性抗体中，TIGIT靶向部分和PD-L1靶向部分之一可以是全长抗体，并且另一个可以是包含重链CDR、轻链CDR或其组合的抗原结合片段(例如scFv)。靶向TIGIT和PD-L1蛋白之一的全长抗体和靶向另一蛋白的抗原结合片段可以直接或通过连接肽以化学方式连接(例如共价连接)。抗原结合片段(例如scFv)可以直接或通过连接肽与全长抗体的N-末端(例如全长抗体的轻链或重链的N-末端)、全长抗体的C-末端(例如全长抗体的重链(或Fc或CH3结构域)的C-末端)或两者连接。
在一个实施方案中，双特异性抗体可以包含全长抗TIGIT抗体、抗PD-L1抗体的抗原结合片段(例如scFab、scFv)以及它们之间的连接肽。在其他实施方案中，双特异性抗体可以包含全长抗TIGIT抗体、抗PD-L1抗体的抗原结合片段(例如scFab、scFv)以及它们之间的连接肽。
在一个实施方案中，双特异性抗体中包含的scFv可以按任何顺序包含重链可变区和轻链可变区。例如，双特异性抗体中包含的scFv可以在从N-末端到C-末端的方向上包含重链可变区和轻链可变区以及任选地在它们之间的连接肽，或者可替代地，双特异性抗体中包含的scFv可以在从N-末端到C-末端的方向上包含轻链可变区和重链可变区以及任选地在它们之间的连接肽。
在一些实施方案中，所述连接肽可包括例如Gly、Asn和/或Ser残基，并且还可以包括中性氨基酸，例如Thr和/或Ala。适用于连接肽的氨基酸序列可以是相关领域中已知的那些。同时，可以在使得融合蛋白功能不受影响的这样的限度内不同地确定连接肽的长度。例如，连接肽可以通过包括总共约1至约100、约2至约50、或约5至约25个选自由Gly、Asn、Ser、Thr、和Ala组成的组的一种或多种来形成。在一个实施方案中，连接肽可以表示为(GmSl)n(m、l和n独立地是约1至约10的整数，特别是约2至约5的整数)。
在另一个实施方案中，PD-L1靶向部分和TIGIT靶向部分可以均是全长抗体或包含重链CDR、轻链CDR或其组合的抗原结合片段。
在另一个实施方案中，双特异性抗体可以是异二聚体形式，其包含第一臂和第二臂，该第一臂包括靶向TIGIT和PD-L1之一的一对第一重链和第一轻链，该第二臂包括靶向另一者的一对第二重链和第二轻链。
在一个实施方案中，全长抗体可以是全长免疫球蛋白形式(例如IgG、IgM、IgA、IgE或IgD，例如人IgG、人IgM、人IgA、人IgE或人IgD)，并且抗原结合片段可以选自由Fab、Fab’、F(ab’) 2、Fd、Fv、scFv、scFab、单链抗体、sdFv等组成的组。例如，全长抗体可以是全长人IgG(人IgG1、人IgG2、人IgG3或人IgG4)形式，并且抗原结合片段可以是scFv。
例如，本文描述的抗体可以包含柔性接头序列，或者可以被修饰以添加功能部分(例如PEG、药物、毒素或标记)。在一些具体的实施方案中，根据本发明的抗TIGIT/抗PD-L1双特异性抗体，所述抗TIGIT抗体或其抗原结合片段的结构 为(VL-CL)-连接肽-(VH)-IgG4CH，所述抗PD-L1抗体或其抗原结合片段的结构为(VL-CL)-连接肽-(VH)-IgG4CH。在另一些具体的实施方案中，根据本发明的抗TIGIT/抗PD-L1双特异性抗体，所述抗TIGIT抗体或其抗原结合片段的结构为(VL-VH)-连接肽-IgG4FC，所述抗PD-L1抗体或其抗原结合片段的结构为(VL-CL)-连接肽-(VH)-IgG4CH。在一些具体的实施方案中，所述连接肽为(GGGGS)n形式，其中n为1-12，优选为3-10，更优选为6-8，例如6、7、8个GGGGS重复序列。在另一些具体的实施方案中，靶向TIGIT部分的(VL-CL)-连接肽-(VH)-IgG4CH中的IgG4FC为含有S228P、L235E突变的IgG4CH段，靶向PD-L1部分的(VL-CL)-连接肽-(VH)-IgG4CH中的IgG4FC为含有S228P、L235E突变的IgG4CH段。在另一些具体的实施方案中，靶向TIGIT部分的(VL-CL)-连接肽-(VH)-IgG4CH中的IgG4FC为含有S228P、S354C、T366W突变形成“Hole”结构的IgG4CH段，靶向PD-L1部分的(VL-CL)-连接肽-(VH)-IgG4CH中的IgG4FC为含有S228P、Y349C、T366S、L368A、Y407V突变形成“Knob”结构的IgG4CH段。在另一些具体的实施方案中，靶向TIGIT部分的(VL-CL)-连接肽-(VH)-IgG4CH中的IgG4FC为含有S228P、L235E、S354C、T366W突变形成“Hole”结构的IgG4CH段，靶向PD-L1部分的(VL-CL)-连接肽-(VH)-IgG4CH中的IgG4FC为含有S228P、L235E、Y349C、T366S、L368A、Y407V突变形成“Knob”结构的IgG4CH段。在一些具体的实施方案中，根据本发明的抗TIGIT/抗PD-L1双特异性抗体，其由两条肽链组成，其中：
肽链1的结构为：
Figure PCTCN2021119496-appb-000001
肽链2的结构为：
Figure PCTCN2021119496-appb-000002
其中IgG4CH/Ks表示IgG4恒定区(S228P、Y349C、T366S、L368A、Y407V突变)，IgG4CH/Hs表示IgG4恒定区(S228P、S354C、T366W突变)。
在另一些具体的实施方案中，根据本发明的抗TIGIT/抗PD-L1双特异性抗体，其由两条肽链组成，其中：
肽链1的结构为：
Figure PCTCN2021119496-appb-000003
肽链2的结构为：
Anti‐PD‐L1抗体VL‐CL
其中IgG4CH/PE表示IgG4恒定区(S228P、L235E突变)。
在一些具体的实施方案中，根据本发明的抗TIGIT/抗PD-L1双特异性抗体，其由两条肽链组成，其中肽链1的氨基酸序列为SEQ ID NO:102，肽链2的氨基酸序列为SEQ ID NO:103。在另一些具体的实施方案中，根据本发明的抗TIGIT/抗PD-L1双特异性抗体，其由两条肽链组成，其中肽链1的氨基酸序列为SEQ ID NO:104，肽链2的氨基酸序列为SEQ ID NO:105。在另一些具体的实施方案中，根据本发明的抗TIGIT/抗PD-L1双特异性抗体，其由两条肽链组成，其中肽链1的氨基酸序列为SEQ ID NO:102，肽链2的氨基酸序列为SEQ ID NO:106。在另一些具体的实施方案中，根据本发明的抗TIGIT/抗PD-L1双特异性抗体，其由两条肽链组成，其中肽链1的氨基酸序列为SEQ ID NO:107，肽链2的氨基酸序列为SEQ ID NO:108。在另一些具体的实施方案中，根据本发明的抗TIGIT/抗PD-L1双特异性抗体，其由两条肽链组成，其中肽链1的氨基酸序列为SEQ ID NO:102，肽链2的氨基酸序列为SEQ ID NO:109。在另一些具体的实施方案中，根据本发明的抗TIGIT/抗PD-L1双特异性抗体，其由两条肽链组成，其中肽链1的氨基酸序列为SEQ ID NO:110，肽链2的氨基酸序列为SEQ ID NO:111。本发明的另一方面提供分离的核酸。在一些实施方案中，根据本发明的分离的核酸编码本发明的抗TIGIT/抗PD-L1抗体。在一些实施方案中，根据本发明的分离的核酸编码本发明的抗TIGIT抗体或其抗原结合片段。在另一些实施方案中，根据本发明的分离的核酸编码本发明的抗PD-L1抗体或其抗原结合片段。
在一个具体的实施方案中，根据本发明的分离的核酸，其包含编码第一重链可变区如SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:98或SEQ ID NO:99的核苷酸序列和编码第一轻链可变区如SEQ ID NO:77、SEQ ID NO:78、SEQ ID NO:100或SEQ ID NO:101的核苷酸序列。在另一个具体的实施方案中，根据本 发明的分离的核酸，其包含编码第二重链可变区如SEQ ID NO:50、SEQ ID NO:52或SEQ ID NO:61的核苷酸序列和编码第二轻链可变区如SEQ ID NO:54、SEQ ID NO:56或SEQ ID NO:64的核苷酸序列。在再一个具体的实施方案中，根据本发明的分离的核酸，其包含编码第一重链可变区如SEQ ID NO:99的核苷酸序列和编码第一轻链可变区如SEQ ID NO:101的核苷酸序列。在再一个具体的实施方案中，根据本发明的分离的核酸，其包含编码第二重链可变区SEQ ID NO:50的核苷酸序列和编码第二轻链可变区SEQ ID NO:54的核苷酸序列。在一个具体的实施方案中，根据本发明的分离的核酸组合，其包含编码第一重链可变区如SEQ ID NO:99的核苷酸序列，编码第一轻链可变区如SEQ ID NO:101的核苷酸序列，编码第二重链可变区如SEQ ID NO:50的核苷酸序列，和编码第二轻链可变区如SEQ ID NO:54的核苷酸序列。在另一个具体的实施方案中，根据本发明的分离的核酸组合，其包含编码第一重链可变区如SEQ ID NO:99的核苷酸序列，编码第一轻链可变区如SEQ ID NO:101的核苷酸序列，编码第二重链可变区如SEQ ID NO:52的核苷酸序列，和编码第二轻链可变区如SEQ ID NO:56的核苷酸序列。在另一个具体的实施方案中，根据本发明的分离的核酸组合，其包含编码第一重链可变区如SEQ ID NO:99的核苷酸序列，编码第一轻链可变区如SEQ ID NO:101的核苷酸序列，编码第二重链可变区如SEQ ID NO:61的核苷酸序列，和编码第二轻链可变区如SEQ ID NO:64的核苷酸序列。
本发明的另一方面提供表达载体。在一些实施方案中，本发明的表达载体表达本发明的抗TIGIT/抗PD-L1双特异性抗体。在一些实施方案中，本发明的表达载体表达本发明的抗TIGIT抗体或其抗原结合片段。在另一些实施方案中，本发明的表达载体表达本发明的抗PD-L1抗体或其抗原结合片段。在一些实施方案中，根据本发明的表达载体，表达本发明的抗TIGIT抗体或其抗原结合片段的载体和表达本发明的抗PD-L1抗体或其抗原结合片段的载体是同种表达载体。根据本发明的表达载体其包含本发明的分离的核酸分子。
本发明的另一方面提供一种如上所述的表达载体转化的宿主细胞。
在一些实施方案中，根据本发明的宿主细胞选自原核细胞和真核细胞。在一些实施方案中，所述的宿主细胞为细菌，优选为大肠杆菌。在另一个优选的实施方案中，所述的宿主细胞为哺乳动物细胞。
本发明的另一方面提供制备本发明的抗TIGIT/抗PD-L1双特异性抗体的方法，包括在所述宿主细胞中表达抗体以及从宿主细胞中分离所述抗体的步骤。
本发明的另一方面提供一种药物组合物，其包含本发明的抗TIGIT/抗PD-L1双特异性抗体和药学可接受的载体。在一些实施方案中，本发明提供药物组合物，其包含本发明的抗TIGIT/抗PD-L1双特异性抗体，还包含其他活性组分，如其他抗体、靶向药物等。在一些实施方案中，所述药学可接受的载体选自抗氧化剂、多肽、蛋白质、亲水性聚合物、氨基酸、糖、螯合剂、糖醇、离子和表面活性剂。在一个具体的实施方案中，所述药学可接受的载体为缓冲水溶液。在另一个具体的实施方案中，所述药学可接受的载体为脂质体的形式。
本发明的另一方面提供一种嵌合抗原受体(CAR)融合蛋白，其包含本发明的抗TIGIT抗体或其抗原结合片段和/或抗PD-L1抗体或其抗原结合片段。在一些实施方案中，所述嵌合抗原受体融合蛋白包含本发明的抗TIGIT抗体或其抗原结合片段，其为针对TIGIT抗原的V H和V L的单链可变片段(scFv)。在另一些实施方案中，所述嵌合抗原受体融合蛋白包含本发明的抗PD-L1抗体或其抗原结合片段，其为针对PD-L1抗原的V H和V L的单链可变片段(scFv)。在另一些实施方案中，所述嵌合抗原受体融合蛋白包含针对TIGIT抗原的V H和V L的第一单链可变片段(scFv)和针对PD-L1抗原的V H和V L的第二单链可变片段(scFv)。所述针对TIGIT抗原的V H和V L的第一scFv具有以上实施方案中描述的第一重链可变区的H1CDR1、H1CDR2和H1CDR3和第一轻链可变区的L1CDR1、L1CDR2和L1CDR3。所述针对PD-L1抗原的V H和V L的第二scFv具有以上实施方案中描述的第二重链可变区的H2CDR1、H2CDR2和H2CDR3和第二轻链可变区的L2CDR1、L2CDR2和L2CDR3。
可以将本发明的抗TIGIT/抗PD-L1双特异性抗体与药学上可接受的载体、稀释剂或赋形剂混合制备成药物制剂，以适合于经口或胃肠外给药。给药方法包括，但不限于经口、皮内、肌内、腹膜内、静脉内、脑内、眼内、气管内、皮下、鼻内途径。所述制剂可以通过任何途径施用，例如通过输注或推注，通过经上皮或皮肤粘膜(例如口腔粘膜或直肠等)吸收的途径施用。给药可以是全身的或局部的。所述制剂可通过本领域已知的方法制备，且包含药物制剂领域常规使用的载体、稀释剂或赋形剂。
本发明的另一方面提供治疗和/或预防与TIGIT、PD-L1或两者相关的疾病的方法，所述方法包括向有此需要的个体施用本发明的抗TIGIT/抗PD-L1双特异性抗体或本发明的药物组合物。
本发明的另一方面提供本发明的抗TIGIT/抗PD-L1双特异性抗体或本发明的药物组合物在制备治疗和/或预防与TIGIT、PD-L1或两者相关的疾病的药物中的应用。在一些实施方案中，所述与TIGIT、PD-L1或两者相关的疾病包括血液肿瘤、淋巴瘤、乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、宫颈癌、肾癌、膀胱癌、胰腺癌、神经胶质瘤和/或黑素瘤。所述肿瘤可以是任何表达PD-L1蛋白的肿瘤，例如膀胱癌、肝癌、结肠癌、直肠癌、子宫内膜癌、白血病、淋巴瘤、胰腺癌、肺癌(例如小细胞肺癌、非小细胞肺癌等)、乳腺癌、尿道癌、头颈癌、胃肠癌、胃癌、食道癌、卵巢癌、肾癌、黑素瘤、前列腺癌、甲状腺癌等。所述肿瘤可以是原发性或转移性肿瘤。在一些实施方案中，本发明提供上述抗TIGIT/抗PD-L1双特异性抗体或本发明的药物组合物在制备抗肿瘤的药物中的应用，例如所述肿瘤选自血液肿瘤、淋巴瘤、乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、宫颈癌、肾癌、膀胱癌、胰腺癌、神经胶质瘤、黑素瘤、骨髓瘤、前列腺癌。
本发明提供的抗TIGIT/抗PD-L1双特异性抗体具有显著的抗肿瘤作用，可明显抑制肿瘤生长，可在制备用于治疗各类肿瘤疾病的药物中应用，具有广阔的市场前景。
定义
除非另有定义，本文中使用的科学和技术术语的含义是本领域技术人员所通常理解的含义。本文中所述的细胞和组织培养、分子生物学以及蛋白质和寡或多核苷酸化学及杂交中使用的命名和技术是本领域公知且普遍使用的。对于重组DNA、寡核苷酸合成和组织培养与转化(如电穿孔、脂质转染)，使用了标准技术。酶促反应和纯化技术根据生产商的说明书或本领域普遍使用或本文所述的方法进行。前述技术和方法通常根据本领域公知且本说明书中引用和讨论的多部综合和较具体的文献中描述的那样使用。参见例如Sambrook等，Molecular Cloning:A Laboratory Manual)(第2版，Cold Spring Harbor Laboratory Press，纽约冷泉港 (1989))。本文所述的分析化学、合成有机化学以及医学和药学化学中使用的命名以及实验室方法和技术是本领域公知且普遍使用的。
在本发明中，术语“至少80％序列同一性”是指至少80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％的序列同一性。在本发明中，术语“至少85％序列同一性”是指至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％的序列同一性。在一些优选的实施方案中，本发明所述的序列同一性可以至少为90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％。两个序列之间的序列比较和同一性百分比测定可以通过National Center For Biotechnology Instutute网站上的BLASTN/BLASTP算法来进行。
在抗体分子中，轻链的三个高变区和重链的三个高变区在三维空间中以相对彼此的位置排列以形成抗原结合表面。抗原结合表面与所结合抗原的三维表面互补，且每条重链和轻链的三个高变区均被称作“互补决定区”或“CDR”。氨基酸向每个结构域的分配是根据Kabat《免疫学感兴趣的蛋白质的序列》(国立卫生研究院，马里兰州贝塞斯达(1987和1991))或Chothia和Lesk，J.Mol.Biol.196:901-917(1987)，Chothia等，Nature 342:878-883(1989)定义。
本发明的“抗体”是指特异性地识别并结合抗原的多肽或多肽复合物。抗体可以是完整抗体和其任何抗原结合片段或单链。本发明的“抗体”包括含有Ig分子的具有结合抗原的生物活性的至少一部分的任何蛋白质或肽。本发明“抗体”的实例包括但不限于重链或轻链的CDR或其配体结合部分、重链或轻链可变区、重链或轻链恒定区、框架区或其任何部分。
本发明所述的“抗原结合片段”包括具有抗原结合活性的Fab片段、Fab’片段、F(ab’)2片段及与人TIGIT或PD-L1结合的Fv片段、scFv片段。Fv片段含有抗体第一重链可变区和第一轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多连接肽，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体或单链Fv(scFv)。本发明的抗TIGIT或抗PD-L1抗体可以是单链可变区片段(scFv)，其源自抗体的单链多肽，保留了结合抗原的 能力。scFv的实例包括通过重组DNA技术形成的抗体多肽，其中免疫球蛋白重链(H链)和轻链(L链)片段的Fv区经由间隔序列连接。制备scFv的各种方法是本领域技术人员所熟知的。
本发明所述的抗体指免疫球蛋白分子或其免疫活性部分，即包含特异性结合抗原(与其免疫反应)的抗原结合位点的分子。“特异性结合”指抗体与抗原的一种或多种抗原决定簇反应而不与其他多肽反应或以很低的亲和性(Kd>10 -6)结合其他多肽。抗体包括但不限于多克隆、单克隆、嵌合、dAb(结构域抗体)、单链、Fab、Fab’和F(ab’)2片段、Fv、scFv及Fab表达文库。单克隆抗体(mAb)是由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的、原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术及合成技术如CDR grafting或其它现有技术进行重组得到。
本发明所述的“鼠源抗体”为根据本领域知识和技能制备的对人TIGIT的单克隆抗体。制备时用TIGIT抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。
本发明所述的“嵌合抗体”是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。
本发明所述的“人源化抗体”也称为CDR移植抗体，是将小鼠的CDR序列移植到人的抗体可变区框架(FR)中产生的抗体。此类可变区框架序列可以从公共的DNA数据库或公开的参考文献获得，例如从ImMunoGeneTics(IMGT)网站http://imgt.cines.fr得到或从免疫球蛋白杂志，2001ISBN012441351上获得。
本发明所述的“双特异性抗体”指对至少两种不同抗原具有结合特异性的单克隆抗体。
本发明所述的“连接肽”可以是包括1至10，特别是2至50个任何氨基酸的那些，并且可以包括任何种类的氨基酸而没有任何限制。
附图说明
图1是ScFab结构的双特异性抗TIGIT/抗PD-L1抗体结构示意图。
图2是ScFv结构的双特异性抗TIGIT/抗PD-L1抗体结构示意图。
具体实施方式
下面代表性的实施例是为了更好地说明本发明，而非用于限制本发明的保护范围。以下实施例中未注明条件的实验方法通常按照常规条件，如冷泉港的抗体技术实验手册、分子克隆手册等，或按照原料或商品制造厂商所建议的条件进行。实施例中使用的材料、试剂如无特殊说明均为商购获得。
实施例1抗人PD-L1抗体的获得
1.动物免疫
采用mFc标签的PD-L1抗原蛋白(购于北京百普赛斯生物科技有限公司，中国)与佐剂共同免疫的方法免疫BALB/c、SJL品系的实验小鼠和SD品系的实验大鼠。
免疫佐剂首次是Freund’s Adjuvant,Complete(SIGMA,F5881-10ML)，后期用Freund’s Adjuvant,Incomplete(SIGMA,F5506-10ML)。将不同标签的PD-L1抗原蛋白样品逐滴加入到佐剂溶液中，边滴加边涡旋以充分混合，佐剂使用剂量参考说明书进行。混合均匀形成油包水的乳状后免疫小鼠或大鼠。免疫方案如表1所示：
表1免疫方案
Figure PCTCN2021119496-appb-000004
Figure PCTCN2021119496-appb-000005
*i.m.肌内注射
2.细胞融合
无菌取小鼠脾脏并制成细胞悬液，按脾细胞：Sp2/0细胞(小鼠骨髓瘤细胞)＝1:1的比例进行细胞融合。将融合后的细胞悬液转入到含有20％FBS的15mL RPMI 1640完全培养基中，室温放置20min。用含1×HAT、1×BIOMYC3、20％FBS的RPMI 1640培养基重悬融合细胞。按100μl/孔将细胞悬液加到若干块96孔细胞培养板中，保证每孔细胞量约为4×10 4个细胞/孔，置于37℃细胞培养箱中培养。5天后补加100μL/孔RPMI 1640完全培养基(含20％FBS，1×HAT，1×BIOMYC-3)。
3.阳性克隆筛选
融合一周后，取细胞上清，通过ELISA筛选出具有结合及阻断能力的杂交瘤母克隆细胞并进行扩大培养，复测结合活性及阻断活性，再次筛选获得具有结合及阻断能力的杂交瘤阳性细胞株。利用有限稀释法将阳性细胞株进行亚克隆，培养一周后用ELISA检测亚克隆上清与PD-L1分子的结合活性以及阻断PD-L1/PD-1相互作用的活性，获得PL-7、PL-15、PL-16、PL-18四株优选双阳性细胞株。
4.抗PD-L1抗体可变区序列的获得
将经亚克隆操作的阳性杂交瘤细胞进行扩大培养，取适量细胞按RNeasy Plus Mini Kit(Qiagen，74134)试剂盒说明书提取总RNA，利用Prime Script 1st strand cDNA Synthesis Kit(Takara，6110A)反转录试剂盒合成cDNA第一条链。
根据小鼠抗体亚型可变区设计特异性引物(5’端含有用于与真核表达载体发生同源重组的同源臂序列)，以cDNA为模板进行抗体可变区基因的PCR扩增，从而分别获得小鼠抗体轻链与重链可变区的基因片段，命名为SHS009PL-7、SHS009PL-15、SHS009PL-16、SHS009PL-18；设计引物(参考文献：1.Anke Krebber,Susanne Bornhauser,Jorg Burmester etal.Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system.Journal of Immunological Methods,1997,201: 35–55；2.Simon KorenMiha
Figure PCTCN2021119496-appb-000006
Colja Venturini etal.Antibody variable-region sequencing as a method for hybridoma cell-line authentication,2008,78:1071–1078)，进行DNA测序获得序列，测序结果见表2。
表2抗PD-L1鼠源单克隆抗体序列
Figure PCTCN2021119496-appb-000007
5.嵌合抗体的构建
将纯化后的小鼠抗体轻链与重链可变区基因片段分别与线性化的含有人抗体轻链或重链恒定区基因片段的真核表达质粒共转化大肠杆菌DH5α感受态细胞，挑取测序正确的嵌合抗体分别标记为PL-7CHI、PL-15CHI、PL-16CHI、PL-18CHI，嵌合抗体测序结果见表3。
PL-7CHI、PL-15CHI、PL-16CHI、PL-18CHI轻重链可变区氨基酸序列，分别同鼠源性抗体SHS009PL-7、SHS009PL-15、SHS009PL-16、SHS009PL-18轻重链可变区氨基酸序列相同。
提取嵌合抗体轻链质粒与重链质粒转染HEK 293F细胞，表达纯化获得大量抗体，进行纯度检测、活性分析及亲和力的检测。
表3抗PD-L1嵌合抗体序列
嵌合抗体 重链可变区氨基酸序列 轻链可变区氨基酸序列
PL-7CHI SEQ ID NO:37 SEQ ID NO:41
PL-15CHI SEQ ID NO:38 SEQ ID NO:42
PL-16CHI SEQ ID NO:39 SEQ ID NO:43
PL-18CHI SEQ ID NO:40 SEQ ID NO:44
6.抗PD-L1抗体人源化改造
根据嵌合抗体的活性分析、亲和力KD值等结果选择PL-7CHI、PL-16CHI进行人源化抗体改造。
鼠源单克隆嵌合抗体PL-7CHI、PL-16CHI的人源化参照经典的CDR移植策略进行，以PL-7CHI、PL-16CHI抗体框架区序列FR1-FR3作为模板，在人框架区库中寻找3D结构相似但是免疫原性较低的全人框架替代PL-7CHI/PL-16CHI的FR1-FR3序列。经同源比对分析，PL-7CHI、PL-16CHI抗体重链可变区FR区分别与人抗体种系基因M99683|IGHV4-31*02(SEQ ID NO:45)、X62109|IGHV1-3*01(SEQ ID NO:46)最为相似，PL-7CHI、PL-16CHI抗体轻链可变区FR区与人抗体种系基因Z00023|IGKV4-1*01(SEQ ID NO:47)最为相似。人源化后的重链/轻链全长序列进行3D建模并和原抗体重链/轻链序列进行结构比对分析，综合考虑抗原性和3D结构相似度，并将在结构模拟中显示对抗体结构稳定起到关键作用的的氨基酸位点回突变为鼠源性氨基酸残基。最后得到PL-7CHI人源化重链5条(PL-7CHI人源化重链可变区序列：VH1-0(SEQ ID NO:48)、VH1-1(SEQ ID NO:49)、VH1-2(SEQ ID NO:50)、VH1-3(SEQ ID NO:51)或VH1-4(SEQ ID NO:52))、人源化轻链4条(PL-7CHI人源化轻链可变区序列：VL1-0(SEQ ID NO:53)、VL1-1(SEQ ID NO:54)、VL1-2(SEQ ID NO:55)或VL1-3(SEQ ID NO:56))；PL-16CHI人源化重链5条(PL-16CHI人源化重链可变区序列：VH1-0(SEQ ID NO:57)、VH1-1(SEQ ID NO:58)、VH1-2(SEQ ID NO:59)、VH1-3(SEQ ID NO:60)或VH1-4(SEQ ID NO:61))、人源化轻链3条(PL-16CHI人源化轻链可变区序列：VL1-0(SEQ ID NO:62)、VL1-1(SEQ ID NO:63)或VL1-2(SEQ ID NO:64))。在此基础上，通过不同的轻重链组合，我们得到了多个人源化抗体。经活性检测后，确定其中评分最高的抗PD-L1人源化抗体序列分别为HuPL7-21、HuPL16-42。
实施例2人源化抗PD-L1抗体生产
将以上设计好的人源化抗体轻链与重链可变区氨基酸序列合成相对应的核苷酸编码序列，并生成相邻片段之间含有互补序列的寡核苷酸片段，通过Overlap PCR将寡核苷酸片段退火后连接起来，再利用特异性引物(5’端含有用于与真核表达载体发生同源重组的同源臂序列)扩增出完整的轻链与重链可变区核苷酸片段；将纯化后的轻链可变区核苷酸片段与线性化的含有IgG4轻链恒定区的真核 表达质粒共转化大肠杆菌DH5α感受态细胞，将纯化后的重链可变区核苷酸片段与含S228P/L235E突变的IgG4重链恒定区的真核表达质粒共转化大肠杆菌DH5α感受态细胞，分别将转化质粒的感受态细胞均匀涂布于含有相应抗生素的琼脂平板表面，于37℃恒温培养箱过夜培养后分别挑取若干单菌落进行DNA测序。
将测序正确的阳性克隆进行质粒提取，从而获得人源化抗体轻链与重链表达质粒，使用核酸定量分析仪检测质粒的浓度与纯度。
将质粒转染HEK293F细胞，表达纯化获得大量抗体，进行纯度检测、活性分析及亲和力的检测。序列见表4。
表4抗PD-L1人源化抗体序列
Figure PCTCN2021119496-appb-000008
实施例3 TIGIT抗体相关抗原蛋白及阳性对照抗体的制备
1、抗原蛋白及阳性对照抗体的表达载体构建
1)抗原蛋白的表达载体构建
人源TIGIT蛋白胞外区氨基酸序列与hIgG1-Fc或mIgG1-Fc标签氨基酸序列，氨基酸序列设计分别如SEQ ID NO:65和SEQ ID NO:66所示。对上述氨基酸序列进行密码子优化后合成带有标签的TIGIT蛋白胞外区基因片段TIGIT-hFc、TIGIT-mFc，并将其分别克隆至真核表达质粒pHR中，获得其表达质粒pHR-TIGIT-hFc、pHR-TIGIT-mFc。
融合人源TIGIT蛋白胞外区氨基酸序列与his氨基酸序列，氨基酸序列设计如SEQ ID NO:67所示。对该氨基酸序列进行密码子优化后合成完整的表达质粒pcDNA3.1-TIGIT-his。
2)配体蛋白的表达载体构建
人源CD155蛋白胞外区氨基酸序列与hIgG1-Fc或mIgG1-Fc标签氨基酸序列，氨基酸序列设计分别如SEQ ID NO:68和SEQ ID NO:69所示。对上述氨基酸序列进行密码子优化后合成带有标签的CD155蛋白胞外区基因片段CD155-hFc、CD155-mFc，并将其分别克隆至真核表达质粒pHR中，获得其表达质粒pHR-CD155-hFc、pHR-CD155-mFc。
融合人源CD155蛋白胞外区氨基酸序列与his氨基酸序列，氨基酸序列设计如SEQ ID NO:70所示。对该氨基酸序列进行密码子优化后合成完整的表达质粒pcDNA3.1(+)-CD155-his。
3)阳性对照抗体的表达载体构建
使用专利申请US 2016/0176963A1中公开的人源化抗体22G2(本文简称22G2)作为阳性对照抗体。参照US 2016/0176963A1中公开的方法制备22G2。22G2的氨基酸序列如下所示：
22G2重链可变区氨基酸序列：SEQ ID NO:71；
22G2轻链可变区氨基酸序列：SEQ ID NO:72；
对22G2抗体所对应的氨基酸序列进行密码子人工优化，将其重链基因片段克隆到含有IgG4轻链恒定区的真核表达质粒pHR上，获得22G2的重链真核表达质粒pHR-22G2-hG4，其轻链表达质粒为pHR-22G2-hk。
2、抗原蛋白及阳性对照抗体的表达与纯化
1)抗原蛋白的稳转细胞株的构建
合成编码TIGIT蛋白全长的基因片段，氨基酸序列设计如SEQ ID NO:73所示，然后克隆到真核表达质粒pTargeT上，获得其表达质粒pTargeT-TIGIT。
将真核表达质粒pTargeT-TIGIT在160V电压，15msec的方形脉冲下以电转的方式转染到CHO-K1细胞(中国科学院上海细胞生物学研究所)，置37℃，5％CO 2的培养箱中培养。24h后采用含500μg/ml G418的培养基加压培养。16天后采用FACS检测pool阳性率，将电转质粒后的细胞铺板(1x10 6个/ml的细胞密度，100ul/孔)，采用PE mouse anti-human TIGIT抗体(BD，556046)与细胞孵育，以流式细胞仪(BD，FACSJazz)读取585nm波长下mean值，使用GraphPad生成进行数据分析。将阳性细胞株进行亚克隆，挑选出克隆化的CHO-K1细胞株，该细胞株高水平表达TIGIT分子，命名为hTIGIT-CHO-K1。
2)配体蛋白的稳转细胞株的构建
合成编码CD155蛋白全长的基因片段，氨基酸序列设计如SEQ ID NO:74所示，然后克隆到真核表达质粒pTargeT上，获得其表达质粒pTargeT-CD155。
将真核表达质粒pTargeT-CD155在160V电压，15msec的方形脉冲下以电转的方式转染到CHO-K1细胞(中国科学院上海细胞生物学研究所)，置37℃，5％CO 2的培养箱中培养。24h后采用含500μg/ml G418的培养基加压培养。16天后采用FACS检测pool阳性率，将电转质粒后的细胞铺板(1x10 6个/ml的细胞密度，100ul/孔)，采用PE mouse anti-human CD155抗体(BD，556046)与细胞孵育，以流式细胞仪(BD，FACSJazz)读取585nm波长下mean值，使用GraphPad生成进行数据分析。将阳性细胞株进行亚克隆，挑选出克隆化的CHO-K1细胞株，该细胞株高水平表达CD155分子，命名为hCD155-CHO-K1。
3)标签蛋白及阳性对照抗体的表达
在1L细胞培养瓶中接种密度为0.5x 10 6个细胞/ml的293F细胞，加入新鲜的预热的FreeStyle 293表达培养基，使接种后总体积达到250mL，置37℃，8％CO 2，加湿的CO 2培养箱中培养过夜。取8.5mL FreeStyle 293表达培养基，加入1mg/ml的PEI溶液500ul，混合均匀，取250μg待转染质粒加入8.5ml FreeStyle 293表达培养基中，混合均匀，其中标签抗原蛋白质粒pHR-TIGIT-hFc、pHR-TIGIT-his、pcDNA3.1(+)-TPA-TIGIT-mIgG1-Fc分别转染；标签配体蛋白质粒pHR-CD155-hFc、pHR-CD155-his、pcDNA3.1(+)-TPA-CD155-mIgG1-Fc分别转染；阳性对照抗体22G2重链质粒pHR-22G2-hG4和轻链质粒pHR-22G2-hk共同转染。将PEI与FreeStyle 293表达培养基的混合溶液加入到质粒中，混合均匀，然后加入细胞培养物中，置37℃，8％CO 2，加湿的CO 2培养箱中培养。在细胞转染后第1天和第3天对细胞进行补料，每瓶加入2.5ml的谷氨酰胺(母液浓度为200mM)和5ml的葡萄糖(母液浓度为180g/L)。当细胞细胞活力降至65％～75％时，收集细胞上清。将细胞培养物1500rpm离心5min，收集上清，再8000rpm离心20min，收集上清。
4)亲和层析柱纯化
利用AKTA(GE，AKTA pure-150)根据蛋白性质采用不同的亲和层析柱进 行纯化(不同蛋白适配的亲和层析柱见表5)，具体纯化步骤如下：
表5不同蛋白适配的亲和层析柱
Figure PCTCN2021119496-appb-000009
清洗：超纯水清洗设备及管路2min，流速10mL/min，后用0.1M NaOH清洗层析系统；
接柱：将层析柱接入层析设备，并用超纯水冲洗5min；后0.1M NaOH冲洗30min，保留时间5min；
平衡：20mM PB+0.15M NaCl，pH 7.2平衡5个CV(柱体积)；
上样：将细胞表达上清上样，保留时间5min；
后平衡：20mM PB+0.15M NaCl，pH 7.2平衡5个CV；
洗脱：50mM醋酸，pH＝3.4洗脱，保留时间5min。UV280至50mAu左右时开始收集，降至50mAu左右时停止收集。用1M Tris-HCl，pH 9.0将样品pH调节至7.0；
再平衡：20mM PB+0.15M NaCl，pH 7.2平衡3个CV，保留时间5min；
在线清洗：0.1M NaOH清洗30min，保留时间5min；
清洗保存：纯化水清洗10min，后20％乙醇2个CV。
实施例4抗TIGIT单克隆抗体的制备
1、杂交瘤单克隆的制备
(1)动物免疫
采用购买的不同标签的TIGIT抗原蛋白(TIGIT-His(Sino Biological，10917-H08H)、TIGIT-hFc(R&D,7898-TG)、TIGIT-mFc(Acro Biosystems,Cat.No.TIT-H5253))与佐剂共同免疫的方法免疫C57、SJL品系的实验小鼠，首次免疫使用50μg抗原，后期使用25μg抗原免疫；采用不同标签的TIGIT抗原蛋白与佐剂共同免疫的方法免疫SD品系的实验大鼠，首次抗原使用 100μg抗原，后期使用50μg抗原免疫。
免疫佐剂可以是Quick Antibody-Mouse5W(北京博奥龙免疫技术有限公司，北京)或Titer Max(Sigma)与CpG(金斯瑞生物科技有限合成)/Alum(thermo)佐剂间隔。将不同标签的TIGIT抗原蛋白样品逐滴加入到佐剂溶液中，边滴加边涡旋以充分混合，佐剂使用剂量参考说明书进行。混合均匀形成油包水的乳状后免疫小鼠及大鼠。
高水平表达TIGIT分析的细胞系如hTIGIT-CHO-K1也用来免疫大鼠，使之产生抗体。用胰蛋白酶消化处理正在培养的实施例1中获得的hTIGIT-CHO-K1阳性单细胞，1000rpm离心5min，弃上清，用PBS重悬细胞沉淀，取样用细胞计数仪计数，剩余样品1000rpm离心5min，弃上清，用PBS重悬细胞沉淀，计入适量的PBS以获得1×10 8个细胞/ml的细胞悬液。实验组小鼠每只免疫1×10 7个细胞。
免疫方案如表6、表7所示：
表6小鼠免疫方案
Figure PCTCN2021119496-appb-000010
表7大鼠免疫方案
Figure PCTCN2021119496-appb-000011
*i.m.肌内注射；s.c.皮下注射；i.p.腹腔注射。
(2)杂交瘤融合
脾细胞的获取和制备：将加强免疫后的小/大鼠处死后浸泡75％的酒精中。解剖取出脾脏，用研磨棒研磨后，经细胞筛网过滤后制备成单细胞悬液。将脾细胞悬液2000rpm离心5min，弃上清。加入2mL红细胞裂解液，室温裂解红细胞2min，加入PBS至20mL，1500rpm离心7min，弃上清，重悬后进行活细胞计数。收集培养瓶中的Sp2/0细胞，1000rpm离心5min后弃上清，重悬后进行活细胞计数。按脾细胞：Sp2/0细胞＝1:1的比例混合细胞，1500rpm离心7min后弃上清。用20mL电转缓冲液重悬细胞，1500rpm离心7min。弃上清，重复一次。分别用适量电转缓冲液重悬细胞，保证细胞浓度2×10 7个细胞/mL左右。把细胞悬液加入9mL电转融合槽中融合。融合后将细胞悬液转入到含有20％FBS的15mL RPMI 1640完全培养基中，室温放置20min。用含1×HAT、1×BIOMYC-3、20％FBS的RPMI 1640培养基重悬融合细胞。按100μl/孔将细胞悬液加到若干块96孔细胞培养板中，保证每孔细胞量约为4×10 4个细胞/孔，置于37℃细胞培养箱中培养。5天后补加100μL/孔RPMI 1640完全培养基(含20％FBS，1×HAT，1×BIOMYC-3)。
(3)杂交瘤及亚克隆上清的筛选
初筛：融合一周后，取细胞上清，通过ELISA筛选出能结合TIGIT-his蛋白或细胞表面TIGIT的杂交瘤上清，利用TIGIT-his筛选针对TIGIT而非hFc、mFc的抗体。然后利用ELISA分析杂交瘤上清阻断TIGIT-CD155相互作用的能力。包被CD155-hFc于酶标板上，加入重组人源蛋白TIGIT-mFc与杂交瘤上清的混合物孵育2h，加入HRP标记的anti mouse IgG Fc特异性抗体(Jackson Immuno Research)孵育1h，利用酶标仪检测450nm处的吸光值。
复测：将筛选获得的具有结合能力及阻断能力的杂交瘤母克隆扩大培养，进行ELISA结合活性的复测；通过FACS筛选出能结合hTIGIT-CHO-K1细胞表面TIGIT的杂交瘤上清；再通过ELISA筛选出能结合cyno-TIGIT-his蛋白的杂交瘤上清；通过三次实验阳性交叉的杂交瘤上清作为候选阳性克隆。
利用有限稀释法将阳性细胞株进行亚克隆，培养一周后利用ELISA检测亚克隆上清与TIGIT分子的结合活性以及阻断TIGIT-CD155相互作用的活性，获得5种双阳性细胞株，分别标记为SHS006-17、SHS006-23。
2、单克隆抗体的制备
根据亚克隆上清活性分析结果确定单克隆抗体母克隆株SHS006-17、SHS006-23，将其扩大培养。培养条件是含有10％胎牛血清、1×NAEE、1×丙酮酸钠、1％青链霉素双抗的1640培养基，待细胞汇合度大于>80％时，进将细胞传代扩培，待培养至约50ml时收集上清，纯化抗体。获得抗体经SDS-PAGE凝胶电泳确定纯度良好。
3、单克隆抗体测序
将经亚克隆操作的阳性杂交瘤细胞进行扩大培养，取适量细胞按RNeasy Plus Mini Kit(Qiagen，74134)试剂盒说明书提取总RNA，利用Prime Script 1st strand cDNA Synthesis Kit(Takara，6110A)反转录试剂盒合成cDNA第一条链。
根据大鼠抗体亚型可变区设计特异性引物(5’端含有用于与真核表达载体发生同源重组的同源臂序列)，以cDNA为模板进行抗体可变区基因的PCR扩增，从而分别获得大鼠抗体轻链与重链可变区的基因片段；设计引物(参考文献：1.Anke Krebber,Susanne Bornhauser,Jorg Burmester etal.Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system.Journal of Immunological Methods,1997,201:35–55；2.Simon KorenMiha
Figure PCTCN2021119496-appb-000012
Colja Venturini etal.Antibody variable-region sequencing as a method for hybridoma cell-line authentication,2008,78:1071–1078)，进行DNA测序获得序列，测序结果见表8。
表8抗TIGIT鼠源单克隆抗体序列
Figure PCTCN2021119496-appb-000013
实施例5抗TIGIT嵌合抗体的构建
将纯化后(纯化步骤参见实施例3)的鼠抗轻链与重链可变区基因片段分别与线性化的含有人抗体轻链或重链恒定区的真核表达质粒共转化大肠杆菌DH5α 感受态细胞，将混合液均匀涂布于含有相应抗生素的琼脂平板表面，于37℃恒温培养箱过夜培养后分别挑取若干单菌落进行DNA测序；将测序正确的嵌合抗体分别标记为SHS006-17CHI、SHS006-23CHI。
将测序正确的阳性克隆接种于含有相应抗生素的2×YT液体培养基中，于37℃振荡培养12小时以上，然后收集菌体进行质粒提取，从而获得嵌合抗体轻链与重链表达质粒，使用核酸定量分析仪检测质粒的浓度与纯度。
将嵌合抗体转染HEK293E细胞，表达纯化获得大量抗体，进行纯度检测、活性分析及亲和力的检测。
嵌合抗体测序结果见表9。
表9抗TIGIT嵌合抗体序列
Figure PCTCN2021119496-appb-000014
实施例6抗TIGIT人源化抗体的构建及生产
根据嵌合抗体的活性分析、亲和力KD值等结果选择SHS006-17CHI、SHS006-23CHI进行人源化抗体改造。
抗体的人源化改造，首先是通过与免疫基因数据库(IMGT)中的小鼠抗体序列进行比对，确认SHS006-17CHI、SHS006-23CHI抗体可变区的鼠源种系，经过同源比对，SHS006-17CHI、SHS006-23CHI抗体的重链可变区序列的FR区分别与小鼠抗体种系基因IGHV2-26*01以及IGHV3-7*01最为相似；抗体轻链可变区的FR序列则分别与小鼠抗体IGKV2-28*01以及IGKV1-39*01最为相似。以SHS006-17CHI/SHS006-23CHI抗体框架区序列FR1-FR3作为模板，在人框架区库中寻找3D结构相似但是免疫原性较低的全人框架替代SHS006-17CHI/SHS006-23CHI的FR1-FR3序列，重链/轻链全长序列进行3D建模并和原抗体重链/轻链序列进行结构比对分析，综合考虑抗原性和3D结构相似度，并将在结构模拟中显示对抗体结构稳定起到关键作用的的氨基酸位点回突变为鼠源性氨基酸残基。最终选择SHS006-17CHI的5条人源化重链可变区(参见SEQ ID NO:79、80、81、82、83)和4条人源化轻链可变区(参见SEQ ID NO:84、 85、86、87)及SHS006-23CHI的5条人源化重链可变区(参见SEQ ID NO:88、89、90、91、92)和5条人源化轻链可变区(参见SEQ ID NO:93、94、95、96、97)进行下一步优化。SHS006-17CHI/SHS006-23CHI人源化抗体非CDR区序列均达到95％以上人源化。
将以上设计好的人源化抗体轻链与重链可变区氨基酸序列反转录成相对应的核苷酸序列，并生成相邻片段之间含有互补序列的寡核苷酸片段，通过Overlap PCR将寡核苷酸片段退火后连接起来，再利用特异性引物(5’端含有用于与真核表达载体发生同源重组的同源臂序列)扩增出完整的轻链与重链可变区核苷酸片段；将纯化后的轻链可变区核苷酸片段与线性化的含有IgG4轻链恒定区的真核表达质粒共转化大肠杆菌DH5α感受态细胞，将纯化后的重链可变区核苷酸片段与含S228P/L235E突变的IgG4重链恒定区的真核表达质粒共转化大肠杆菌DH5α感受态细胞，分别将转化质粒的感受态细胞均匀涂布于含有相应抗生素的琼脂平板表面，于37℃恒温培养箱过夜培养后分别挑取若干单菌落进行DNA测序。
将测序正确的阳性克隆接种于含有相应抗生素的2×YT液体培养基中，于37℃振荡培养12小时以上，然后收集菌体进行质粒提取，从而获得人源化抗体轻链与重链表达质粒，使用核酸定量分析仪检测质粒的浓度与纯度。
将质粒转染HEK293E细胞，表达纯化获得大量抗体，进行纯度检测、活性分析及亲和力的检测。
挑选纯度、活性和亲和力均较好的人源化抗体，标记为SHS006-T-Hu17-31、SHS006-T-Hu23-53，序列见表10。
表10抗TIGIT人源化抗体序列
Figure PCTCN2021119496-appb-000015
实施例7抗TIGIT抗体细胞功能测定
采用荧光素酶报告基因系统(购于BPS Bioscience公司，美国)对本发明的抗TIGIT抗体进行细胞功能测定，使用抗体22G2作为对照。
实验步骤如下：
①靶细胞准备：将CD155/TCR Activator-CHO细胞以2.5x 10 4cells/ml的密度铺板(检测孔及NTC)，100ul/孔，细胞培养板放入于37℃、5％CO2培养箱中培养过夜；
②效应细胞准备：取TIGIT/NFAT-reporter-Jurkat细胞，用分析培养基洗一遍，调整密度至4x 10 5cells/ml；
③抗体与效应细胞孵育：将按梯度稀释好的本发明抗TIGIT抗体与效应细胞(V:V＝1:1，各60μl)于37℃孵育30min；
④与靶细胞孵育：去除靶细胞培养板中的培养基，加入TIGIT/NFAT-reporter-Jurkat细胞和抗体复合物，100μl/孔，BG孔加入100ul分析培养基，将细胞培养板放入培养箱孵育5-6个小时；
⑤检测：水浴融化试剂A及试剂B，在避光的条件下，用试剂A将试剂B稀释，体积比100:1，混匀，在每孔中加入100ul一步法荧光素酶试剂，室温轻微晃动15-30min，使用荧光酶标仪检测。
根据每个样品的吸光度与抗体浓度关系绘图，以展示抗体在不同浓度下诱导荧光素酶的表达相对空白对照组的倍数，并对数据计算其Top值及EC50值。Top值及EC50值的结果如表11所示。
表11抗TIGIT抗体诱导的荧光素酶表达
Figure PCTCN2021119496-appb-000016
实验结果显示本发明的抗TIGIT抗体诱导荧光素酶表达的EC50值显著低于对照抗体22G2。这表明本发明的抗TIGIT抗体能够更有效地通过阻断TIGIT-CD155的结合来活化T细胞，在活化T细胞进而杀伤肿瘤方面表现出显著优于22G2的功能。
实施例8抗TIGIT抗体与人TIGIT结合活性测定(ELISA)
采用ELISA分析抗体的结合活性。将人TIGIT-His蛋白(1μg/孔，Sino Biological，10917-H08H)包被到96孔酶标板，37℃孵育2h。用1×PBST清洗3次后用5％的脱脂牛奶4℃封闭过夜。用1×PBST清洗3次后，本发明提供的抗TIGIT抗体作为一抗从2μg/mL开始，5倍梯度稀释加入酶标板，共8个浓度，浓度分别为2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL、0.128ng/mL、0ng/mL，37℃孵育1.5h；用1×PBST清洗5次后，二抗使用Anti-Human IgG HRP(Jackson，109-035-003，1:10000)，37℃孵育40min。用1xPBST清洗5次后，加入显色液TMB，终止后利用酶标仪(thermo，Multiskan FC)读取OD450值。使用GraphPad生成EC50，结果如表12所示。
表12抗TIGIT抗体与人TIGIT结合活性
Figure PCTCN2021119496-appb-000017
实验结果显示本发明的抗TIGIT抗体SHS006-17CHI、SHS006-T-Hu17-31、SHS006-23CHI、SHS006-T-Hu23-53均具有较好的与人TIGIT结合能力。
实施例9抗TIGIT抗体与细胞表面TIGIT结合活性测定(FACS)
采用FACS分析抗体的结合活性。hTIGIT-CHO-K1细胞以每孔1x10 5个细胞的方式铺细胞板，置于37℃，5％CO 2的条件下过夜培养；第二天用Cell stanining buffer洗涤2遍；本发明提供的抗TIGIT抗体作为一抗从2μg/mL开始，梯度稀释加入细胞板，共8个浓度，浓度分别为2000ng/mL、400ng/mL、80ng/mL、40ng/mL、20ng/mL、4ng/mL、0.8ng/mL、0.16ng/mL，阳性对照抗体为22G2，37℃孵育1h；Cell stanining buffer洗涤2遍；二抗使用PE anti-human IgG Fc(Biolegend，409304，1.2ul/孔)，4℃避光孵育30min；Cell stanining buffer洗涤2遍后利用流式细胞仪(ACEABIO，Novocyte)测定585nm波长下mean值。使用GraphPad生成EC50，结果如表13所示。
表13抗TIGIT抗体与细胞表面TIGIT结合活性
Figure PCTCN2021119496-appb-000018
Figure PCTCN2021119496-appb-000019
实验结果显示，本发明的人源化抗TIGIT抗体SHS006-T-Hu23-53、SHS006-T-Hu17-31均可与细胞表面TIGIT结合，且结合能力优于抗体22G2。
实施例10抗TIGIT抗体与人TIGIT蛋白的亲和力测定
利用Fortebio Octet对实施例1、2中制得的人源化抗TIGIT抗体结合抗原TIGIT-his的亲和力进行测定。将所述人源化抗TIGIT抗体用SD缓冲液(PBST+0.02％Tween20+0.1％BSA)稀释到浓度5μg/ml，抗原TIGIT-His用SD缓冲液4倍浓度梯度稀释，使其浓度为1μg/ml、0.25μg/ml、0.0625μg/ml、0μg/ml，选用SA传感器固化该抗原，按Fortebio Octet RED96的操作规程进行亲和力测定，具体参数及实验结果如表14所示。
表14与人TIGIT蛋白的亲和力测定
Figure PCTCN2021119496-appb-000020
实验结果显示，本发明的人源化抗TIGIT抗体SHS006-T-Hu17-31、SHS006-T-Hu23-53与人TIGIT蛋白具有较高的亲和力。
实施例11 ELISA检测阻断TIGIT与CD155的结合活性
采用ELISA分析抗体的阻断活性。将人CD155-hFc蛋白(1μg/孔)包被到96孔酶标板，37℃孵育2h。用1×PBST清洗3次后用5％的脱脂牛奶4℃封闭过夜。用1×PBST清洗3次后，本发明提供的抗TIGIT抗体与TIGIT-mFc混合作为一抗，其中抗TIGIT抗体从5μg/mL开始，梯度稀释加入酶标板，共8个浓度，浓度分别为5000ng/mL、1667ng/mL、556ng/mL、278ng/mL、139ng/mL、46ng/mL、15ng/mL、0ng/mL，阳性对照抗体为22G2，TIGIT-mFc浓度恒定为1μg/ml，37℃孵育2h；用1×PBST清洗5次后，二抗使用Goat anti mouse IgG-HRP antibody(abcam，Ab6789，1:10000)，37℃孵育40min。用1xPBST清洗5次后，加入显色液TMB，终止后利用酶标仪(thermo，Multiskan FC)读取OD450值。使 用GraphPad生成IC50，结果如表15所示。
表15抗TIGIT抗体阻断TIGIT与CD155的结合活性
Figure PCTCN2021119496-appb-000021
实验结果显示本发明的人源化抗TIGIT抗体SHS006-T-Hu23-53、SHS006-T-Hu17-31均具有阻断人TIGIT与CD155结合的能力，且阻断能力优于抗体22G2。
实施例12 FACS检测阻断TIGIT与CD155的结合活性
采用FACS检测本发明提供的抗TIGIT抗体阻断TIGIT结合到细胞表面CD155的能力。hCD155-CHO-K1阳性细胞株作为CD155提供者，在梯度稀释的抗TIGIT抗体存在的情况下，观察TIGIT-mFc与CD155-CHO-K1的结合能力。二抗使用PE Goat anti mouse IgG(Biolegend，405307，1.2ul/孔)来监测TIGIT-mFc的变化。22G2作为阻断TIGIT结合到细胞表面CD155的阳性对照。流式细胞仪(ACEABIO，Novocyte)读取585nm波长下mean值，使用GraphPad生成IC50，结果如表16所示。
表16抗TIGIT抗体阻断TIGIT与CD155的结合活性
Figure PCTCN2021119496-appb-000022
实验结果显示本发明的人源化抗TIGIT抗体SHS006-T-Hu17-31、SHS006-T-Hu23-53均具有阻断人TIGIT与CD155结合的能力，且阻断能力优于抗体22G2。
实施例13抗TIGIT/抗PD-L1双特异性抗体表达载体的构建及蛋白表达纯化
1.抗TIGIT/抗PD-L1双特异性抗体的表达载体构建
本发明所用抗PD-L1抗体是通过用人PD-L1-his蛋白免疫小鼠后筛选得到的小鼠单克隆抗体PL-7(重链可变区序列为SEQ ID NO:37，轻链可变区序列为SEQ  ID NO:41)、PL-16(重链可变区序列为SEQ ID NO:39，轻链可变区序列为SEQ ID NO:43)，将其人源化后筛选获得人源化单克隆抗体HuPL7-21(重链可变区序列SEQ ID NO:50，轻链可变区序列SEQ ID NO:54)、HuPL7-43(重链可变区序列SEQ ID NO:52，轻链可变区序列SEQ ID NO:56)、HuPL16-42(重链可变区序列SEQ ID NO:61，轻链可变区序列SEQ ID NO:64)。
本发明所用抗TIGIT抗体是通过用人TIGIT-his蛋白免疫小鼠后筛选得到的小鼠单克隆抗体T-23(重链可变区序列为SEQ ID NO:76，轻链可变区序列为SEQ ID NO:78)，将其人源化后筛选获得人源化单克隆抗体HuT23-53(重链可变区序列为SEQ ID NO:99，轻链可变区序列为SEQ ID NO:101)。
本发明利用上述抗PD-L1和抗TIGIT的人源化抗体的可变区序列，构建了涉及两种结构的抗TIGIT/抗PD-L1双特异性抗体。这两种结构分别是以ScFab为元件构成的非对称结构(如本实施例中双特异性抗体HuPL721-T2353-ScFab)及以ScFv为元件构成的对称结构(如本实施例中双特异性抗体HuPL721-T2353-ScFv)。其中ScFab结构的双特异性抗体如图1所示，具体为该双特异性抗体是由两条分别抗PD-L1和抗TIGIT的抗体单链通过异源二聚化而形成；区别于天然IgG抗体，该双特异性抗体中抗PD-L1或抗TIGIT抗体的轻链都通过额外添加的柔性连接肽连接至抗体重链N端，该连接肽包含甘氨酸(G)和丝氨酸(S)残基，包含GGGGS重复序列，优选包含8个GGGGS的重复序列；此外，为促进异源二聚体的形成，在上述S228P或S228P/L235E突变的基础上，在抗TIGIT抗体单链的CH3domain再增加S354C/T366W突变，在抗PD-L1抗体单链的CH3domain再增加Y349C/T366S/L368A/Y407V突变。ScFv结构的双特异性抗体如图2所示，具体为将TIGIT抗体的ScFv重链通过一段柔性连接肽连接至完整的抗PD-L1抗体重链C端，该连接肽包含甘氨酸(G)和丝氨酸(S)残基，包含GGGGS重复序列，优选包含8个GGGGS的重复序列。此外，在上述的两种结构双特异性抗体中，均可在重链可变区VH44位和轻链可变区VL100位同时引入半胱氨酸突变，使得其形成VH44-VL100的二硫键，以增加抗体结构的稳定性。
本发明的非对称结构双特异性抗体序列示意图:
肽链1：
Figure PCTCN2021119496-appb-000023
肽链2：
Figure PCTCN2021119496-appb-000024
本发明的对称结构双特异性抗体序列示意图:
肽链1：
Figure PCTCN2021119496-appb-000025
肽链2：
Anti-PD-L1抗体VL-CL
其中IgG4CH/Ks表示IgG4恒定区(S228P、Y349C、T366S、L368A、Y407V突变)，IgG4CH/Hs表示IgG4恒定区(S228P、S354C、T366W突变)，IgG4CH/PE表示IgG4恒定区(S228P、L235E突变)。
根据上述非对称结构形式，利用抗PD-L1抗体HuPL7-21和抗TIGIT抗体HuT23-53的可变区序列，构建双特异性抗体HuPL721-T2353-ScFab的表达载体ScFab-T2353-Knob-PHR(序列为SEQ ID NO:102所示)和HuPL721-scfab-hole-PHR(序列为SEQ ID NO:103所示)；
根据上述对称结构形式，利用抗PD-L1抗体HuPL7-21和抗TIGIT抗体HuT23-53的可变区序列，构建双特异性抗体HuPL721-T2353-ScFv的表达载体PL721-T2353-scFv-HC-PHR(序列为SEQ ID NO:104所示)和HuPL721-VL1-PHR(序列为SEQ ID NO:105所示)；
根据上述非对称结构形式，利用抗PD-L1抗体HuPL7-43和抗TIGIT抗体HuT23-53的可变区序列，构建双特异性抗体HuPL743-T2353-ScFab的表达载体ScFab-T2353-Knob-PHR(序列为SEQ ID NO:102所示)和HuPL743-scfab-hole-PHR(序列为SEQ ID NO:106所示)；
根据上述对称结构形式，利用抗PD-L1抗体HuPL7-43和抗TIGIT抗体HuT23-53的可变区序列，构建双特异性抗体HuPL743-T2353-ScFv的表达载体 PL743-T2353-scFv-HC-PHR(序列为SEQ ID NO:107所示)和HuPL743-VL3-PHR(序列为SEQ ID NO:108所示)；
根据上述非对称结构形式，利用抗PD-L1抗体HuPL16-42和抗TIGIT抗体HuT23-53的可变区序列，构建双特异性抗体HuPL1642-T2353-ScFab的表达载体ScFab-T2353-Knob-PHR(序列为SEQ ID NO:102所示)和HuPL1642-scfab-hole-PHR(序列为SEQ ID NO:109所示)；
根据上述对称结构形式，利用抗PD-L1抗体HuPL16-42和抗TIGIT抗体HuT23-53的可变区序列，构建双特异性抗体HuPL1642-T2353-ScFv的表达载体PL1642-T2353-scFv-HC-PHR(序列为SEQ ID NO:110所示)和HuPL1642-VL2-PHR(序列为SEQ ID NO:111所示)；
2.抗TIGIT/抗PD-L1双特异性抗体的表达和纯化
1)抗TIGIT/抗PD-L1双特异性抗体的表达
在1L细胞培养瓶中接种密度为1.0×10 6个/ml的HEK293E细胞，加入新鲜的预热的FreeStyle 293表达培养基，使接种后总体积达到250mL，置37℃，8％CO 2，加湿的CO 2培养箱中培养过夜。取8mL FreeStyle 293表达培养基，加入1mg/ml的PEI溶液500ul，混合均匀，取250μg待转染的表达载体加入8.0ml FreeStyle 293表达培养基中，混合均匀，其中双特异性抗体按照两载体比为1:1共同转染。将PEI与FreeStyle 293表达培养基的混合溶液加入到质粒中，混合均匀，然后加入细胞培养物中，置37℃，8％CO 2，加湿的CO 2培养箱中培养。在细胞转染后第1天和第3天对细胞进行补料，每瓶加入2.5ml的谷氨酰胺(母液浓度为200mM)、每250mL加入12.5mL的OPM-CHO PFF05和5ml的葡萄糖(母液浓度为180g/L)。当细胞细胞活力降至65％～75％时，收集细胞上清。将细胞培养物1500rpm离心5min，收集上清，再8000rpm离心20min，收集上清。
2)亲和层析柱纯化
利用AKTA(GE，AKTA pure-150)根据蛋白性质采用不同的亲和层析柱进行纯化(抗TIGIT/抗PD-L1双特异性抗体适配的亲和层析柱见表17)，具体纯化步骤如下：
表17抗TIGIT/抗PD-L1双特异性抗体适配的亲和层析柱
Figure PCTCN2021119496-appb-000026
Figure PCTCN2021119496-appb-000027
清洗：超纯水清洗设备及管路2min，流速10mL/min，后用0.1M NaOH清洗层析系统；
接柱：将层析柱接入层析设备，并用超纯水冲洗5min；后0.1M NaOH冲洗30min，保留时间5min；
平衡：20mM PB+0.15M NaCl，pH 7.2平衡5个CV(柱体积)；
上样：将细胞表达上清上样，保留时间5min；
后平衡：20mM PB+0.15M NaCl，pH 7.2平衡5个CV；
洗脱：50mM醋酸，pH＝3.4洗脱，保留时间5min。UV280至50mAu左右时开始收集，降至50mAu左右时停止收集。用1M Tris-HCl，pH 9.0将样品pH调节至7.0；
再平衡：20mM PB+0.15M NaCl，pH 7.2平衡3个CV，保留时间5min；
在线清洗：0.1M NaOH清洗30min，保留时间5min；
清洗保存：纯化水清洗10min，然后加入20％乙醇2个CV。
纯化获得的抗体HuPL721-T2353-ScFab/ScFv、HuPL743-T2353-ScFab/ScFv、HuPL1642-T2353-ScFab/ScFv经SDS-PAGE和SEC-HPLC鉴定纯度。如若纯度低于95％，则进行进一步精纯，从而获得SEC纯度＞95％的双特异性抗体分子用于后续实验。
实施例14抗TIGIT/抗PD-L1双特异性抗体与人PD-L1蛋白结合活性的测定
采用FACS法分析抗体的结合活性。hPD-L1-CHO-K1细胞以每孔1.5×10 5个细胞的方式铺细胞板；本发明提供的抗TIGIT/抗PD-L1双特异性抗体作为一抗从10μg/mL开始，梯度稀释加入细胞板，共8个浓度，浓度分别为10000ng/mL、2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL、0.128ng/mL，4℃孵育1.5h；稀释液(PBS+2％FBS)洗涤2遍；二抗使用PE anti-human IgG Fc(Abcam，ab98596，0.8ul/孔)，4℃避光孵育60min；稀释液洗涤2遍后利用流式细胞仪(ACEABIO，Novocyte)测定585nm波长下mean值。数据处理时将质量浓度换算为摩尔浓度后使用GraphPad生成EC50，结果如表18所示。
表18抗TIGIT/抗PD-L1双特异性抗体与人PD-L1蛋白结合活性
Figure PCTCN2021119496-appb-000028
实验结果显示本发明提供的抗TIGIT/抗PD-L1双特异性抗体HuPL721-T2353-ScFab、HuPL1642-T2353-ScFv、HuPL721-T2353-ScFv和HuPL743-T2353-ScFv均具有较好的与PD-L1结合能力。
实施例15抗TIGIT/抗PD-L1双特异性抗体与人TIGIT结合活性的测定
采用FACS法分析抗体的结合活性。hTIGIT-CHO-K1细胞以每孔1.5×10 5个细胞的方式铺细胞板；本发明提供的抗TIGIT/抗PD-L1双特异性抗体作为一抗从2μg/mL开始，梯度稀释加入细胞板，共8个浓度，浓度分别为2000ng/mL、400ng/mL、80ng/mL、40ng/mL、20ng/mL、4ng/mL、0.8ng/mL、0.16ng/mL，4℃孵育1.5h；稀释液(PBS+2％FBS)洗涤2遍；二抗使用PE anti-human IgG Fc(Abcam，ab98596，0.8ul/孔)，4℃避光孵育60min；稀释液洗涤2遍后利用流式细胞仪(ACEABIO，Novocyte)测定585nm波长下mean值。数据处理时将质量浓度换算为摩尔浓度后使用GraphPad生成EC50，结果如表19所示。
表19抗TIGIT/抗PD-L1双特异性抗体与人TIGIT结合活性
Figure PCTCN2021119496-appb-000029
实验结果显示本发明的抗TIGIT/抗PD-L1双特异性抗体HuPL721-T2353-ScFab、HuPL721-T2353-ScFv均具有较好的与PD-L1结合的能力。
实施例16抗TIGIT/抗PD-L1双特异性抗体阻断PD-L1与PD-1结合的活性检测
采用FACS法分析抗体的结合活性。hPD-L1-CHO-K1细胞以每孔2.5×10 5个细胞的方式铺细胞板；本发明提供的抗TIGIT/抗PD-L1双特异性抗体梯度稀释后与终浓度为2μg/ml的PD-1-mFc混合均匀后作为一抗，其中抗体的梯度稀释方法为：从20μg/mL开始，3倍梯度稀释加入细胞板，共8个浓度，浓度分别为 20000ng/mL、6666.66ng/mL、2222.22ng/mL、740.74ng/mL、246.9ng/mL、82.3ng/mL、27.4ng/mL、9.14ng/mL，4℃孵育1.5h；稀释液(PBS+2％FBS)洗涤2遍；二抗使用PE anti-Mouse IgG Fc(Biolegend，405307，0.8ul/孔)，4℃避光孵育60min；稀释液洗涤2遍后利用流式细胞仪(ACEABIO，Novocyte)测定585nm波长下mean值。数据处理时将质量浓度换算为摩尔浓度后使用GraphPad生成IC50，结果如表20所示。
表20抗TIGIT/抗PD-L1双特异性抗体阻断PD-L1与PD-1结合的活性
Figure PCTCN2021119496-appb-000030
实验结果显示本发明的抗TIGIT/抗PD-L1双特异性抗体HuPL721-T2353-ScFab、HuPL721-T2353-ScFv均具有阻断PD-L1与PD-1结合的能力。
实施例17抗TIGIT/抗PD-L1双特异性抗体阻断TIGIT与CD155结合的活性检测
采用FACS法分析抗体的阻断活性。hTIGIT-CHO-K1细胞以每孔2.5×10 5个细胞的方式铺细胞板；本发明提供的抗TIGIT/抗PD-L1双特异性抗体梯度稀释后与终浓度为0.4μg/ml的TIGIT-mFc混合均匀后作为一抗，其中抗体的梯度稀释方法为：从20μg/mL开始，梯度稀释加入细胞板，共8个浓度，浓度分别为20000ng/mL、4000ng/mL、800ng/mL、400ng/mL、200ng/mL、100ng/mL、33.33ng/mL、11.11ng/mL，4℃孵育1.5h；稀释液(PBS+2％FBS)洗涤2遍；二抗使用PE anti-Mouse IgG Fc(Biolegend，405307，0.8ul/孔)，4℃避光孵育60min；稀释液洗涤2遍后利用流式细胞仪(ACEABIO，Novocyte)测定585nm波长下mean值。数据处理时将质量浓度换算为摩尔浓度后使用GraphPad生成IC50，结果如表21所示。
表21抗TIGIT/抗PD-L1双特异性抗体阻断TIGIT与CD155结合的活性
Figure PCTCN2021119496-appb-000031
实验结果显示本发明的抗TIGIT/抗PD-L1双特异性抗体HuPL721-T2353-ScFab、HuPL721-T2353-ScFv、HuPL1642-T2353-ScFab和HuPL1642-T2353-ScFv均具有较好的阻断TIGTI与CD155结合的能力。
实施例18抗TIGIT/抗PD-L1双特异性抗体的亲和力检测
利用Fortebio Octet对实施例15中制得的人源化抗TIGIT/抗PD-L1抗体结合抗原PD-L1-his和TIGIT-his的亲和力分别进行测定。先将抗体HuPL721-T2353-ScFab/ScFv、HuPL743-T2353-ScFab/ScFv、HuPL1642-T2353-ScFab/ScFv稀释到67nM，选用AHC传感器固化上述抗体，将PD-L1-his用SD缓冲液(0.02％Tween20+0.1％BSA溶液)梯度稀释到2μg/ml、0.4μg/ml、0.08μg/ml，TIGIT-his用SD缓冲液梯度稀释到0.5μg/ml、0.1μg/ml、0.02μg/ml，按Fortebio Octet RED96的操作规程进行亲和力测定，具体参数及实验结果如表22、表23所示。
表22抗TIGIT/抗PD-L1双特异性抗体与人PD-L1蛋白的亲和力测定
Figure PCTCN2021119496-appb-000032
表23抗TIGIT/抗PD-L1双特异性抗体与人TIGIT蛋白的亲和力测定
Figure PCTCN2021119496-appb-000033
Figure PCTCN2021119496-appb-000034
实验结果显示，本发明的抗TIGIT/抗PD-L1双特异性抗体HuPL1642-T2353-ScFab、HuPL1642-T2353-ScFv、HuPL721-T2353-ScFab和HuPL721-T2353-ScFv具有较好的与人PD-L1蛋白的亲和力以及与人TIGIT蛋白的亲和力。
实施例19抗TIGIT/抗PD-L1双特异性抗体的体外药效检测
采用SEB(金黄色葡萄球菌肠毒素B)两次刺激PBMC(外周血单核细胞)的方法来检测本发明的双特异性抗体的体外药效。
按所需细胞量复苏PBMC，加到8-9ml的IMDM完全培养基中，1200rpm离心10min，弃上清；用适量培养基重悬，用血球计数板计数，加入到6孔板中，同时加入终浓度为100ng/ml的SEB溶液，孵育48h；48h后，1200rpm离心10min，弃上清，使用IMDM完全培养基洗涤1-2次，用适量培养基重悬，用血球计数板计数，并重悬为1M/mL，100μL/孔加入96孔板中；按4倍浓度(即80μg/mL)，50μL/孔，用IMDM完全培养基配制对照IgG4(Isotype)，做好标记，涡旋；将抗体溶液加入对应孔中，对照组加入50μL/孔的培养基，96孔板置于37℃孵箱中，细胞和抗体孵育1h；1h后，按4倍浓度(400ng/ml)，50μL/孔用量，用IMDM完全培养基配制SEB溶液，加入对应孔中；96孔板置于37℃，5％CO 2孵箱中孵育48h后，离心去除上清收集无细胞上清150μL，按一定比例稀释后，按照hIFN-γ(R&D system Cat：DY285B)ELISA检测试剂盒说明书检测上清中IFN-γ的浓度。结果如表24所示。
表24本发明抗体对IFN-γ释放的影响
Figure PCTCN2021119496-appb-000035
Figure PCTCN2021119496-appb-000036
实验结果显示，本发明的抗体具有较好的促IFN-γ释放能力。抗体HuPL16-42和HuPL7-21与抗体HuT23-53联用效果优于抗体单用。双特异性抗体HuPL721-2353促IFN-γ释放具有剂量依赖性。HuPL721-T2353-ScFv在一半摩尔数的情况下依然优于联用组[对于单抗联用组20μg/ml+20μg/ml、scFab双抗组40μg/ml和scFv双抗组26.8μg/ml，这三组浓度是针对TIGIT和PD-L1两种抗原结合臂的数量均相等的浓度(称“等臂”浓度)；相对于单抗联用组20μg/ml+20μg/ml，scFab双抗组20μg/ml和scFv双抗组13.4μg/ml的浓度相当于是结合臂数量减半的浓度；从摩尔数的角度来说，单抗联用组20μg/ml+20μg/ml中两种分子的总摩尔数与scFab双抗组40μg/ml的摩尔数相等，scFv双抗组26.8ul/ml摩尔数仅为联用组20μg/ml+20μg/ml和scFab双抗组40μg/ml的一半]。
实施例20抗PD-L1/抗TIGIT双特异性抗体对小鼠体内移植瘤生长抑制的检测(Raji-PBMC-NSG模型)
本发明利用NSG小鼠(购自北京百奥赛图基因生物技术有限公司，中国)、Raji-PD-L1肿瘤细胞(购自宜明昂科生物医药技术有限公司，中国)建立肿瘤移植模型——Raji-PBMC-NSG模型，研究本发明的抗体在Raji-hPD-L1淋巴瘤皮下移植模型中的抗肿瘤作用。抗PD-L1阳性对照抗体为Atezolizumab(Sino Biological，Cat:68049-H001)。表25为测试药物在Raji-PBMC-NSG肿瘤模型中的抗肿瘤作用实验设计方案。
表25Raji-PBMC-NSG模型测试药物实验设计方案
Figure PCTCN2021119496-appb-000037
*：N是指每组小鼠的数量；i.p.：腹腔注射；BIW指每周给药2次。
各组的肿瘤抑制率(TGI TV)％如表26所示。
表26各抗体对Raji-PBMC-NSG模型小鼠肿瘤体积的影响
Figure PCTCN2021119496-appb-000038
本发明的抗体HuPL7-21、HuPL721-T2353-ScFab体内抑瘤效果显著优于Atezolizumab。
实施例21抗PD-L1/抗TIGIT双特异性抗体对小鼠体内移植瘤生长抑制的检测(MC38-hPD-L1结肠癌模型)
采用B-hPD-L1/hTIGIT双人源化小鼠(购于北京百奥赛图基因生物技术有限公司，中国)MC38-hPD-L1结肠癌(细胞购于舜冉上海生物科技有限公司，中国)的动物模型进行药效实验。将PBS重悬的MC38-hPD-L1结肠癌细胞以5×10 5个/0.1mL浓度，0.1mL/只体积接种于B-hPD-L1/hTIGIT人源化小鼠的右侧皮下。当平均肿瘤体积达到98mm 3时，根据小鼠肿瘤体积和体重选择合适的小鼠入组，平均分配到6个实验组中，每组8只，分组当天开始给药，具体给药方案见表27。
表27 B-hPD-L1/hTIGIT双人源化小鼠MC38-hPD-L1结肠癌动物模型实验给药方案
Figure PCTCN2021119496-appb-000039
注：PBS：生理盐水对照组；
a：给药体积依实验动物体重按10μL/g计算；
b：BIW指每周给药2次。
记录结果并计算肿瘤抑制率(TGI TV)。
在实验终点(即分组给药第24天)，Atezolizumab在3mg/kg剂量下的抑瘤率为35.3％；HuPL721-T2353-ScFab在3.14mg/kg和6.27mg/kg剂量下的抑瘤率分别为30.6％和50.7％。实验结果表明抗TIGIT/抗PD-L1双特异性抗体HuPL721-T2353-ScFab对MC38-hPD-L1肿瘤皮下移植瘤生长有明显的抑制作用，且具有剂量依赖性。
以上实施例证明本发明提供的抗TIGIT抗体、抗PD-L1抗体、抗TIGIT/抗PD-L1双特异性抗体可明显抑制肿瘤增长，具有显著的抗肿瘤作用，提示这些抗体可在制备抗肿瘤药物中应用，具有好的市场前景。
尽管以上已经对本发明作了详细描述，但是本领域技术人员理解，在不偏离本发明的精神和范围的前提下可以对本发明进行各种修改和改变。本发明的权利范围并不限于上文所作的详细描述，而应归属于权利要求书。

Claims (20)

  1. 一种抗TIGIT抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
    (1)所述重链可变区包含选自如下组的H1CDR1、H1CDR2和H1CDR3：
    (a1)如SEQ ID NO：25、26和27所示的氨基酸序列；
    (a2)如SEQ ID NO：28、29和30所示的氨基酸序列；和
    (a3)与(a1)或(a2)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
    (2)所述轻链可变区包含选自如下组的L1CDR1、L1CDR2和L1CDR3：
    (a4)如SEQ ID NO：31、32和33所示的氨基酸序列；
    (a5)如SEQ ID NO：34、35和36所示的氨基酸序列；和
    (a6)与(a4)或(a5)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。
  2. 如权利要求1所述的抗TIGIT抗体或其抗原结合片段，其具有：
    所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO：25、26和27或与SEQ ID NO：25、26和27所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO：31、32和33或与SEQ ID NO：31、32和33所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的轻链可变区；或
    所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO：28、29和30或与SEQ ID NO：28、29和30所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ IDNO：34、35和36或与SEQ ID NO：34、35和36所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的轻链可变区。
  3. 如权利要求1或2所述的抗TIGIT抗体或其抗原结合片段，其中：
    (1)所述重链可变区的氨基酸序列选自：
    (b1)如SEQ ID NO：75、SEQ ID NO：76、SEQ ID NO：79、SEQ ID NO：80、SEQ ID NO：81、SEQ ID NO：82、SEQ ID NO：83、SEQ ID NO：88、SEQ ID NO：89、SEQ ID NO：90、SEQ ID NO：91、SEQ ID NO：92、SEQ ID NO：98或SEQ ID NO：99所示的氨基酸序列；
    (b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
    (2)所述轻链可变区的氨基酸序列选自：
    (b4)如SEQ ID NO：77、SEQ ID NO：78、SEQ ID NO：84、SEQ ID NO：85、SEQ  ID NO：86、SEQ ID NO：87、SEQ ID NO：93、SEQ ID NO：94、SEQ ID NO：95、SEQ ID NO：96、SEQ ID NO：97、SEQ ID NO：100或SEQ ID NO：101所示的氨基酸序列；
    (b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
  4. 如权利要求1-3之任一项所述的抗TIGIT抗体或其抗原结合片段，其中
    所述重链可变区的氨基酸序列为SEQ ID NO：75，SEQ ID NO：75经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO：75功能相同的氨基酸序列或与SEQ ID NO：75具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO：77，SEQ ID NO：77经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO：77功能相同的氨基酸序列或与SEQ ID NO：77具有至少85％序列同一性的氨基酸序列；
    所述重链可变区的氨基酸序列为SEQ ID NO：76，SEQ ID NO：76经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO：76功能相同的氨基酸序列或与SEQ ID NO：76具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO：78，SEQ ID NO：78经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO：78功能相同的氨基酸序列或与SEQ ID NO：78具有至少85％序列同一性的氨基酸序列；
    所述重链可变区的氨基酸序列为SEQ ID NO：98，SEQ ID NO：98经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO：98功能相同的氨基酸序列或与SEQ ID NO：98具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO：100，SEQ ID NO：100经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO：100功能相同的氨基酸序列或与SEQ ID NO：100具有至少85％序列同一性的氨基酸序列；或者
    所述重链可变区的氨基酸序列为SEQ ID NO：99，SEQ ID NO：99经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO：99功能相同的氨基酸序列或与SEQ ID NO：99具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO：101，SEQ ID NO：101经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO：101功能相同的氨基酸序列或与SEQ ID NO：101具有至少85％序列同一性的氨基酸序列。
  5. 一种抗TIGIT/抗PD-L1抗体，其包括抗TIGIT抗体或其抗原结合片段和抗PD-L1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包含第一重链可变区和第一轻链可变区，其中：
    (1)所述第一重链可变区包含选自如下组的H1CDR1、H1CDR2和H1CDR3：
    (a1)如SEQ ID NO：25、26和27所示的氨基酸序列；
    (a2)如SEQ ID NO：28、29和30所示的氨基酸序列；和
    (a3)与(a1)或(a2)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
    (2)所述第一轻链可变区包含选自如下组的L1CDR1、L1CDR2和L1CDR3：
    (a4)如SEQ ID NO：31、32和33所示的氨基酸序列；
    (a5)如SEQ ID NO：34、35和36所示的氨基酸序列；和
    (a6)与(a4)或(a5)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
    所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和第二轻链可变区，其中：
    (1)所述第二重链可变区包含选自如下组的H2CDR1、H2CDR2和H2CDR3：
    (A1)如SEQ ID NO：1、2和3所示的氨基酸序列；
    (A2)如SEQ ID NO：4、5和6所示的氨基酸序列；
    (A3)如SEQ ID NO：7、8和9所示的氨基酸序列；和
    (A4)如SEQ ID NO：10、11和12所示的氨基酸序列；
    (A5)与(A1)、(A2)、(A3)或(A4)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
    (2)所述第二轻链可变区包含选自如下组的L2CDR1、L2CDR2和L2CDR3：
    (A6)如SEQ ID NO：13、14和15所示的氨基酸序列；
    (A7)如SEQ ID NO：16、17和18所示的氨基酸序列；
    (A8)如SEQ ID NO：19、20和21所示的氨基酸序列；
    (A9)如SEQ ID NO：22、23和24所示的氨基酸序列；
    (A10)与(A6)、(A7)、(A8)或(A9)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。
  6. 如权利要求5所述的抗TIGIT/抗PD-L1抗体，其中
    所述抗TIGIT抗体或其抗原结合片段包含所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO：28、29和30或与SEQ ID NO：28、29和30所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO：34、35和36或与SEQ ID NO：34、35和36所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一轻链可变区。
  7. 如权利要求5或6所述的抗TIGIT/抗PD-L1抗体，其中
    所述抗PD-L1抗体或其抗原结合片段包含所述H2CDR1、H2CDR2和H2CDR3分别为SEQ ID NO：1、2和3或与SEQ ID NO：1、2和3所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二重链可变区，和所述L2CDR1、L2CDR2和L2CDR3分别为SEQ ID NO：13、14和15或与SEQ ID NO：13、14和15所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二轻链可变区；或者
    所述抗PD-L1抗体或其抗原结合片段包含所述H2CDR1、H2CDR2和H2CDR3分别为SEQ ID NO：4、5和6或与SEQ ID NO：4、5和6所示的氨基酸序列具有至 少85％序列同一性的氨基酸序列的第二重链可变区，和所述L2CDR1、L2CDR2和L2CDR3分别为SEQ ID NO：16、17和18或与SEQ ID NO：16、17和18所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二轻链可变区；或者
    所述抗PD-L1抗体或其抗原结合片段包含所述H2CDR1、H2CDR2和H2CDR3分别为SEQ ID NO：7、8和9或与SEQ ID NO：7、8和9所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二重链可变区，和所述L2CDR1、L2CDR2和L2CDR3分别为SEQ ID NO：19、20和21或与SEQ ID NO：19、20和21所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二轻链可变区。
  8. 如权利要求5-7之任一项所述的抗TIGIT/抗PD-L1抗体，其中：
    所述抗TIGIT抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区，其中：
    (1)所述第一重链可变区的氨基酸序列选自：
    (b1)如SEQ ID NO：98或SEQ ID NO：99所示的氨基酸序列；
    (b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
    (2)所述第一轻链可变区的氨基酸序列选自：
    (b4)如SEQ ID NO：100或SEQ ID NO：101所示的氨基酸序列；
    (b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
    所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和第二轻链可变区，其中：
    (1)所述第二重链可变区的氨基酸序列选自：
    (B1)如SEQ ID NO：37、SEQ ID NO：38、SEQ ID NO：39、SEQ ID NO：40、SEQ ID NO：50、SEQ ID NO：52或SEQ ID NO：61所示的氨基酸序列；
    (B2)(B1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(B1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (B3)与(B1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
    (2)所述第二轻链可变区的氨基酸序列选自：
    (B4)如SEQ ID NO：41、SEQ ID NO：42、SEQ ID NO：43、SEQ ID NO：44、SEQ ID NO：54、SEQ ID NO：56或SEQ ID NO：64所示的氨基酸序列；
    (B5)(B4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(B4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (B6)与(B4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
  9. 如权利要求5-8之任一项所述的抗TIGIT/抗PD-L1抗体，其中：
    所述第一重链可变区的氨基酸序列为SEQ ID NO：99，SEQ ID NO：99经取代、 缺失或添加一个或多个氨基酸获得的且与SEQ ID NO：99功能相同的氨基酸序列或与SEQ ID NO：99具有至少85％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO：28、29和30所示的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO：101，SEQ ID NO：101经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO：101功能相同的氨基酸序列或与SEQ ID NO：101具有至少85％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO：34、35和36所示的氨基酸序列。
  10. 如权利要求5-9之任一项所述的抗TIGIT/抗PD-L1抗体，其中：
    所述第二重链可变区的氨基酸序列为SEQ ID NO：50所示的氨基酸序列，SEQ ID NO：50经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO：50功能相同的氨基酸序列或与SEQ ID NO：50具有至少85％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO：1、2和3所示的氨基酸序列，且所述第二轻链可变区的氨基酸序列为SEQ ID NO：54，SEQ ID NO：54经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO：54功能相同的氨基酸序列或与SEQ ID NO：54具有至少85％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO：13、14和15所示的氨基酸序列；
    所述第二重链可变区的氨基酸序列为SEQ ID NO：52所示的氨基酸序列，SEQ ID NO：52经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO：52功能相同的氨基酸序列或与SEQ ID NO：52具有至少85％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO：1、2和3所示的氨基酸序列，且所述第二轻链可变区的氨基酸序列为SEQ ID NO：56，SEQ ID NO：56经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO：56功能相同的氨基酸序列或与SEQ ID NO：56具有至少85％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO：13、14和15所示的氨基酸序列；或者
    所述第二重链可变区的氨基酸序列为SEQ ID NO：61所示的氨基酸序列，SEQ ID NO：61经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO：61功能相同的氨基酸序列或与SEQ ID NO：61具有至少85％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO：7、8和9所示的氨基酸序列，且所述第二轻链可变区的氨基酸序列为SEQ ID NO：64，SEQ ID NO：64经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO：64功能相同的氨基酸序列或与SEQ ID NO：64具有至少85％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO：19、20和21所示的氨基酸序列。
  11. 如权利要求5-10之任一项所述的抗TIGIT/抗PD-L1抗体，其中所述抗体是双特异性抗体。
  12. 如权利要求1-4之任一项所述的抗TIGIT抗体或如权利要求5-11之任一项所述的抗TIGIT/抗PD-L1抗体，其中所述抗体是人源化抗体或完全人抗体。
  13. 一种分离的核酸，其编码如权利要求1-4之任一项所述的抗TIGIT抗体或如权利要求5-11之任一项所述的抗TIGIT/抗PD-L1抗体。
  14. 如权利要求13所述的核酸，其包含：
    (1)编码第一重链可变区如SEQ ID NO：75、SEQ ID NO：76、SEQ ID NO：98或SEQ ID NO：99的核苷酸序列；
    (2)编码第一轻链可变区如SEQ ID NO：77、SEQ ID NO：78、SEQ ID NO：100或SEQ ID NO：101的核苷酸序列；和/或
    (3)编码第二重链可变区如SEQ ID NO：50、SEQ ID NO：52或SEQ ID NO：61的核苷酸序列；
    (4)编码第二轻链可变区如SEQ ID NO：54、SEQ ID NO：56或SEQ ID NO：64的核苷酸序列。
  15. 一种表达载体，其包含如权利要求13或14所述的核酸。
  16. 一种宿主细胞，其转化如权利要求15所述的表达载体，所述宿主细胞选自原核细胞和真核细胞，优先为哺乳动物细胞。
  17. 制备权利要求1-4之任一项所述的抗TIGIT抗体或权利要求5-11之任一项所述的抗TIGIT/抗PD-L1抗体的方法，包括在如权利要求16所述的宿主细胞中表达抗体，以及从宿主细胞中分离所述抗体的步骤。
  18. 一种药物组合物，其包含权利要求1-4之任一项所述的抗TIGIT抗体或权利要求5-11之任一项所述的抗TIGIT/抗PD-L1抗体和药学可接受的载体。
  19. 如权利要求1-4之任一项所述的抗TIGIT抗体或权利要求5-11之任一项所述的抗TIGIT/抗PD-L1抗体或如权利要求18的药物组合物在制备用于抑制TIGIT和/或PD-L1活性的药物中的应用。
  20. 如权利要求19所述的应用，所述抑制TIGIT和/或PD-L1活性的药物用于治疗肿瘤。
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