WO2023045370A1 - 靶向tigit的单克隆抗体 - Google Patents

靶向tigit的单克隆抗体 Download PDF

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WO2023045370A1
WO2023045370A1 PCT/CN2022/094422 CN2022094422W WO2023045370A1 WO 2023045370 A1 WO2023045370 A1 WO 2023045370A1 CN 2022094422 W CN2022094422 W CN 2022094422W WO 2023045370 A1 WO2023045370 A1 WO 2023045370A1
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sequence
amino acid
acid sequence
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陈羿
蔡则玲
何巧巧
王雅玲
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Shanghai Celgen Bio Pharmaceutical Co Ltd
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Shanghai Celgen Bio Pharmaceutical Co Ltd
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Priority to CN202280053939.7A priority patent/CN117999284A/zh
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Definitions

  • This disclosure is in the fields of biotechnology and medicine. Specifically, the present disclosure relates to antibodies or antigen-binding fragments thereof capable of binding to human TIGIT (T cell immunoreceptor with immunoglobulin and ITIM domains), and encoding nucleic acid molecules, vectors and Host cells, immunoconjugates and compositions comprising the antibodies or fragments, and applications of the above products.
  • human TIGIT T cell immunoreceptor with immunoglobulin and ITIM domains
  • T cells are the key mediators of the immune response, and the activation of T cells depends on TCR signaling and co-stimulatory signals.
  • some co-stimulatory signals play a positive stimulating role, and some perform a negative regulatory function, that is, the immune response is not only positively regulated by co-stimulatory molecules (such as OX40, GITR and 4-1BB), but also by immune response.
  • Responses are negatively regulated by checkpoint molecules such as PD-1 and CTLA-4 (Rudd C.E., Unifying concepts in CD28, ICOS and CTLA4 co-receptor signaling.
  • Tumor immunotherapy is the frontier and hot field of basic and clinical research on tumor treatment in recent years.
  • the first-generation immune checkpoint inhibitors represented by CTLA-4 and PD-1/PD-L1 monoclonal antibodies, can exert anti-tumor effects by activating T cell-mediated immune responses.
  • the first generation of immune checkpoint antibodies still faces problems such as limited benefit patients, poor efficacy of most tumors, and drug resistance.
  • a considerable number of patients with effective early treatment may experience tumor recurrence during or after treatment.
  • it is crucial to discover and validate new immune checkpoint inhibitory molecules.
  • T cell immunoreceptor with immunoglobulin and ITIM domains TAGIT, also known as WUCAM, Vstm3 or VSIG9
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • TIGIT is known to interact with CD155 (also known as PVR and necl-5), CD112 (also known as PVRL2 and cohesin-2), and possibly CD113 (also known as PVRL3 and cohesin-3), where CD155 is The high-affinity core ligand of TIGIT, its interaction with TIGIT is much higher than other paired molecules in the pathway (Martinet et al. Nat. Rev. Immunol. 15:243-254, 2015).
  • TIGIT can inhibit the body's immune response through various mechanisms.
  • TCR T cell antigen receptor
  • TIGIT directly blocks the activation, proliferation and acquisition of effector functions of naive T cells (Tn), and can inhibit the cell proliferation of CD4+ T cells and the production of inflammatory cytokines Generated (Fourcade J et al., JCI Insight, 2018, 3(14):e121157).
  • TIGIT can indirectly suppress T cells by modulating cytokine production by dendritic cells (Yu et al., Nat. Immunol. 10:48-57, 2009).
  • TIGIT can also enhance the stability of Tregs and its inhibitory function on the proliferation of IFN- ⁇ -producing T cells (Fourcade J et al., JCI Insight, 2018, 3(14): e121157).
  • TIGIT is highly expressed on the surface of NK cells and effector T cells related to tumor infiltration or migration, and CD155 is highly expressed on the surface of tumor cells. Tumor cells directly act on NK cells and effector T cells through the combination of CD155/TIGIT, inhibiting Its activity (Li et al., J. Biol. Chem. 289: 17647-17657, 2014).
  • TIGIT is a potential new target for immunotherapy for enhancing immune response and preventing and/or treating tumors, infections or infectious diseases.
  • CD155-blocking antibodies targeting TIGIT especially anti-human TIGIT antibodies, can release the tumor-killing activity of immune effector cells, and are expected to achieve good anti-tumor efficacy.
  • Indications include small cell lung cancer, esophageal cancer, and esophageal squamous cell carcinoma , non-small cell lung cancer, squamous cell carcinoma of the head and neck, liver cancer, cervical cancer, triple-negative breast tumors, B-cell lymphoma, multiple myeloma, non-Hodgkin's lymphoma, melanoma, ovarian cancer, colon cancer, breast cancer, etc.
  • TIGIT antibodies are in clinical trials, including Roche's Tiragolumab, which has entered phase III clinical trials.
  • TIGIT-blocking monoclonal antibody drug on the market at home and abroad, so it is urgent to develop a TIGIT monoclonal antibody with high affinity, high specificity and better effect.
  • the present disclosure just provides a TIGIT monoclonal antibody with high affinity, high specificity and better effect.
  • monoclonal antibodies or antigen-binding fragments thereof targeting T cell immunoglobulin and ITIM structural protein (TIGIT) that compete with CD155 for binding to TIGIT are provided.
  • monoclonal antibodies or antigen-binding fragments thereof targeting T cell immunoglobulin and ITIM domain proteins comprising specific heavy chain complementarity determining regions (VH) disclosed herein are provided.
  • nucleic acid molecules encoding the disclosed monoclonal antibodies or antigen-binding fragments thereof, or vectors or host cells comprising such nucleic acid molecules are provided.
  • an immunoconjugate comprising a monoclonal antibody of the present disclosure or an antigen-binding fragment thereof is provided.
  • composition comprising a monoclonal antibody or antigen-binding fragment thereof, nucleic acid molecule, vector or host cell or immunoconjugate thereof of the present disclosure is provided.
  • a chimeric antigen receptor comprising an extracellular domain and an intracellular domain, wherein the extracellular domain comprises the monoclonal antibody of the present application or an antigen-binding fragment thereof.
  • nucleic acid molecules and constructs or vectors comprising the nucleic acid molecules are provided, wherein the nucleic acid molecules comprise the coding sequence of the chimeric antigen receptor of the present application.
  • a transformed immune cell which expresses a chimeric antigen receptor according to the present application, or is transformed with a nucleic acid molecule, construct or vector of the present application, for example, the immune cell is selected from T Cells such as ⁇ T cells, ⁇ T cells or NK T cells or T cells derived from pluripotent cells.
  • monoclonal antibodies or antigen-binding fragments thereof nucleic acid molecules, vectors or host cells of the present disclosure, immunoconjugates, chimeric antigen receptors of the present disclosure or nucleic acid molecules encoding them, constructs
  • a method for positively regulating immune cell activity and/or improving immune response which includes administering to a subject in need the monoclonal antibody or antigen-binding fragment thereof, nucleic acid molecule, Vectors or host cells, immunoconjugates, chimeric antigen receptors of the present disclosure or nucleic acid molecules encoding them, constructs or vectors, transformed immune cells and/or compositions of the present disclosure, e.g. for preventing and/or Treatment of tumors, infections or infectious diseases.
  • a monoclonal antibody of the present disclosure for positively regulating immune cell activity and/or improving immune response (for example, for preventing and/or treating tumors, infections or infectious diseases) or an antigen-binding fragment thereof, a nucleic acid molecule, a vector or a host cell, an immunoconjugate, a chimeric antigen receptor of the disclosure or a nucleic acid molecule encoding it, a construct or a vector, a transformed immune cell, and/or a combination of the disclosure things.
  • monoclonal antibodies or antigen-binding fragments thereof nucleic acid molecules, vectors or host cells of the present disclosure, immunoconjugates, chimeric antigen receptors of the present disclosure or nucleic acid molecules encoding them, constructs Use of the body or vector, transformed immune cells, and/or the composition of the present disclosure in the manufacture of a product that reduces or eliminates TIGIT-expressing cells (eg, Treg cells).
  • TIGIT-expressing cells eg, Treg cells
  • a method for reducing or eliminating TIGIT-expressing cells (such as Treg cells) is provided, which includes administering to a subject in need a monoclonal antibody or an antigen-binding fragment thereof, a nucleic acid molecule, or a carrier of the present disclosure Or host cells, immunoconjugates, chimeric antigen receptors of the disclosure or nucleic acid molecules encoding them, constructs or vectors, transformed immune cells, and/or compositions of the disclosure.
  • monoclonal antibodies or antigen-binding fragments thereof nucleic acid molecules, vectors or host cells, immunoconjugates, A chimeric antigen receptor of the present disclosure or its encoding nucleic acid molecule, construct or vector, a transformed immune cell and/or a composition of the present disclosure.
  • Figure 1 shows the SDS-PAGE of eight anti-human TIGIT mouse monoclonal antibodies (No. Electrophoresis analysis research: upper panel: non-reducing 4-12% SDS-PAGE electrophoresis analysis; lower panel: reducing 4-12% SDS-PAGE electrophoresis analysis.
  • the molecular weight of the non-reduced antibody is about 150KD, and the molecular weight of the reduced antibody is about 50KD and 25KD respectively.
  • FIG. 2 shows the in vitro and recombination of eight anti-human TIGIT mouse monoclonal antibodies (No. ELISA study of human TIGIT binding.
  • FIG. 3 shows the pairing of 8 anti-human TIGIT mouse monoclonal antibodies (No. ELISA study of competition for binding of recombinant human TIGIT.
  • FIG. 4 shows eight anti-human TIGIT mouse monoclonal antibodies (No. ELISA study of TIGIT binding in cynomolgus monkeys.
  • Figure 5 shows flow cytometry of five anti-human TIGIT mouse monoclonal antibodies (No. 7103-01, 7103-03, 7103-04, 7105-06, 7103-07) bound to the membrane TIGIT of CHO cells overexpressing TIGIT Technical analysis research.
  • Figure 6 shows five anti-human TIGIT human-mouse chimeric monoclonal antibodies (No. hFc)) SDS-PAGE electrophoresis analysis. Among them, the molecular weight of the non-reduced antibody is about 150KDa, and the molecular weight of the reduced antibody is 55kDa and 25KDa respectively.
  • Figure 7 shows five anti-human TIGIT human-mouse chimeric monoclonal antibodies (No. hFc), 7103-07(hFc)) combined with recombinant human TIGIT in vitro by ELISA.
  • Figure 8 shows five strains of human-mouse chimeric TIGIT monoclonal antibodies (No. , 7103-07 (hFc)) combined with membrane TIGIT flow cytometry analysis.
  • Figure 9 shows five human-mouse chimeric TIGIT monoclonal antibodies (No. , 7103-07(hFc)) competition with CD155 for binding of recombinant human TIGIT by ELISA.
  • Figure 10 shows the ELISA of the competition of anti-human TIGIT murine monoclonal antibodies (No. 7103-01, 7103-03, 7103-04, 7105-06, 7103-07) with tisrelumab for the binding of recombinant human TIGIT Research.
  • the isotype in the figure is the negative control of the isotype antibody.
  • Figure 11 shows the three anti-human TIGIT mouse monoclonal antibodies (No. ELISA study of the competition of , 7103-04(hFc) for binding of recombinant human TIGIT.
  • Figure 12 shows 5 strains of human-mouse chimeric TIGIT monoclonal antibody (No. , 7103-07(hFc)) promotes the production of IFN ⁇ by PHA-activated PBMCs.
  • Figure 13 shows the ELISA study on the binding of six 7103-01 anti-human TIGIT humanized monoclonal antibodies (numbers P03486, P03487, P03490, P03491, P03492, P03493) to recombinant human TIGIT in vitro.
  • Human-mouse chimeric 7301-01 (hFc) antibody and tisreliumab were used as positive controls.
  • Figure 14A shows the ELISA study of the binding of nine 7103-07 anti-human TIGIT humanized monoclonal antibodies (numbers P03476, P03477, P03478, P03479, P03480, P03481, P03482, P03483 and P03484) to recombinant human TIGIT in vitro.
  • Human-mouse chimeric antibody 7103-07 (hFc) and tisreliumab were used as positive controls.
  • Figure 14B shows the ELISA study on the binding of another batch of 5 anti-human TIGIT humanized monoclonal antibodies (numbers P04662, P04663, P04664, P04665, P04666 and P04667) to recombinant human TIGIT in vitro.
  • Human-mouse chimeric antibody 7103-07 (hFc) and tisrelumab (or Tira) were used as positive controls.
  • Figure 15 shows the flow cytometry analysis of the binding of three 7103-01 anti-human TIGIT humanized monoclonal antibodies (No. P03489, P03492, P03493) to membrane TIGIT.
  • Figure 16 shows the flow cytometric analysis of the binding of nine 7103-07 anti-human TIGIT humanized monoclonal antibodies (numbers P03476, P03477, P03478, P03479, P03480, P03481, P03482, P03483)) to membrane TIGIT.
  • Figure 17A shows the ELISA of the competition of nine 7103-07 anti-human TIGIT humanized monoclonal antibodies (No. P03476, P03477, P03478, P03479, P03480, P03481, P03482, P03483 and P03484) with the binding of CD155 to recombinant human TIGIT Research.
  • Figure 17B shows an ELISA study of another batch of 4 strains of 7103-07 anti-human TIGIT humanized monoclonal antibody numbers (numbers P04662, P04663, P04666 and P04667) competing with CD155 for binding to recombinant human TIGIT.
  • Figure 18 shows the promoting effect of seven humanized TIGIT monoclonal antibodies (numbers P03479, P03480, P03481, P03488, P03489, P03491 and P03493) on the production of IFN ⁇ by PBMCs activated by PHA.
  • Figure 19 Shows the effect of anti-human TIGIT humanized monoclonal antibody in inhibiting the growth of mouse tumors in mice:
  • Figure 19A The relationship between the tumor volume and time of each mouse in each group
  • FIG. 19B Tumor weight per mouse in each group at the end of the experiment (D17).
  • the present disclosure provides antibodies, antibody fragments, and related compositions and molecules that can specifically bind to human TIGIT. Further, the present disclosure relates to the murine antibody, human-mouse chimeric antibody and humanized antibody of the TIGIT antibody. The present disclosure also relates to a pharmaceutical composition comprising the TIGIT antibody, and its use as a diagnostic agent and therapeutic drug for related diseases.
  • the anti-TIGIT antibody of the present disclosure has high specificity and high affinity to human TIGIT, can effectively block the binding of TIGIT and its ligand CD155, enhance the cytokine release of immune cells, improve the activity of immune cells, and enhance immune response, and is used for prevention and treatment of /or to treat tumors, infections or infectious diseases.
  • Anti-TIGIT antibodies of the disclosure are also capable of reducing or eliminating TIGIT-expressing cells (eg, Treg cells) or their activity.
  • the present disclosure also provides a method for enhancing immune response, preventing and/or treating tumors, infections or infectious diseases, which comprises administering to a subject a therapeutically effective amount of the pharmaceutical composition described herein, wherein the pharmaceutical composition
  • the compound blocks the binding of human TIGIT to the ligand CD155.
  • “comprising”, “having” or “comprising” includes “comprising”, “consisting essentially of”, “consisting essentially of”, and “consisting of. 7-8consists of”; “consisting essentially of”, “consisting essentially of” and “consisting of” belong to “contains”, “has “ or the subconcept of "include”.
  • the term "monoclonal antibody (mAb)” refers to an antibody obtained from a substantially homogeneous population, ie, the individual antibodies comprised in the population are identical except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized by hybridoma cultures and are not contaminated by other immunoglobulins. The modifier "monoclonal” indicates the identity of the antibody, which is obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring any particular method for producing the antibody.
  • antibody or "immunoglobulin” is a heterotetrameric protein of about 150,000 Daltons with the same structural characteristics, consisting of two identical light (L) chains and two identical heavy chains (H) Composition. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable region (VH) at one end followed by constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain .
  • VL variable region
  • Specific amino acid residues form the interface between the variable domains of the light and heavy chains.
  • variable means that certain portions of the variable regions among antibodies differ in sequence, which contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout antibody variable domains. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable domains are called the framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • the variable domains of native heavy and light chains each contain four FR regions in a roughly ⁇ -sheet configuration connected by three CDRs forming connecting loops and, in some cases, partial b-sheet structures.
  • the CDRs in each chain are in close proximity through the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, for example involved in the antibody-dependent cytotoxicity of the antibody.
  • the present disclosure provides antibodies that specifically bind human TIGIT, including murine antibodies, chimeric antibodies, and humanized antibodies. Monoclonal antibodies of the present disclosure may also be chimeric or humanized antibodies. In the present disclosure, if not specified otherwise, "m” denotes a murine portion, and “h” denotes a human portion.
  • the antibody includes a heavy chain variable region (VH), the heavy chain variable region includes 3 complementarity determining regions (CDR), the antibody includes a light chain variable region (VL), and The light chain variable region includes 3 CDRs.
  • sequence identity refers to the number of identical residues in a candidate sequence and a reference sequence (e.g. amino acids or nucleic acids).
  • reference sequence e.g. amino acids or nucleic acids.
  • at least 70% sequence identity means that the sequence identity between the candidate sequence and the reference sequence reaches more than 70%, such as 75, 80, 85, 90, 91, 92, 93, 94, 95 , 96, 97, 98, 99, 100% or any numerical point therein.
  • the VH amino acid sequence of the antibody is selected from the group consisting of: SEQ ID NO: 27, 35, 43, 51, 59, 67, 75, 83, 124, 125, 126, 127, 131, 132, 134, 135, 136 or a sequence having at least 70% sequence identity thereto.
  • the VL amino acid sequence of the antibody is selected from the group consisting of: SEQ ID NO: 31, 39, 47, 55, 63, 71, 79, 87, 129, 130, 137, 138, 139, 140, 141 or A sequence having at least 70% sequence identity thereto.
  • the antibody or antigen-binding fragment thereof comprises a VH CDR1 selected from the group consisting of: SEQ ID NO: 28, 36, 44, 52, 60, 68, 76, 84; a VH CDR2 selected from the group : SEQ ID NO: 29, 37, 45, 53, 61, 69, 77, 85, 128, 133, 142, 143.
  • the antibody or antigen-binding fragment thereof comprises a VH CDR3 selected from the group consisting of SEQ ID NO: 30, 38, 46, 54, 62, 70, 78, 86.
  • the antibody or antigen-binding fragment thereof comprises a VL CDR1 selected from the group consisting of SEQ ID NO: 32, 40, 48, 56, 64, 72, 80, 88. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL CDR2 selected from the group consisting of SEQ ID NO: 33, 41, 49, 57, 65, 73, 81, 89. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL CDR3 selected from the group consisting of SEQ ID NO: 34, 42, 50, 58, 66, 74, 72, 90.
  • certain changes can be made to the CDR sequence of the antibody, for example, to meet the requirements of humanization and the like.
  • the altered CDRs may have at least 90% to 99% or more sequence identity to the parent sequence.
  • an altered CDR may differ from the parental sequence by 1, 2 or 3 amino acid residues.
  • one or more of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, VL CDR3 comprises altered amino acid residues.
  • VH CDR2 is altered.
  • the VH CDR2 of SEQ ID NO: 29 is replaced by the sequence of SEQ ID NO: 128 or SEQ ID NO: 142; for example, in some embodiments, the VH CDR2 of SEQ ID NO: 77 is replaced by SEQ ID NO: ID NO: 133 or the sequence of SEQ ID NO: 143 is replaced.
  • VL CDR2 of SPSYRYT (SEQ ID NO: 33, 57, and 65) can be combined with that of SEQ ID NO: 32 and 34, or SEQ ID NO: 56 and 58, or SEQ ID NO: 64 and 66 Combination of VL CDR1 and VL CDR3.
  • VL CDR3 of QQHYSTPWT (SEQ ID NO: 34, 50, and 66) can be combined with that of SEQ ID NO: 32 and 33, or SEQ ID NO: 48 and 49, or SEQ ID NO: 64 and 65 Combination of VL CDR1 and VL CDR2.
  • the VH CDR1 of TYAMS (SEQ ID NO: 68 and 76), the VH CDR3 of KTLDYYALDY (SEQ ID NO: 70 and 78), the VL CDR2 of SGSRLQS (SEQ ID NO: 73 and 81), and VL CDR3 of QQHNEYPWT (SEQ ID NO:74 and 82) in combination with VH CDR2 of SEQ ID NO:69 or 77 and VH CDR1 of SEQ ID NO:72 or 80.
  • the antibody comprises any combination of VH amino acid sequences and VL amino acid sequences selected from the above.
  • the amino acid sequence combinations of VH and VL of the antibody are: SEQ ID NO: 27 and SEQ ID NO: 31; SEQ ID NO: 35 and 39; SEQ ID NO: 43 and 47; SEQ ID NO: 43 and 47; NO: 51 and 55; SEQ ID NO: 59 and 63; SEQ ID NO: 67 and 71; SEQ ID NO: 75 and 79; SEQ ID NO: 83 and 87; selected from any one of SEQ ID NO: 124-126 Combination of derived VH and any VL sequence selected from SEQ ID NO: 31, 39, 47, 55, 63, 71, 79, 87, 129, 130, 137, 138, 139, 140, 141; ID NO: 27, 35, 43, 51, 59, 67, 75, 83, 124, 125, 126, 127, 131, 132, 134, 135,
  • the amino acid sequences of VH CDR1, 2 and 3 of the antibody are SYNIY (SEQ ID NO: 28), GVNPSNGNTNFNENFKS (SEQ ID NO: 29) and GNYYGYEFAY (SEQ ID NO: 30), respectively.
  • the amino acid sequences of VLCDR1, 2 and 3 of the antibody are KASQHVSTAVA (SEQ ID NO: 32), SPSYRYT (SEQ ID NO: 33) and QQHYSTPWT (SEQ ID NO: 34), respectively.
  • the VH amino acid sequence of the antibody is SEQ ID NO: 27 or a sequence having at least 70% sequence identity thereto, and the amino acid sequences of VH CDR1, 2 and 3 of the antibody are SYNIY (SEQ ID NO: 28), GVNPSNGNTNFNENFKS (SEQ ID NO: 29) and GNYYGYEFAY (SEQ ID NO: 30).
  • the VL amino acid sequence of the antibody is SEQ ID NO: 31 or a sequence having at least 70% sequence identity thereto, and the amino acid sequences of VL CDR1, 2 and 3 of the antibody are respectively KASQHVSTAVA (SEQ ID NO: 32), SPSYRYT (SEQ ID NO: 33) and QQHYSTPWT (SEQ ID NO: 34).
  • the amino acid sequences of VH CDR1, 2 and 3 of the antibody are SYVMH (SEQ ID NO: 36), YINPYNDGTKYNEKFKG (SEQ ID NO: 37) and WNPYYYAMDY (SEQ ID NO: 38), respectively.
  • the amino acid sequences of VL CDR1, 2 and 3 of the antibody are SANSSARYVH (SEQ ID NO: 40), EISKLAS (SEQ ID NO: 41) and QQWNYPLIT (SEQ ID NO: 42), respectively.
  • the amino acid sequence of the VH of the antibody is SEQ ID NO: 35 or a sequence having at least 70% sequence identity thereto, and the amino acid sequences of CDR1, 2 and 3 are respectively SYVMH (SEQ ID NO: 36) , YINPYNDGTKYNEKFKG (SEQ ID NO: 37) and WNPYYYAMDY (SEQ ID NO: 38).
  • the amino acid sequence of VL is SEQ ID NO: 39 or a sequence having at least 70% sequence identity therewith, and the amino acid sequences of CDR1, 2 and 3 are SANSSARYVH (SEQ ID NO: 40), EISKLAS (SEQ ID NO: 41) and QQWNYPLIT (SEQ ID NO: 42).
  • the amino acid sequences of VH CDR1, 2 and 3 of the antibody are EYTMH (SEQ ID NO: 44), GINPNNGGTSYNQKFKG (SEQ ID NO: 45) and GAYYRYDFDY (SEQ ID NO: 46), respectively.
  • the amino acid sequences of VLCDR1, 2 and 3 of the antibody are KASQDVRTAVV (SEQ ID NO: 48), SASYRYT (SEQ ID NO: 49) and QQHYSTPWT (SEQ ID NO: 50), respectively.
  • the amino acid sequence of the VH of the antibody is SEQ ID NO: 43 or a sequence having at least 70% sequence identity thereto, and the amino acid sequences of CDR1, 2 and 3 are EYTMH (SEQ ID NO: 44) , GINPNNGGTSYNQKFKG (SEQ ID NO: 45) and GAYYRYDFDY (SEQ ID NO: 46).
  • the amino acid sequence of VL is SEQ ID NO: 47 or a sequence having at least 70% sequence identity therewith, and the amino acid sequences of CDR1, 2 and 3 are KASQDVRTAVV (SEQ ID NO: 48), SASYRYT (SEQ ID NO: 49) and QQHYSTPWT (SEQ ID NO: 50).
  • the amino acid sequences of VH CDR1, 2 and 3 of the antibody are DYFVY (SEQ ID NO: 52), GINPSNGGATFNEKFKN (SEQ ID NO: 53) and GNFYGYEFAY (SEQ ID NO: 54), respectively.
  • the amino acid sequences of VLCDR1, 2 and 3 of the antibody are KASQHVSTAVV (SEQ ID NO: 56), SPSYRYT (SEQ ID NO: 57) and QQHYITPWT (SEQ ID NO: 58), respectively.
  • the amino acid sequence of the VH of the antibody is SEQ ID NO: 51 or a sequence having at least 70% sequence identity thereto, and the amino acid sequences of CDR1, 2 and 3 are DYFVY (SEQ ID NO: 52) , GINPSNGGATFNEKFKN (SEQ ID NO: 53) and GNFYGYEFAY (SEQ ID NO: 54).
  • the amino acid sequence of VL is SEQ ID NO: 55 or a sequence having at least 70% sequence identity therewith, and the amino acid sequences of CDR1, 2 and 3 are KASQHVSTAVV (SEQ ID NO: 56), SPSYRYT (SEQ ID NO: 57) and QQHYITPWT (SEQ ID NO: 58).
  • the amino acid sequences of VH CDR1, 2 and 3 of the antibody are EYTMY (SEQ ID NO: 60), GINPNNGGTRYNQKFKG (SEQ ID NO: 61) and GGHYDYGFAY (SEQ ID NO: 62), respectively.
  • the amino acid sequences of VLCDR1, 2 and 3 of the antibody are KASQDVKTAVA (SEQ ID NO: 64), SPSYRYT (SEQ ID NO: 65) and QQHYSTPWT (SEQ ID NO: 66), respectively.
  • the amino acid sequence of the VH of the antibody is SEQ ID NO: 59 or a sequence having at least 70% sequence identity thereto, and the amino acid sequences of CDR1, 2 and 3 are EYTMY (SEQ ID NO: 60) , GINPNNGGTRYNQKFKG (SEQ ID NO: 61) and GGHYDYGFAY (SEQ ID NO: 62).
  • the amino acid sequence of VL is SEQ ID NO: 63 or a sequence having at least 70% sequence identity therewith, and the amino acid sequences of CDR1, 2 and 3 are KASQDVKTAVA (SEQ ID NO: 64), SPSYRYT (SEQ ID NO: 65) and QQHYSTPWT (SEQ ID NO: 66).
  • the amino acid sequences of VH CDR1, 2 and 3 of the antibody are TYAMS (SEQ ID NO: 68), EISSGGSYTYYSDTVTG (SEQ ID NO: 69) and KTLDYYALDY (SEQ ID NO: 70), respectively.
  • the amino acid sequences of VLCDR1, 2 and 3 of the antibody are RANKTISKYLA (SEQ ID NO: 72), SGSRLQS (SEQ ID NO: 73) and QQHNEYPWT (SEQ ID NO: 74), respectively.
  • the amino acid sequence of the VH of the antibody is SEQ ID NO: 67 or a sequence having at least 70% sequence identity thereto, and the amino acid sequences of CDR1, 2 and 3 are respectively TYAMS (SEQ ID NO: 68) , EISSGGSYTYYSDTVTG (SEQ ID NO: 69) and KTLDYYALDY (SEQ ID NO: 70).
  • the amino acid sequence of VL is SEQ ID NO: 71 or a sequence having at least 70% sequence identity therewith, and the amino acid sequences of CDR1, 2 and 3 are RANKTISKYLA (SEQ ID NO: 72), SGSRLQS (SEQ ID NO: 73) and QQHNEYPWT (SEQ ID NO: 74).
  • the amino acid sequences of VH CDR1, 2 and 3 of the antibody are TYAMS (SEQ ID NO: 76), EISSGGSHTFYSDTVTG (SEQ ID NO: 77) and KTLDYYALDY (SEQ ID NO: 78), respectively.
  • the amino acid sequences of VLCDR1, 2 and 3 of the antibody are RASKSISKYLA (SEQ ID NO: 80), SGSRLQS (SEQ ID NO: 81) and QQHNEYPWT (SEQ ID NO: 82), respectively.
  • the amino acid sequence of the VH of the antibody is SEQ ID NO: 75 or a sequence having at least 70% sequence identity thereto, and the amino acid sequences of CDR1, 2 and 3 are respectively TYAMS (SEQ ID NO: 76) , EISSGGSHTFYSDTVTG (SEQ ID NO: 77) and KTLDYYALDY (SEQ ID NO: 78).
  • the amino acid sequence of VL is SEQ ID NO: 79 or a sequence having at least 70% sequence identity therewith, and the amino acid sequences of CDR1, 2 and 3 are RASKSISKYLA (SEQ ID NO: 80), SGSRLQS (SEQ ID NO: 81) and QQHNEYPWT (SEQ ID NO: 82).
  • the amino acid sequences of VH CDR1, 2 and 3 of the antibody are GYYML (SEQ ID NO: 84), RINPYNGATTYNQNFKD (SEQ ID NO: 85) and WFGDFDF (SEQ ID NO: 86), respectively.
  • the amino acid sequences of VLCDR1, 2 and 3 of the antibody are RASQEISGYLS (SEQ ID NO: 88), ATSTLDS (SEQ ID NO: 89) and LQYASYPFT (SEQ ID NO: 90), respectively.
  • the amino acid sequence of the VH of the antibody is SEQ ID NO: 83 or a sequence having at least 70% sequence identity thereto, and the amino acid sequences of CDR1, 2 and 3 are respectively GYYML (SEQ ID NO: 84) , RINPYNGATTYNQNFKD (SEQ ID NO: 85) and WFGDFDF (SEQ ID NO: 86).
  • the amino acid sequence of VL is SEQ ID NO: 87 or a sequence having at least 70% sequence identity therewith, and the amino acid sequences of CDR1, 2 and 3 are RASQEISGYLS (SEQ ID NO: 88), ATSTLDS (SEQ ID NO: 89) and LQYASYPFT (SEQ ID NO: 90).
  • the antibody is a humanized antibody
  • the amino acid sequence of its VH is the heavy chain variable region shown in any one of SEQ ID NO: 124, 125, 126, and 127, and the amino acids of CDR1, 2, and 3
  • the sequences are CDR1: SYNIY (SEQ ID NO: 28), CDR2: GVNPSNGNTNFNENFKS (SEQ ID NO: 29) or GVNPSNGNTNFNENFKG (SEQ ID NO: 142) or GVNPSNGNTNFNENFQG (SEQ ID NO: 128) and CDR3: GNYYGYEFAY (SEQ ID NO :30).
  • the antibody is a humanized antibody
  • the amino acid sequence of its VL is the light chain variable region shown in any one of SEQ ID NO: 129, 130
  • the amino acid sequences of CDR1, 2 and 3 are respectively KASQHVSTAVA (SEQ ID NO: 32), SPSYRYT (SEQ ID NO: 33) and QQHYSTPWT (SEQ ID NO: 34).
  • the antibody is a humanized antibody
  • the amino acid sequence of its VH is the heavy chain variable region shown in any one of SEQ ID NO: 131, 132, 134, 135, 136, CDR1, 2 and 3
  • the amino acid sequences are CDR1: TYAMS (SEQ ID NO: 76), CDR2: EISSGGSHTFYSDTVTG (SEQ ID NO: 77) or EISSGGSHTFYADTVKG (SEQ ID: 133) or EISSGGSHTFYADTVTG (SEQ ID NO: 143), and CDR3: KTLDYYALDY (SEQ ID NO: 78).
  • the antibody is a humanized antibody, and the amino acid sequence of its VL is the light chain variable region shown in any one of SEQ ID NO: 137, 138, 139, 140, 141, CDR1, 2 and 3
  • the amino acid sequences of are RASKSISKYLA (SEQ ID NO: 80), SGSRLQS (SEQ ID NO: 81) and QQHNEYPWT (SEQ ID NO: 82).
  • non-human monoclonal antibodies can be humanized through the following pathways: (1) homologous replacement, using human FRs that have greater homology to the non-human counterpart; (2) surface Reshaping, reshaping the surface amino acid residues of non-human CDR and FR to make it similar to the outline of human antibody CDR or FR type; (3) compensating for changes, reorganizing amino acid residues at key positions to compensate for CDR transplantation; (4) Positioning is conserved.
  • the humanized monoclonal antibody is humanized using the FR conservative sequence as a template, but retains the key amino acid residues of the variable region of the non-human monoclonal antibody.
  • immunologically active antibody fragments such as Fab, Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-linked Fv , V-NAR domain, IgNar, intrabody, IgG ⁇ CH2, small antibody, F(ab')3, tetrabody, triabody, bispecific antibody, single domain antibody, DVD-Ig, Fcab, mAb2, ( scFv)2 or scFv-Fc, etc.
  • immunologically active antibody fragments such as Fab, Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-linked Fv , V-NAR domain, IgNar, intrabody, IgG ⁇ CH2, small antibody, F(ab')3, tetrabody, triabody, bispecific antibody, single domain antibody, DVD-Ig, Fcab, mAb2, ( scFv)2 or scFv-F
  • an antibody of the disclosure does not compete with tisleliumab for binding to TIGIT. In some embodiments, an antibody of the disclosure binds to a different epitope than tisreliumab. In some embodiments, the aforementioned antibodies are, for example, 7103-06, 7103-07.
  • an antibody of the disclosure competes with tisleliumab for binding to TIGIT and partially blocks tisleliumab binding. In some embodiments, an antibody of the disclosure binds to an overlapping epitope with tisleliumab. In some embodiments, the aforementioned antibodies are, for example, 7103-01, 7103-03, 7103-04.
  • antibodies of the disclosure bind the same epitope.
  • antibodies 7103-01, 7103-03, 7103-04 mutually block each other's TIGIT binding and bind the same epitope.
  • an antibody of the disclosure binds human or other primate TIGIT, but does not bind mouse TIGIT.
  • Monoclonal antibodies and fragments thereof can be prepared by various methods well known to those skilled in the art.
  • monoclonal antibodies can be prepared by the hybridoma method (first described by Kohler et al., Nature, 256:495 (1975)), or by recombinant DNA methods (US Patent No. 4,816,567).
  • Monoclonal antibodies can also be isolated from phage antibody libraries using techniques such as those described by Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).
  • Nucleic acid molecules encoding said anti-TIGIT monoclonal antibodies or fragments thereof are also provided herein.
  • the sequences of these nucleic acid molecules can be obtained by conventional techniques, such as PCR amplification or genomic library screening.
  • the coding sequences for the light and heavy chains can be fused together to form single-chain antibodies.
  • This article also discloses expression vectors containing the above nucleic acid molecules, such as pcDNA3.4, pcDNA3.1, etc.; this article also discloses host cells transformed by the above expression vectors, which can be, for example, CHO cells, COS cells, etc.
  • recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.
  • related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.
  • nucleic acid sequence encoding the monoclonal antibody (or its fragment, or its derivative) described herein can be obtained completely by chemical synthesis. This sequence can then be introduced into various existing molecules (or eg vectors) and cells known in the art. In addition, mutations may also be introduced into the protein sequences herein by chemical synthesis.
  • This document also relates to vectors comprising the coding sequences described above together with appropriate promoter or control sequences.
  • These vectors can be used to transform appropriate host cells so that they express the protein.
  • the host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells, such as CHO cells and COS cells.
  • Mammalian cell lines are commonly used as host cells for the expression of eukaryotic-derived polypeptides. Propagation of mammalian cells in culture is well known in the art. See Tissue Culture, Academic Press, eds. Kruse and Patterson (1973), which is incorporated herein by reference.
  • Preferred mammalian cells are the many immortalized cell lines available commercially. These immortal cell lines include, but are not limited to, Chinese hamster ovary (CHO) cells, Vero cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (such as Hep G2) and many other cell lines. They provide post-translational modifications to protein molecules, including correct folding, correct disulfide bond formation, and glycosylation at the correct sites.
  • CHO Chinese hamster ovary
  • Vero cells Vero cells
  • HeLa cells HeLa cells
  • BHK baby hamster kidney cells
  • COS monkey kidney cells
  • Hep G2 human hepatocellular carcinoma cells
  • hybridoma cell lines that produce the monoclonal antibodies herein. After obtaining the hybridoma producing the monoclonal antibody herein, those skilled in the art can easily know the structure of the antibody herein (such as the heavy chain variable region and light chain variable region of the antibody), and then prepare the monoclonal antibody herein.
  • Methods for introducing heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, Polybrene (1,5-dimethyl-1,5-di Nidecylmethylidene)-mediated transfection, protoplast fusion, electroporation, liposome-mediated transfection, and microinjection of DNA directly into the nucleus.
  • the preferred method is electroporation or liposome-mediated method, etc.
  • the liposome method kit from Invitrogen can be used to transfect host cells such as COS and CHO cells.
  • the transformed host cells are cultured under conditions suitable for the expression of the monoclonal antibodies herein. Then use conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc.
  • the monoclonal antibody herein was purified by conventional separation and purification methods well known to personnel.
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined using immunoprecipitation or an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the binding affinity of monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
  • the monoclonal antibody herein may be expressed intracellularly, on the cell membrane, or secreted extracellularly.
  • the recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the present application also provides conjugates comprising the antibodies or antigen-binding fragments thereof herein, chimeric antigen receptors, and immune cells expressing the chimeric antigen receptors. Due to the specificity of the antibody or antigen-binding fragment thereof of the present application to TIGIT, the conjugate, chimeric antigen receptor, and immune cells expressing the chimeric antigen receptor can play a targeted role in expressing or overexpressing TIGIT cells or tissues. Moreover, since the antibody or antigen-binding fragment thereof of the present application itself has biological activity (for example, has immunomodulatory activity), through its conjugate, chimeric antigen receptor and the antibody or antigen in immune cells expressing chimeric antigen receptor Binding fragment moieties may also play a corresponding role.
  • an antibody conjugate may comprise: an antibody described herein, or an antigen-binding fragment thereof; a conjugating moiety, such as a drug, toxin, cytokine, radionuclide, or enzyme; and, optionally, a linker.
  • the coupling is achieved by enzymatic coupling, chemical coupling, fusion, and the like.
  • the linker can be a "cleavable linker" that facilitates release of the conjugated moiety in the cell, such as acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, and disulfide-containing linkers.
  • a conjugation moiety attached to an antibody can be, for example, a biologically active agent, which can be any synthetic, recombinantly produced or naturally occurring substance that enhances and/or modulates a desired biological effect.
  • the effects that the bioactive agent can provide include, but are not limited to: regulating, stimulating, and/or inhibiting growth signals; regulating, stimulating, and/or inhibiting anti-apoptotic signals; regulating, stimulating, and/or inhibiting Or inhibit apoptosis or necrosis signal; regulate, stimulate, and/or inhibit ADCC cascade; regulate, stimulate, and/or inhibit CDC cascade, etc.
  • it can be a chemotherapeutic agent, toxin, cytokine, isotope, enzyme, etc.
  • the present application also provides a chimeric antigen receptor (CAR), which comprises an extracellular domain and an intracellular domain, wherein the extracellular domain comprises the antibody of the present application or an antigen-binding fragment thereof.
  • CAR of the present application further comprises a transmembrane domain connecting the extracellular domain and the intracellular domain, such as the transmembrane part of CD28.
  • the CAR of the present application can be a first-generation, second-generation, third-generation or fourth-generation CAR.
  • the antibody or antigen-binding fragment thereof in the CAR of the present application is scFv or VH sdAb, or consists of it.
  • the intracellular domains of the CAR include those that mimic or approximate signaling through native antigen receptors, signaling through such receptors in combination with co-stimulatory receptors, and/or signaling through co-stimulatory receptors alone .
  • the intracellular domain includes one or more co-stimulatory domains and activation domains, such as one or more selected from the group consisting of ITAM domains, CD3 ⁇ , CD28, 4-1BB, OX40, CD27, ICOS or combinations thereof, such as (CD28+CD3 ⁇ ), (CD28+CD27+CD3 ⁇ ), (CD28+OX40+CD3 ⁇ ), (CD28+4-1BB+CD3 ⁇ ), (CD28+CD27+OX40+CD3 ⁇ ), (CD28+4-1BB+CD27+CD3 ⁇ ), (CD28+4-1BB+OX40+CD3 ⁇ ), (4-1BB+CD3 ⁇ ), (4-1BB+OX40+CD3 ⁇ ), (4-1BB+CD27+CD3 ⁇ ), (CD27+CD3 ⁇ ), (CD27+OX 40+CD3 ⁇ ), (CD28 ⁇ +CD3 ⁇ ), (CD28 ⁇ +CD27+CD3 ⁇ ), (CD28 ⁇ +OX40+CD3 ⁇ ), (CD28 ⁇ +4-1BB+CD
  • the extracellular domain of the CAR further comprises a hinge region, for example, the hinge region is derived from the hinge of IgG or the extracellular region of CD8 ⁇ /CD28, such as selected from: IgG4 Fc ⁇ EQ, IgG4 Fc ⁇ Q, (t-12AA+ t-20AA), mKate, phiLov, dsRed, Venus, eGFP, CH3 HA, (CD8 ⁇ +t-20AA), dual t-20AA, (t-20AA+CD8 ⁇ ), (CD8 ⁇ +leucine zipper Basep1), (CD8 ⁇ + Leucine Zipper Acid1), 2D3, CD8 ⁇ or IgG4 Fc.
  • the hinge region is derived from the hinge of IgG or the extracellular region of CD8 ⁇ /CD28, such as selected from: IgG4 Fc ⁇ EQ, IgG4 Fc ⁇ Q, (t-12AA+ t-20AA), mKate, phiLov, dsRed, Venus, eGFP
  • the present application also provides a transformed immune cell, which expresses the chimeric antigen receptor of the present application, for example, the immune cell is selected from T cells, such as ⁇ T cells, ⁇ T cells or NK T cells or from pluripotent cells Derived T cells.
  • T cells such as ⁇ T cells, ⁇ T cells or NK T cells or from pluripotent cells Derived T cells.
  • Immune cells can be from the individual to be treated or from other sources.
  • Transformed (optionally expanded) immune cells can be administered to an individual to be treated, eg, for adoptive therapy.
  • this application also includes CAR constructs, expression vectors, preparation methods and applications for transforming immune cells.
  • This article also provides a pharmaceutical composition, which contains a pharmaceutically effective amount of the monoclonal antibody or immunoconjugate thereof and a pharmaceutically acceptable carrier.
  • the term "pharmaceutically acceptable” means that the molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.
  • the "pharmaceutically acceptable carrier” used herein should be compatible with the active substance herein, that is, it can be blended with it without greatly reducing the effect of the pharmaceutical composition under normal circumstances. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
  • Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • auxiliary substances in these carriers such as wetting agents or emulsifying agents, pH buffering substances and the like.
  • composition herein can be administered orally, intravenously, intramuscularly or subcutaneously; oral administration or intravenous injection is preferred.
  • the pharmaceutical composition herein can be made into various dosage forms according to needs, and the doctor can determine the dosage beneficial to the patient according to the patient's type, age, body weight and general disease condition, administration method and other factors for administration.
  • a safe and effective amount of the monoclonal antibody or immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually about 0.1 ⁇ g-5 mg/kg body weight, and in most cases does not exceed about 3 mg/kg body weight, preferably the dose is about 1-10 microgram/kg body weight to about 1 mg/kg body weight.
  • the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.
  • unit dosage form refers to the preparation of the composition of the present disclosure into a dosage form required for single administration for the convenience of administration, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, capsules , Sustained release agent.
  • the composition is in unit dosage form or multiple dosage form, and the content of the active substance is 0.01-2000 mg/dose, preferably 0.1-1500 mg/dose, more preferably 1-1000 mg/dose.
  • 1-6 doses of the composition of the present disclosure are administered per day, preferably 1-3 doses are administered; most preferably, the daily dose is 1 dose.
  • the monoclonal antibody or immunoconjugate herein can effectively block the binding of TIGIT and its ligand CD155, enhance the cytokine release of immune cells, improve the activity of immune cells, and enhance immune response, and are used for the prevention and/or treatment of tumors, infections or infectious disease.
  • the active substances herein can be used in the treatment of various types of tumors such as (including but not limited to): adenocarcinoma, leukemia, lymphoma, melanoma, sarcoma; from the adrenal gland, gallbladder, bone, bone marrow, brain, breast , bile duct, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, skin, salivary gland, spleen, testis, thymus, thyroid, or uterus; central nervous system Tumors, such as glioblastoma, or astrocytoma; tumors of the eye (such as basal cell carcinoma, squamous cell carcinoma, or melanoma), endocrine gland tumors, neuroendocrine system tumors, gastrointestinal pancreatic endocrine system tumors, Tumors of the reproductive system or head and neck.
  • tumors such as (including but not limited to): aden
  • This article also provides a kit for detecting TIGIT, which contains the anti-TIGIT monoclonal antibody or its active fragment and immunoconjugate described herein.
  • the test kit herein can be used to detect whether there is TIGIT or its content in a biological sample, and the detection method includes the steps of: (a) contacting the sample with the anti-TIGIT monoclonal antibody or its immunoconjugate in the test kit herein; (b ) to detect whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of TIGIT in the sample or quantitatively detect the amount of the formed antigen-antibody complex to reflect the content of TIGIT in the sample.
  • the sample may or may not have been pretreated, eg, may have been extracted, purified or concentrated, among others.
  • the kit contains a container, the monoclonal antibody herein located in the container, or a detection plate with the monoclonal antibody, and instructions for use.
  • the kit may also contain other reagents required for detection, such as buffers, indicators and the like. Those skilled in the art can adjust the contents of the kit according to specific needs.
  • mice Three Balb/c mice (6-8 weeks old, weighing about 18g, female, purchased from the Experimental Animal Management Department of the Institute of Family Planning Science) were treated with 50 ⁇ g recombinant human TIGIT (Human TIGIT His, Sino Biological, 10917-H08H Accession #NP 776160.2.Met1-Phe138, C-terminal expression polyhistidine tag) in the presence of complete Freund's adjuvant CFA (complete Freund's adjuvant CFA), emulsified by 100 ⁇ l protein and 100 ⁇ l CFA, and multi-point immunization was performed intraperitoneally and subcutaneously (Injection volume per point is 50 ⁇ l).
  • complete Freund's adjuvant CFA complete Freund's adjuvant CFA
  • the same emulsification was carried out according to 100 ⁇ l protein and 100 ⁇ l IFA.
  • the mice were boosted 4 times with the same immunogen hTIGIT at two weeks, three weeks, four weeks and five weeks after the first immunization.
  • the serum titer was detected to be above 10W, and the same 25 ⁇ g immunogen hTIGIT was injected subcutaneously for sprint immunization and prepared for myeloma cell fusion.
  • mice The splenocytes and lymphocytes of the mouse were fused with mouse myeloma cells (purchased from the Cell Bank of the Chinese Academy of Sciences, the article number is TCM18), and the specific steps were as follows:
  • the medium is IMDM medium containing 10% FBS (containing 1 ⁇ HAT and 1 ⁇ double antibody), 200 ⁇ l/well, 37°C, 5% CO2 incubation.
  • FBS containing 1 ⁇ HAT and 1 ⁇ double antibody
  • ELISA detection was performed according to the cell growth density. The detected positive well cells were subcloned for the first and second time. The ELISA detection steps are as follows:
  • TIGIT-Fc Human TIGIT(Fc Tag): Sino Biological, 10917-H02H
  • Enzyme plate blocking wash twice with 250 ⁇ l/well PBS and pat dry. Then add 150 ⁇ l/well of blocking solution, seal the plate and place it at 37°C for 1.5 hours.
  • Primary antibody incubation Take 100 ⁇ l/well of the hybridoma supernatant and add it to the microtiter plate, seal the plate with a sealing film and place it at 37°C for 1 hour.
  • TMB incubation Discard the blocking solution, wash 3 times with 250 ⁇ l PBST, and pat dry. TMB 50ul//well was added to the enzyme labeling plate, sealed with the sealing film and placed at 37°C for 15-25 minutes.
  • Microplate reader detection dual wavelength 450nm/655nm detection, the result is calculated by OD 450 -OD 655 .
  • the resulting cell line is cultured, and the cells are expanded. Centrifuge the cell culture medium, collect the supernatant, purify the fusion protein from the supernatant with Protein-A affinity chromatography column, run gel analysis, load 2 ⁇ g of reduced and non-reduced samples on each lane respectively. Voltage 150V, 1 hour. The molecular weight of the non-reduced antibody is about 150KD, and the molecular weight of the reduced antibody is about 50KD and 25KD respectively, which is in line with the molecular weight characteristics of antibodies ( Figure 1).
  • TIGIT(His), Sino Biological, 10917-H08H The binding of eight TIGIT monoclonal antibodies to recombinant human TIGIT antigen (Human TIGIT(His), Sino Biological, 10917-H08H) was tested by ELISA using standard methods and procedures. The specific steps are as follows:
  • TIGIT coating Dilute TIGIT with the coating solution to a concentration of 1 ⁇ g/ml, mix well and pipette into a 96-well ELISA plate, 50 ⁇ l/well, seal with a sealing film and place in a 4°C refrigerator overnight .
  • Enzyme plate blocking wash twice with 250 ⁇ l/well PBS and pat dry. Then add 150 ⁇ l/well of blocking solution, seal the plate and place it at 37°C for 1.5 hours.
  • TMB incubation Discard the blocking solution, wash 3 times with 250 ⁇ l PBST, and pat dry. Add 50 ⁇ l/well of TMB to the enzyme labeling plate, seal the plate with a sealing film and place it at 37°C for 15-25 minutes.
  • Microplate reader detection dual wavelength 450nm/655nm detection, the result is calculated by OD 450 -OD 655 .
  • the results of the ELISA test ( Figure 2): As shown in Table 1, the eight anti-human TIGIT mouse monoclonal antibodies (7103-01-7103-08) can specifically bind to human TIGIT, and the binding affinity EC50 is respectively: 0.0647 nM, 0.0987nM, 0.0674nM, 0.0727nM, 0.0571nM, 0.0393nM, 0.075nM and 0.0372nM.
  • TIGIT coating Dilute TIGIT with the coating solution to a concentration of 0.8 ⁇ g/ml, mix well and use a multi-well pipette to inhale into a 96-well microplate, 50 ⁇ l/well, seal with a sealing film and place in a 4°C refrigerator overnight.
  • Enzyme plate blocking wash twice with 250 ⁇ l/well PBS and pat dry. Then add 150 ⁇ l/well of blocking solution, seal the plate and place it at 37°C for 1.5 hours.
  • PNPP incubation Discard the blocking solution, wash 3 times with 250 ⁇ l PBST, and pat dry. Take 1 PNPP (SouthemBiotech, 0201-01) in 1ml 5 ⁇ diethanolamine substrate, then add 4ml deionized water to dissolve, add 50 ⁇ l/well to the microplate, seal the plate and place it at 37°C for 15-25 minutes.
  • Microplate reader detection dual wavelength 405nm/490nm detection, the result is calculated as OD 405 -OD 490 .
  • Anti-human TIGIT mouse monoclonal antibodies of five of the eight strains can recognize cynomolgus TIGIT, and the binding EC 50 is: 1.066 nM, 0.398nM, 0.334nM, 0.174nM, 0.704nM.
  • there were 3 anti-human TIGIT mouse monoclonal antibodies No. 7103-02, 7103-05, 7103-08) that did not bind to cynomolgus monkey TIGIT.
  • the CHO-K (CHO-K-TIGIT) cell line stably expressing full-length human TIGIT was constructed as follows: a plasmid (purchased from Sino Biological) containing full-length human TIGIT cDNA (NM_173799.2) was used to clone the cDNA into The mammalian cell expression vector pcDNA3.1 (Invitrogen) of aminoamide synthetase gene, then use electroporation (Bio-Rad, Gene Pulser Xcell) method to transfect the above-mentioned constructed plasmid into CHO-K cells, the transfected cells After 24-48 hours of cultivation in OptiCHO medium (Invitrogen), the medium was changed to selection medium.
  • OptiCHO medium Invitrogen
  • Selection medium contained OptiCHO, 5 ⁇ g/ml recombinant human insulin and 10 ⁇ M sulfomethionine (MSX). Cells were cultured at 37°C in an 8% CO 2 incubator. After 3 weeks, the cultured cells were sorted by flow cytometry (FACS) with anti-TIGIT antibody, and the CHO-K monoclonal cell line expressing human TIGIT on the cell membrane was obtained.
  • FACS flow cytometry
  • the CHO-TIGIT was counted and centrifuged at 1000 rpm for 5 min. Resuspend with 0.5% BSA PBS, take 5 ⁇ 10 5 cells/well and add to 96-well sharp bottom plate. Centrifuge at 400 g for 5 min, and discard the supernatant.
  • the anti-TIGIT antibody was serially diluted and added to a 96-well sharp-bottom plate at 100 ⁇ l/well, at 4°C for 30 minutes. Wash twice with 0.5% BSAPBS at 250 ⁇ l/well, add 1:100 diluted Anti-mouse-IgG-PE (Jackson Immuno Research, 115-116-071) at 100 ⁇ l/well to a 96-well sharp bottom plate, 4°C, 30min. Wash twice with 0.5% BSA PBS at 250 ⁇ l/well, and add 0.5% BSA PBS at 200 ⁇ l/well. Samples were detected with a Beckman CytoFLEX.
  • RNA extraction About 1-2 ⁇ 10 5 hybridoma cells were taken for RNA extraction (Sigma, GenElute TM Mammalian Total RNA Miniprep Kit, Cat. No. RTN70). Take about 8 ⁇ L of total RNA for subsequent reverse transcription PCR, first at 65°C for 5 minutes to eliminate the secondary structure of RNA, then add 2 ⁇ L of 5 ⁇ gDNA wiper at 42°C for 2 minutes to remove genomic DNA contamination. Finally, add 2 ⁇ L of 10 ⁇ RT Mix, 2 ⁇ L of HiscriptIII Enzyme Mix and 1 ⁇ L of random hexamers (Random Hexamers, Novozyme, R312). Gently flick the tube wall to mix, centrifuge briefly to throw the liquid to the bottom of the tube, and finally carry out the reverse transcription reaction under the following conditions: 25°C, 5min; 42°C, 45min; 85°C, 5s.
  • the VH and VL genes of the hybridoma antibody were amplified by PCR with different primer combinations.
  • the main references for the primers used in this experiment were von Boehmer, L. et al., Sequencing and cloning of antigen-specific antibodies from mouse memory B cells.
  • the obtained PCR products were either directly sequenced or sequenced by TA cloning.
  • the sequencing primer for the heavy chain was SEQ ID NO: 14
  • the sequencing primer for the light chain was SEQ ID NO: 26.
  • Table 3 V region sequences of 8 strains of TIGIT mouse monoclonal antibodies.
  • the V region of the hybridoma antibody is constructed on a chimeric antibody expression vector (pcDNA3.1), that is, the V region sequence is connected to an expression vector whose constant region is human IgG1 or human ⁇ / ⁇ .
  • pcDNA3.1 chimeric antibody expression vector
  • the PCR reaction conditions were as follows: after denaturation at 94°C for 3 minutes, 40 cycles at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 35 seconds, and incubation at the end of the cycle at 72°C for 5 minutes.
  • the fragment to be cloned was successfully retrieved.
  • the HEK293F cells, medium and transfection reagents used in this experiment were all purchased from Sino Biotech.
  • the specific usage method is as follows: the day before transfection, take a sample to calculate the cell density and viability, and inoculate it into a fresh cell at a density of 2E6 cells/mL.
  • the culture medium is placed in a constant temperature shaker incubator at 37° C., 5% CO 2 , and a rotation speed of 150-175 rpm.
  • On the day of transfection calculate the cell viability and adjust the cells to 3E6 cells/mL.
  • transfection solution Preparation of transfection solution: Taking a transfection volume of 20mL as an example, dilute 20 ⁇ g of DNA with 150mM NaCl to a total volume of 500 ⁇ L and mix gently; dilute 100 ⁇ L of sinofection transfection reagent with 150mM NaCl to a total volume of 500 ⁇ L and mix gently Uniformly; the diluted DNA and transfection reagent were left alone for 5 minutes and then mixed gently. The total volume was 1 mL, and then stood for 10 minutes. Finally, they were added dropwise to the cell culture medium, mixed well and continued to cultivate. After the third day after transfection, the viability of the cells was detected every day, and the supernatant was harvested when the viability of the cells was about 60%.
  • the results of SDS-PAGE analysis after purification are shown in FIG. 6 .
  • the molecular weight of the non-reduced antibody is about 150KDa, and the molecular weight of the reduced antibody is 55kDa and 25KDa respectively, which is in line with the molecular weight characteristics of the antibody.
  • the antigen hTIGIT Human TIGIT(His), Sino Biological, 10917-H08H was diluted with NaHCO3 solution at pH 9.6 to 1 ⁇ g/mL, 50 ⁇ L per well was added to a 96-well microtiter plate, and refrigerated overnight at 4 °C.
  • ELISA test results show: 5 anti-TIGIT human-mouse chimeric monoclonal antibodies (No. 7103-07(hFc)) can specifically bind human TIGIT, and its binding affinity EC 50 is 0.499nM, 0.380nM, 0.554nM, 0.554nM, 0.310nM, while the EC 50 of the positive control tisleliumab is 1.194nM.
  • the dissociation constant (Kd) of human-mouse chimeric TIGIT monoclonal antibody was detected by GATOR (ProbeLife) detection instrument and HFC (LOT#2007065Tray 2), Human antibody Capture probe.
  • Kinetic correlation assays are started by placing the antibody capture sensor in the above serially diluted antigen solution. Open the Octet Data Acquisition software and select New Kinetics Experiment-Basic Kinetics model;
  • FACS buffer solution that is, 1 ⁇ PBS+0.5% BSA solution. Take the required number of cells.
  • the amount of cells required for one sample is about 1 ⁇ 10 ⁇ 5 ⁇ 5 ⁇ 10 ⁇ 5.
  • the volume is about 100 ⁇ L.
  • Incubate at 4°C refrigerator for 30min wash the cells once with FACS buffer, resuspend the cells with FITC-anti-human IgG (Abcam: 6854) diluted in FACS buffer, and incubate at 4°C refrigerator for 30min, finally, FACS buffer After washing the cells, resuspend the cells with 200 ⁇ L of FACS buffer, and detect on the machine.
  • the antibody of the present disclosure not only has a strong binding ability to solid-phase antigen (ELISA method), but also has a high binding activity to TIGIT on the cell membrane, and the EC 50 value is 1/3-1/2 of tisleliumab , indicating that the binding of the antibody of the present invention to TIGIT on the cell membrane is significantly better than the binding of tisrelumab to TIGIT on the cell membrane surface.
  • the primary antibody and CD155-mFC (6 ⁇ g/mL) (Human CD155 (Fc Tag): Sino Biological, 10109-H02H) in the serial dilution were mixed in equal volumes, then added to the blocked and washed microtiter plate, incubated at 37°C for 2 hours, and continued After washing 4 times with PBST, add a certain proportion of diluted secondary antibody HRP anti-mFC (Jackson Immuno Research, 115-035-164), incubate at 37°C for 1.5h, and finally wash with PBST 4 times, add TMB chromogenic solution, 37°C After incubation for 10-15 min, read with a microplate reader (wavelength 450nm and 655nm).
  • the experimental results show five kinds of chimeric TIGIT monoclonal antibodies (No. hFc)) can specifically block the binding of CD155 to human hTIGIT.
  • IC 50 were: 4.029nM, 1.455nM, 3.298mM, 2.029nM, 1.698nM, and the IC 50 of the positive control tisrelumab was 5.235nM.
  • the antibody of the present disclosure can block the binding of TIGIT and its ligand CD155, and the blocking ability of the antibody of the present disclosure is better than tisrelumab, among which 7103-03 (hFc), 7103-06
  • the IC 50 values of (hFc) and 7103-07(hFc) are 1/3-1/2 of that of tisleliumab, and the blocking ability is significantly better than that of tisleliumab.
  • the antigen TIGIT Human TIGIT(His), Sino Biological, 10917-H08H was diluted to 1 ⁇ g/mL with NaHCO 3 solution at pH 9.6, and 50 ⁇ L per well was added to a 96-well microtiter plate and refrigerated overnight at 4°C. The next day, after washing twice with PBS, block with 3% BSA, 100 ⁇ L per well, incubate at 37° C. for 1.5 h, and wash 4 times with PBST.
  • Equal volumes of serially diluted murine antibody of the present disclosure and tisrelumab (final concentration 0.83 ⁇ g/mL, equivalent to the EC 80 of tisrelumab binding to TIGIT-His) were mixed and added to the blocked and washed In the ELISA plate, incubate at 37°C for 2h, continue to wash with PBST for 4 times, add a certain proportion of diluted secondary antibody HRP anti-mFC (Jackson Immuno Research, 115-035-164), incubate at 37°C for 1.5h, and finally wash with PBST for 4 After three times, add TMB chromogenic solution, incubate at 37°C for 10-15min, and read with microplate reader (wavelength 450nm and 655nm).
  • the experimental results show the basic situation of the binding of the antibody of the present disclosure and tisleliumab to the TIGIT epitope.
  • 7103-06, 7103-07 did not compete with Tireliumab, indicating that their binding epitopes are different from Tireliumab.
  • 7103-01, 7103-03, and 7103-04 partially blocked the combination of tisleliumab and TIGIT, indicating that the epitopes combined by 7103-01, 7103-03, and 7103-04 had a certain affinity with tisleliumab. overlap, but are different.
  • the antibodies of the present invention can be divided into 2 groups:
  • Antibodies 7103-06, 7103-07 that did not compete with tisleliumab, which bind to a different epitope than tisleliumab;
  • Group B Antibodies 7103-01, 7103-03, and 7103-04 that partially blocked the binding of tisleliumab to TIGIT.
  • the binding epitopes overlapped with tisleliumab, but were different.
  • the antibodies 7103-01, 7103-03, 7103-04 mutually blocked each other's TIGIT binding, indicating that they bind the same epitope.
  • Example 6 Human-mouse chimeric TIGIT monoclonal antibody enhances INF ⁇ production of PBMCs in vitro
  • lymphocytes secrete the cytokine IFN ⁇ .
  • TIGIT human-mouse chimeric TIGIT monoclonal antibody
  • Figure 12 shows that five strains of human and mouse chimeric TIGIT monoclonal antibodies of the present disclosure (No. 07(hFc)) can promote PHA-activated PBMC to produce IFN ⁇ , and its effects are better than that of tisleliumab, and the percentage of increase in release amount reaches 42%-62% of that of tisleliumab.
  • the humanized antibody sequence was designed. The specific steps are as follows: First, use Discovery Studio and Antibody Modeling, using the homology modeling method to construct a three-dimensional molecular model of the variable region. Next, by comparing the existing antibody structures in the database, the structure simulations of the variable regions and CDRs of the parental antibodies were performed. At the same time, human germline (Germline) sequences derived from cDNAs with high homology to the murine parental antibody VH and VL were selected for comparison. The heavy chain VH selects IGHV1 with the highest degree of homology as the humanized design template and sequence design. IGKV1 was selected as the humanized design template and sequence for light chain VL.
  • the original heavy chain mVH sequence of murine parental antibody No. 7103-01 was designed as 4 humanized sequences: 7103-01-huVH1 (SEQ ID NO: 124), 7103-01-huVH2 (SEQ ID NO: 125), 7103 -01-huVH3 (SEQ ID NO: 126) and 7103-01-huVH4 (SEQ ID NO: 127); the antibody original light chain mVL was designed as 2 humanized sequences: 7103-01-huVL1 (SEQ ID: 129) and 7103-01-huVL2 (SEQ ID: 130).
  • the original heavy chain mVH sequence of the murine parental antibody 7103-07 was designed as 5 humanized sequences: 7103-07-huVH1 (SEQ ID: 131), 7103-07-huVH2 (SEQ ID: 132), 7103-07- huVH3 (SEQ ID: 134), 7103-07-huVH4 (SEQ ID: 135), 7103-07-huVH5 (SEQ ID: 136); the original light chain mVL was designed as 5 humanized sequences: 7103-07-huVL1 (SEQ ID NO: 137), 7103-07-huVL2 (SEQ ID NO: 138), 7103-07-huVL3 (SEQ ID NO: 139), 7103-07-huVL4 (SEQ ID NO: 140), 7103-07 -huVL5 (SEQ ID NO: 141).
  • the sequence was constructed on the pcDNA3.4 vector, and the plasmid was obtained after PCR, digestion, ligation, transformation, identification, sequencing, comparison and extraction.
  • the above plasmids were expressed in ExpiCHO-S cells for 7 days as required, and on the 6th day of expression, by HPLC Measure the expression level. Finally, one-step purification by ProteinA affinity chromatography to obtain the antibody protein to be tested.
  • Biofilm optical interferometry was used to study the binding activity of humanized TIGIT monoclonal antibody to TIGIT.
  • the results of ELISA ( FIG. 13 ) showed that six 7103-01-anti-human TIGIT humanized monoclonal antibodies (numbers P03486, P03487, P03490, P03491, P03492, and P03493) could specifically bind to human TIGIT.
  • the binding affinities EC 50 are 0.511nM, 0.532nM, 0.501nM, 0.471nM, 0.463nM, 0.444nM, respectively, compared with 0.540nM of tisrelumab, the humanized monoclonal antibody of the present invention has a higher binding affinity to human TIGIT high.
  • the results of the ELISA test ( FIG. 14A ) showed that nine 7103-07 anti-human TIGIT humanized monoclonal antibodies (numbers P03476, P03477, P03478, P03479, P03480, P03481, P03482, P03483 and P03484) could specifically bind to human TIGIT.
  • the binding affinities EC 50 are 0.221nM, 0.194nM, 0.161nM, 0.0858nM, 0.252nM, 0.122nM, 0.182nM, 0.212nM, 0.187nM, compared with human-mouse chimeric antibody EC 50 is 0.206nM, and the EC of Tira 50 is 0.528nM, indicating that the recombinantly expressed humanized antibody has a good activity in binding to the antigen TIGIT, which is equivalent to that of human-mouse chimeric antibody and better than that of tiraliumab (Tira).
  • the binding affinities EC 50 are 0.0419nM, 0.0330nM, 0.0913nM, 0.0915nM, 0.0836nM respectively, compared with human-mouse chimeric antibody EC 50 is 0.0640nM, and Tira’s EC 50 is 0.1619nM, which also shows that the recombinantly expressed human Antibody binding activity of TIGIT antigen is very good, equivalent to human-mouse chimeric antibody, better than Tiraliumab (Tira).
  • the three 7103-01 anti-human TIGIT humanized monoclonal antibodies (numbers P03489, P03492, and P03493) all bind well to CHO-TIGIT, and the EC 50 for binding to CHO-TIGIT cell membranes are: 0.761 nM, 0.437nM, 0.557nM, significantly better than Tira's 1.356nM.
  • the 8 strains of 7103-07 anti-human TIGIT humanized monoclonal antibodies (No. The EC 50 on the TIGIT cell membrane are: 1.518nM, 1.738nM, 2.582nM, 0.963nM, 2.193nM, 2.824nM, 2.376nM, 2.607nM, compared with 1.347nM of human-mouse chimeric monoclonal antibody and 3.844 of Tira.
  • the affinity of the invented humanized antibody is higher.
  • the humanized antibody of the present disclosure not only has strong binding ability to solid-phase antigen (ELISA method), but also has high binding activity to TIGIT on the cell membrane, and is also superior to Tira in binding to TIGIT on the cell membrane surface.
  • FIG. 17A The experimental results ( FIG. 17A ) showed that nine 7103-07 anti-human TIGIT humanized monoclonal antibodies (numbers P03476, P03477, P03478, P03479, P03480, P03481, P03482, P03483 and P03484) could specifically block the interaction between CD155 and human The combination of TIGIT.
  • IC 50 are: 1.316nM, 0.928nM, 0.989nM, 0.714nM, 1.239nM, 0.620nM, 0.791nM, 0.836nM, 1.288nM, the blocking ability is equivalent to the IC 50 value of human mouse chimeric monoclonal antibody 1.033nM or Better, and significantly better than Tireliumab (IC 50 value 2.534nM).
  • IC 50 are: 0.630nM, 0.594nM, 0.127nM, 0.631nM, the blocking ability is equivalent to the IC 50 value of human mouse chimeric monoclonal antibody 0.273nM, and better than tisrelumab (IC 50 value 0.775nM ).
  • lymphocytes secrete the cytokine IFN ⁇ .
  • TIGIT monoclonal antibody as a positive regulator of T cell activation, we conducted the following experiments to illustrate the enhanced effect of the disclosed antibody on TIGIT signal blockade on PBMC cell IFN ⁇ production.
  • Experimental method thaw frozen human peripheral blood mononuclear cell PBMC, adjust the cell density to 5 ⁇ 10 ⁇ 6 cells/mL, and dilute the antibody with RPMI 1640+10% FBS medium to an antibody concentration of 20 ⁇ g/mL and a PHA concentration of 6 ⁇ g /mL, respectively add 50 ⁇ L of PHA dilution and 50 ⁇ L of antibody dilution to the 96-well plate, and finally add 100 ⁇ L of cell suspension to the 96-well plate, mix gently and place in a 37°C, 5% CO2 incubator cultured for 4 days. After 4 days, 100 ⁇ L of the supernatant was drawn to detect the content of IFN ⁇ .
  • seven humanized TIGIT monoclonal antibodies can promote the release of IFN ⁇ from PBMCs activated by PHA.
  • the release of IFN ⁇ after adding humanized TIGIT antibody was 31%-81% higher than that without adding humanized TIGIT monoclonal antibody, and the effects were all better than tisrelumab (16%).
  • Example 10 Functional research on inhibiting tumor growth in mice in vivo
  • mice C57BL/6
  • Human TIGIT gene knock-in mice were raised in an SPF environment.
  • the mouse tumor model is colon cancer MC38.
  • mice in each group were given PBS (control group), recombinant human IgG110 mg/kg (control group) and TIGIT humanized antibody P0347910 mg/kg by intraperitoneal injection. Administered 2 times a week, 4 times in total. The tumor volume (ie, long diameter ⁇ short diameter 2 /2) was measured at each administration, and the mice were weighed. The experiment was terminated on D17. The mice were sacrificed by cervical dislocation, and the tumors were weighed.
  • Figure 19A shows the tumor volume per mouse in each group versus time
  • Figure 19B shows the tumor weight per mouse in each group at the end of the experiment (D17).
  • the experimental results showed that TIGIT humanized antibody P03479 could effectively inhibit the growth of mouse tumors ( Figure 19A and 19B), and at the end of the experiment (D17), the average weight of mouse tumors in the P03479 group was significantly smaller than that in the hIgG1 group (p ⁇ 0.05) .

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Abstract

提供了结合人TIGIT（含Ig和ITIM结构域的T细胞免疫受体，T cell immunoreceptor with immunoglobulin and ITIM domains）的抗体或其抗原结合片段；还提供了编码该抗体或其抗原结合片段的核酸分子、包含该核酸分子的载体和宿主细胞，以及包含该抗体或其抗原结合片段的免疫偶联物、组合物，以及该抗体或其抗原结合片段在制备正向调节免疫细胞活性和/或提高免疫应答的产品、减少或消除表达TIGIT的细胞(如Treg细胞)的免疫抑制作用的产品、检测TIGIT存在与否、其水平或活性的试剂盒中的应用。

Description

靶向TIGIT的单克隆抗体 技术领域
本公开属于生物技术和医学领域。具体而言，本公开涉及能够结合人TIGIT(含Ig和ITIM结构域的T细胞免疫受体，T cell immunoreceptor with immunoglobulin and ITIM domains)的抗体或其抗原结合片段，及其编码核酸分子、载体和宿主细胞，以及包含所述抗体或片段的免疫偶联物、组合物，以及上述产品的应用。
背景技术
T细胞是免疫应答的关键介导者，T细胞的活化依赖TCR信号和共刺激信号。免疫应答中，共刺激信号有的发挥正向刺激作用，有的行使负向调节功能，即免疫应答既会受到共刺激分子的正调节(如OX40、GITR和4-1BB)，也会受到免疫应答受检查点分子(如PD-1和CTLA-4)的负调节(Rudd C.E.，Unifying concepts in CD28，ICOS and CTLA4 co-receptor signalling.Nat Rev Immunol 3：544-556，2003；Rudd C.E.，The reverse stop-signal model for CTLA4function，Nat Rev Immunol 8：153-160，2008)。抑制性共刺激可以预防免疫反应的发作或下调免疫反应，然而，它们也限制了免疫系统对抗癌症和感染性疾病的能力。
肿瘤免疫治疗是近年来肿瘤治疗基础与临床研究的前沿及热门领域。以CTLA-4和PD-1/PD-L1单抗为代表的第一代免疫检查点抑制剂，能通过激活T细胞介导的免疫应答而发挥抗肿瘤作用。然而，第一代免疫检查点抗体仍然面临着受益患者有限、多数肿瘤效果不佳、药物耐受等问题。同时，相当一部分早期治疗有效的患者在治疗过程中或之后可能出现肿瘤复发的情况。为了解决以上问题，发现和验证新的免疫检查点抑制性分子变得至关重要。
多种新的免疫检查点分子已经被报道，例如TIM-3、LAG-3、TIGIT、BTLA和VISTA(Mercier等，Front.Immunol.6：418，2015)。含Ig和ITIM结构域的T细胞免疫受体(T cell immunoreceptor with immunoglobulin and ITIM domains，TIGIT，也称WUCAM、Vstm3或VSIG9)，是一种主要表达在NK细胞、T细胞、Treg细胞等免疫细胞表面的检查点分子，其在细胞质尾部具有基于免疫受体酪氨酸的抑制基序(ITIM)，是典型的抑制性受体蛋白(Yu等，Nat.Immunol.10：48-57，2009)。
已知TIGIT可以与CD155(也称为PVR和necl-5)、CD112(也称为PVRL2和粘连蛋白-2)以及可能与CD113(也称为PVRL3和粘连蛋白-3)相互作用，其中CD155是TIGIT的高亲和力核心配体，其与TIGIT的相互作用力远高于通路中的其它配对分子(Martinet等.Nat.Rev.Immunol.15：243-254，2015)。
TIGIT可通过多种机制抑制机体的免疫反应。通过靶向T细胞抗原受体(TCR)的信号通路分子，TIGIT直接阻断初始T细胞(Tn)活化、增殖和效应功能的获得，并且能够抑制CD4+T细胞的细胞增殖和炎症细胞因子的产生(Fourcade J等，JCI Insight，2018，3(14)：e121157)。TIGIT可以通过调节树突细胞的细胞因子产生来间接抑制T细胞(Yu等，Nat.Immunol.10：48-57，2009)。TIGIT还可增强Tregs稳定性及其对产生IFN-γ的T细胞增殖的抑制功能(Fourcade J等，JCI Insight，2018，3(14)：e121157)。在肿瘤环境中，TIGIT高表达于肿瘤浸润或迁移相关的NK细胞与效应T细胞表面，CD155在肿瘤细胞表面高表达，肿瘤细胞通过CD155/TIGIT的结合直接作用于NK细胞和效应T细胞，抑制其活性(Li等，J.Biol.Chem.289：17647-17657，2014)。
综上所述，对于增强免疫应答以及预防和/或治疗肿瘤、感染或感染性疾病，TIGIT是极具潜力的免疫治疗新靶点。靶向TIGIT的CD155阻断型抗体，特别是抗人TIGIT抗体，能释放免疫效应细胞的肿瘤杀伤活性，有望取得良好的抗肿瘤疗效，适应症包括小细胞肺癌、食道癌、食管鳞状细胞癌、非小细胞肺癌、头颈部鳞状细胞癌、肝癌、宫颈癌、三阴性乳腺肿瘤、B细胞淋巴瘤、多发性骨髓癌、非霍奇金淋巴瘤、黑色素瘤、卵巢癌、结肠癌、乳腺癌等。
目前，有几个TIGIT抗体处在临床试验中，包括罗氏的替瑞利尤单抗(Tiragolumab)已经进入三期临床实验。然而，目前国内外还没有阻断TIGIT的单克隆抗体药物上市，因此亟须开发出高亲和力、高特异性且效果更优的TIGIT单克隆抗体。
发明内容
本公开正是提供了一种高亲和力、高特异性且效果更优的TIGIT单克隆抗体。
在本公开的一些方面中，提供了靶向T细胞免疫球蛋白和ITIM结构蛋白(TIGIT)的单克隆抗体或其抗原结合片段，其与CD155竞争性结合TIGIT。
在本公开的一些方面中，提供了靶向T细胞免疫球蛋白和ITIM结构域蛋白(TIGIT)的单克隆抗体或其抗原结合片段，其包含本文中所公开的特定重链互补决定区(VH CDR)1～3和轻链互补决定区(VL CDR)1～3，或与其具有至少95％序列同一性的序列。
在本公开的一些方面中，提供了编码本公开单克隆抗体或其抗原结合片段的核酸分子，或包含所述核酸分子的载体或宿主细胞。
在本公开的一些方面中，提供了一种免疫偶联物，其包含本公开的单克隆抗体或其抗原结合片段。
在本公开的一些方面中，提供了一种组合物，其包含本公开的单克隆抗体 或其抗原结合片段、核酸分子、载体或宿主细胞或免疫偶联物。
在本发明的一些方面中，提供了嵌合抗原受体，其包含胞外结构域和胞内结构域，其中，所述胞外结构域包含本申请的单克隆抗体或其抗原结合片段。
在本发明的一些方面中，提供了核酸分子以及包含所述核苷酸分子的构建体或载体，其中，所述核酸分子包含本申请嵌合抗原受体的编码序列。
在本发明的一些方面中，提供了经转化的免疫细胞，其表达根据本申请的嵌合抗原受体，或采用本申请的核酸分子、构建体或载体转化，例如所述免疫细胞选自T细胞，如αβT细胞、γδT细胞或NK T细胞或从多能细胞衍生的T细胞。
在本公开的一些方面中，提供了本公开的单克隆抗体或其抗原结合片段、核酸分子、载体或宿主细胞、免疫偶联物、本公开的嵌合抗原受体或其编码核酸分子、构建体或载体、经转化的免疫细胞和/或本公开的组合物在制备正向调节免疫细胞活性和/或提高免疫应答的药物中的应用，例如用于制备预防和/或治疗肿瘤、感染或感染性疾病的药物中的应用。
在本公开的一些方面中，提供了一种在正向调节免疫细胞活性和/或提高免疫应答的方法，其包括给予有需要的对象本公开的单克隆抗体或其抗原结合片段、核酸分子、载体或宿主细胞、免疫偶联物、本公开的嵌合抗原受体或其编码核酸分子、构建体或载体、经转化的免疫细胞和/或本公开的组合物，例如用于预防和/或治疗肿瘤、感染或感染性疾病。
在本公开的一些方面中，提供了一种用于正向调节免疫细胞活性和/或提高免疫应答(例如用于预防和/或治疗肿瘤、感染或感染性疾病)的本公开的单克隆抗体或其抗原结合片段、核酸分子、载体或宿主细胞、免疫偶联物、本公开的嵌合抗原受体或其编码核酸分子、构建体或载体、经转化的免疫细胞和/或本公开的组合物。
在本公开的一些方面中，提供了本公开的单克隆抗体或其抗原结合片段、核酸分子、载体或宿主细胞、免疫偶联物、本公开的嵌合抗原受体或其编码核酸分子、构建体或载体、经转化的免疫细胞和/或本公开的组合物在制备减少或消除表达TIGIT的细胞(例如Treg细胞)的产品中的应用。
在本公开的一些方面中，提供了一种减少或消除表达TIGIT的细胞(例如Treg细胞)的方法，其包括给予有需要的对象本公开的单克隆抗体或其抗原结合片段、核酸分子、载体或宿主细胞、免疫偶联物、本公开的嵌合抗原受体或其编码核酸分子、构建体或载体、经转化的免疫细胞和/或本公开的组合物。
在本公开的一些方面中，提供了用于减少或消除表达TIGIT的细胞(例如Treg细胞)的本公开的单克隆抗体或其抗原结合片段、核酸分子、载体或宿主细胞、免疫偶联物、本公开的嵌合抗原受体或其编码核酸分子、构建体或载体、 经转化的免疫细胞和/或本公开的组合物。
本领域的技术人员可对前述的技术方案和技术特征进行任意组合而不脱离本公开的发明构思和保护范围。本公开的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
下面结合附图对本公开作进一步说明，其中这些显示仅为了图示说明本公开的实施方案，而不是为了局限本公开的范围。
图1显示8株抗人TIGIT鼠单克隆抗体(编号7103-01、7103-02、7103-03、7103-04、7103-05、7105-06、7103-07、7103-08)的SDS-PAGE电泳分析研究：上图：非还原4-12％SDS-PAGE电泳分析；下图：还原4-12％SDS-PAGE电泳分析。非还原的抗体的分子量在150KD左右，还原抗体的分子量分别在50KD和25KD左右。
图2显示了8株抗人TIGIT鼠单克隆抗体(编号7103-01、7103-02、7103-03、7103-04、7103-05、7105-06、7103-07、7103-08)体外与重组人TIGIT结合的ELISA研究。
图3显示了8株抗人TIGIT鼠单克隆抗体(编号7103-01、7103-02、7103-03、7103-04、7103-05、7105-06、7103-07、7103-08)与CD155对重组人TIGIT的结合的竞争的ELISA研究。
图4显示了8株抗人TIGIT鼠单克隆抗体(编号7103-01、7103-02、7103-03、7103-04、7103-05、7105-06、7103-07、7103-08)体外与重组食蟹猴TIGIT结合的ELISA研究。
图5显示了五株抗人TIGIT鼠单克隆抗体(编号7103-01、7103-03、7103-04、7105-06、7103-07)与过表达TIGIT的CHO细胞的膜TIGIT结合的流式细胞技术分析研究。
图6显示了5株抗人TIGIT人鼠嵌合单克隆抗体(编号7103-01(hFc)、7103-03(hFc)、7103-04(hFc)、7103-06(hFc)、7103-07(hFc))的SDS-PAGE电泳分析研究。其中，非还原的抗体的分子量在150KDa左右，还原抗体的分子量分别在55kDa和25KDa。
图7显示了5株抗人TIGIT人鼠嵌合单克隆抗体(编号7103-01(hFc)、7103-03(hFc)、7103-04(hFc)、7103-05(hFc)、7103-06(hFc)、7103-07(hFc))体外与重组人TIGIT结合的ELISA研究。
图8显示了五株人鼠嵌合TIGIT单克隆抗体(编号7103-01(hFc)、7103-03(hFc)、7103-04(hFc)、7103-05(hFc)、7103-06(hFc)、7103-07(hFc))与膜TIGIT结合的流式细胞技术分析研究。
图9显示了5株人鼠嵌合TIGIT单克隆抗体(编号7103-01(hFc)、7103-03(hFc)、7103-04(hFc)、7103-05(hFc)、7103-06(hFc)、7103-07(hFc))与CD155对重组人TIGIT的结合的竞争的ELISA研究。
图10显示了抗人TIGIT鼠单克隆抗体(编号7103-01、7103-03、7103-04、7105-06、7103-07)与替瑞利尤单抗对重组人TIGIT的结合的竞争的ELISA研究。图中isotype为同型抗体阴性对照。
图11显示了3株抗人TIGIT鼠单克隆抗体(编号7103-01、7103-03、7103-04)分别与人鼠嵌合TIGIT单克隆抗体7103-01(hFc)、7103-03(hFc)、7103-04(hFc)对重组人TIGIT的结合的竞争的ELISA研究。
图12显示了5株人鼠嵌合TIGIT单克隆抗体(编号7103-01(hFc)、7103-03(hFc)、7103-04(hFc)、7103-05(hFc)、7103-06(hFc)、7103-07(hFc))对PHA激活的PBMC产生IFNγ的促进作用。
图13显示了6株7103-01抗人TIGIT人源化单克隆抗体(编号P03486、P03487、P03490、P03491、P03492、P03493)体外与重组人TIGIT结合的ELISA研究。将人鼠嵌合7301-01(hFc)抗体和替瑞利尤单抗用作阳性对照。
图14A显示了9株7103-07抗人TIGIT人源化单克隆抗体(编号P03476、P03477、P03478、P03479、P03480、P03481、P03482、P03483和P03484)体外与重组人TIGIT结合的ELISA研究。将人鼠嵌合抗体7103-07(hFc)和替瑞利尤单抗用作阳性对照。
图14B显示了另一批次5株7103-07抗人TIGIT人源化单克隆抗体(编号P04662、P04663、P04664、P04665、P04666和P04667)体外与重组人TIGIT结合的ELISA研究。将人鼠嵌合抗体7103-07(hFc)和替瑞利尤单抗(或称Tira)用作阳性对照。
图15显示了3株7103-01抗人TIGIT人源化单克隆抗体(编号P03489、P03492、P03493)与膜TIGIT结合的流式细胞技术分析研究。
图16显示了9株7103-07抗人TIGIT人源化单克隆抗体(编号P03476、P03477、P03478、P03479、P03480、P03481、P03482、P03483))与膜TIGIT结合的流式细胞技术分析研究。
图17A显示了9株7103-07抗人TIGIT人源化单克隆抗体(编号P03476、P03477、P03478、P03479、P03480、P03481、P03482、P03483和P03484)与CD155对重组人TIGIT的结合的竞争的ELISA研究。
图17B显示了另一批次4株7103-07抗人TIGIT人源化单克隆抗体编号(编号P04662、P04663、P04666和P04667)与CD155对重组人TIGIT的结合的竞争的ELISA研究。
图18显示了7株人源化TIGIT单克隆抗体(编号P03479、P03480、P03481、 P03488、P03489、P03491和P03493)对PHA激活的PBMC产生IFNγ的促进作用。
图19：显示了抗人TIGIT人源化单克隆抗体在小鼠体内抑制小鼠肿瘤生长的作用：
图19A：各组每只小鼠的肿瘤体积与时间的关系；
图19B：试验结束时(D17)各组每个小鼠的肿瘤重量。
以上图中，*表示p＜0.05，**表示p＜0.01。
具体实施方式
本公开提供了可以特异性结合于人TIGIT的抗体、抗体片段以及相关的组合物和分子。进一步地，本公开涉及所述TIGIT抗体的鼠源抗体、人鼠嵌合抗体以及人源化抗体。本公开还涉及包含所述TIGIT抗体的药物组合物，以及其用于相关疾病诊断剂和治疗药物的用途。本公开的抗TIGIT抗体对人TIGIT具有高特异性和高亲和力，能够有效阻断TIGIT与其配体CD155的结合，增强免疫细胞的细胞因子释放，提高免疫细胞活性、增强免疫应答，用于预防和/或治疗肿瘤、感染或感染性疾病。本公开的抗TIGIT抗体还能够减少或消除表达TIGIT的细胞(例如Treg细胞)或其活性。
本公开还提供了一种增强免疫应答，预防和/或治疗肿瘤、感染或感染性疾病的方法，其包括向受试者使用治疗有效量的本文所述的药物组合物，其中所述药物组合物阻断人TIGIT与配体CD155的结合。
本文中提供的所有数值范围旨在清楚地包括落在范围端点之间的所有数值及它们之间的数值范围。可对本公开提到的特征或实施例提到的特征进行组合。本说明书所揭示的所有特征可与任何组合物形式并用，说明书中所揭示的各个特征，可以任何可提供相同、均等或相似目的的替代性特征取代。因此除有特别说明，所揭示的特征仅为均等或相似特征的一般性例子。
如本文所用，“含有”、“具有”或“包括”包括了“包含”、“主要由......构成”、“基本上由......构成”、和“由......构成”；“主要由......构成”、“基本上由......构成”和“由......构成”属于“含有”、“具有”或“包括”的下位概念。
单克隆抗体及其制备
本文所用的术语“单克隆抗体(单抗)”指从一类基本均一的群体获得的抗体，即该群体中包含的单个抗体是相同的，除少数可能存在的天然发生的突变外。单克隆抗体高特异性地针对单个抗原位点。而且，与常规多克隆抗体制剂(通常是具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们是通过杂交 瘤培养来合成的，不会被其它免疫球蛋白污染。修饰语“单克隆”表示了抗体的特性，是从基本均一的抗体群中获得的，这不应被解释成需要用任何特殊方法来生产抗体。
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150,000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分b折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等，NIH Publ.No.91-3242，卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
本公开提供了特异性结合人TIGIT的抗体，包括鼠抗、嵌合抗体以及人源化抗体。本公开的单克隆抗体也可以是嵌合抗体或人源化抗体。在本公开中，如果没有特别说明，“m”表示鼠源部分，“h”表示人源部分。在一些实施方式中，所述抗体包括重链可变区(VH)，所述重链可变区包括3个互补决定区(CDR)，所述抗体包括轻链可变区(VL)，所述轻链可变区包括3个CDR。
如本文所用，术语“序列同一性”或“同一性％”是指在比对序列并且(必要时)引入空位以获得的最大序列相同性百分比之后，候选序列与参考序列中相同残基(例如氨基酸或核酸)的百分比。例如，如本文所用，“至少70％的序列同一性”是指候选序列与参考序列间的序列同一性达到70％以上，如75、80、85、90、91、92、93、94、95、96、97、98、99、100％或其中任意数值点的同一性。
在一些实施方式中，所述抗体的VH氨基酸序列选自：SEQ ID NO：27、35、43、51、59、67、75、83、124、125、126、127、131、132、134、135、136或与其具有至少70％序列同一性的序列。
在一些实施方式中，所述抗体的VL氨基酸序列选自：SEQ ID NO：31、39、47、55、63、71、79、87、129、130、137、138、139、140、141或与其具有 至少70％序列同一性的序列。
在一些实施方式中，所述抗体或其抗原结合片段包含选自下组的VH CDR1：SEQ ID NO：28、36、44、52、60、68、76、84；选自下组的VH CDR2：SEQ ID NO：29、37、45、53、61、69、77、85、128、133、142、143。在一些实施方式中，所述抗体或其抗原结合片段包含选自下组的VH CDR3：SEQ ID NO：30、38、46、54、62、70、78、86。在一些实施方式中，所述抗体或其抗原结合片段包含选自下组的VL CDR1：SEQ ID NO：32、40、48、56、64、72、80、88。在一些实施方式中，所述抗体或其抗原结合片段包含选自下组的VL CDR2：SEQ ID NO：33、41、49、57、65、73、81、89。在一些实施方式中，所述抗体或其抗原结合片段包含选自下组的VL CDR3：SEQ ID NO：34、42、50、58、66、74、72、90。
在一些实施方式中，可对所述抗体的CDR序列进行一定改变以例如满足人源化等需求。例如，经改变的CDR可与亲本序列具有至少90％～99％或更高的序列同一性。例如，经改变的CDR可与亲本序列具有1个、2个或3个氨基酸残基的差异。例如，VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2、VL CDR3的一者或多者中包含改变的氨基酸残基。在一些实施方式中，VH CDR2被改变。例如，在一些实施方式中，SEQ ID NO：29的VH CDR2被SEQ ID NO：128或SEQ ID NO：142的序列所替代；例如在一些实施方式中，SEQ ID NO：77的VH CDR2被SEQ ID NO：133或SEQ ID NO：143的序列所替代。
在一些实施方式中，可对本文公开的CDR序列进行不同组合以获得具有所需结合和/或靶向和/或活性的抗体或抗原结合片段。在一些实施方式中，可将SPSYRYT(SEQ ID NO：33、57和65)的VL CDR2与SEQ ID NO：32和34、或SEQ ID NO：56和58、或SEQ ID NO：64和66的VL CDR1和VL CDR3组合。在一些实施方式中，可将QQHYSTPWT(SEQ ID NO：34、50和66)的VL CDR3与SEQ ID NO：32和33、或SEQ ID NO：48和49、或SEQ ID NO：64和65的VL CDR1和VL CDR2组合。在一些实施方式中，可将TYAMS(SEQ ID NO：68和76)的VH CDR1、KTLDYYALDY(SEQ ID NO：70和78)的VH CDR3、SGSRLQS(SEQ ID NO：73和81)的VL CDR2以及QQHNEYPWT(SEQ ID NO：74和82)的VL CDR3与SEQ ID NO：69或77的VH CDR2和SEQ ID NO：72或80的VH CDR1组合。
在一些实施方式中，所述抗体包含选自以上的VH氨基酸序列和VL氨基酸序列的任意组合。在一些实施方式中，所述抗体的VH和VL的氨基酸序列组合分别为：SEQ ID NO：27和SEQ ID NO：31；SEQ ID NO：35和39；SEQ ID NO：43和47；SEQ ID NO：51和55；SEQ ID NO：59和63；SEQ ID NO：67和71；SEQ ID NO：75和79；SEQ ID NO：83和87；选自SEQ ID NO：124-126的 任一人源化VH与选自SEQ ID NO：31、39、47、55、63、71、79、87、129、130、137、138、139、140、141的任一VL序列的组合；选自SEQ ID NO：27、35、43、51、59、67、75、83、124、125、126、127、131、132、134、135、136的任一VH序列与选自SEQ ID NO：129、130、137、138、139、140、141中任一人源化VL序列的组合。
在一些实施方式中，所述抗体的VH CDR1、2和3的氨基酸序列分别是SYNIY(SEQ ID NO：28)、GVNPSNGNTNFNENFKS(SEQ ID NO：29)和GNYYGYEFAY(SEQ ID NO：30)。在一些实施方式中，所述抗体的VL CDR1、2和3的氨基酸序列分别是KASQHVSTAVA(SEQ ID NO：32)、SPSYRYT(SEQ ID NO：33)和QQHYSTPWT(SEQ ID NO：34)。
在一些实施方式中，所述抗体其VH氨基酸序列为SEQ ID NO：27或与其具有至少70％序列同一性的序列，所述抗体的VH CDR1、2和3的氨基酸序列分别是SYNIY(SEQ ID NO：28)、GVNPSNGNTNFNENFKS(SEQ ID NO：29)和GNYYGYEFAY(SEQ ID NO：30)。在一些实施方式中，所述抗体其VL氨基酸序列为SEQ ID NO：31或与其具有至少70％序列同一性的序列，所述抗体的VL CDR1、2和3的氨基酸序列分别是KASQHVSTAVA(SEQ ID NO：32)、SPSYRYT(SEQ ID NO：33)和QQHYSTPWT(SEQ ID NO：34)。
在一些实施方式中，所述抗体的VH CDR1、2和3的氨基酸序列分别是SYVMH(SEQ ID NO：36)、YINPYNDGTKYNEKFKG(SEQ ID NO：37)和WNPYYYAMDY(SEQ ID NO：38)。在一些实施方式中，所述抗体的VL CDR1、2和3的氨基酸序列分别是SANSSARYVH(SEQ ID NO：40)、EISKLAS(SEQ ID NO：41)和QQWNYPLIT(SEQ ID NO：42)。
在一些实施方式中，所述抗体其VH的氨基酸序列为SEQ ID NO：35或与其具有至少70％序列同一性的序列，CDR1、2和3的氨基酸序列分别是SYVMH(SEQ ID NO：36)、YINPYNDGTKYNEKFKG(SEQ ID NO：37)和WNPYYYAMDY(SEQ ID NO：38)。VL的氨基酸序列为SEQ ID NO：39或与其具有至少70％序列同一性的序列，CDR1、2和3的氨基酸序列分别是SANSSARYVH(SEQ ID NO：40)、EISKLAS(SEQ ID NO：41)和QQWNYPLIT(SEQ ID NO：42)。
在一些实施方式中，所述抗体其VH CDR1、2和3的氨基酸序列分别是EYTMH(SEQ ID NO：44)、GINPNNGGTSYNQKFKG(SEQ ID NO：45)和GAYYRYDFDY(SEQ ID NO：46)。在一些实施方式中，所述抗体其VL CDR1、2和3的氨基酸序列分别是KASQDVRTAVV(SEQ ID NO：48)、SASYRYT(SEQ ID NO：49)和QQHYSTPWT(SEQ ID NO：50)。
在一些实施方式中，所述抗体其VH的氨基酸序列为SEQ ID NO：43或 与其具有至少70％序列同一性的序列，CDR1、2和3的氨基酸序列分别是EYTMH(SEQ ID NO：44)、GINPNNGGTSYNQKFKG(SEQ ID NO：45)和GAYYRYDFDY(SEQ ID NO：46)。VL的氨基酸序列为SEQ ID NO：47或与其具有至少70％序列同一性的序列，CDR1、2和3的氨基酸序列分别是KASQDVRTAVV(SEQ ID NO：48)、SASYRYT(SEQ ID NO：49)和QQHYSTPWT(SEQ ID NO：50)。
在一些实施方式中，所述抗体其VH CDR1、2和3的氨基酸序列分别是DYFVY(SEQ ID NO：52)、GINPSNGGATFNEKFKN(SEQ ID NO：53)和GNFYGYEFAY(SEQ ID NO：54)。在一些实施方式中，所述抗体的VL CDR1、2和3的氨基酸序列分别是KASQHVSTAVV(SEQ ID NO：56)、SPSYRYT(SEQ ID NO：57)和QQHYITPWT(SEQ ID NO：58)。
在一些实施方式中，所述抗体其VH的氨基酸序列为SEQ ID NO：51或与其具有至少70％序列同一性的序列，CDR1、2和3的氨基酸序列分别是DYFVY(SEQ ID NO：52)、GINPSNGGATFNEKFKN(SEQ ID NO：53)和GNFYGYEFAY(SEQ ID NO：54)。VL的氨基酸序列为SEQ ID NO：55或与其具有至少70％序列同一性的序列，CDR1、2和3的氨基酸序列分别是KASQHVSTAVV(SEQ ID NO：56)、SPSYRYT(SEQ ID NO：57)和QQHYITPWT(SEQ ID NO：58)。
在一些实施方式中，所述抗体其VH CDR1、2和3的氨基酸序列分别是EYTMY(SEQ ID NO：60)、GINPNNGGTRYNQKFKG(SEQ ID NO：61)和GGHYDYGFAY(SEQ ID NO：62)。在一些实施方式中，所述抗体的VL CDR1、2和3的氨基酸序列分别是KASQDVKTAVA(SEQ ID NO：64)、SPSYRYT(SEQ ID NO：65)和QQHYSTPWT(SEQ ID NO：66)。
在一些实施方式中，所述抗体其VH的氨基酸序列为SEQ ID NO：59或与其具有至少70％序列同一性的序列，CDR1、2和3的氨基酸序列分别是EYTMY(SEQ ID NO：60)、GINPNNGGTRYNQKFKG(SEQ ID NO：61)和GGHYDYGFAY(SEQ ID NO：62)。VL的氨基酸序列为SEQ ID NO：63或与其具有至少70％序列同一性的序列，CDR1、2和3的氨基酸序列分别是KASQDVKTAVA(SEQ ID NO：64)、SPSYRYT(SEQ ID NO：65)和QQHYSTPWT(SEQ ID NO：66)。
在一些实施方式中，所述抗体的VH CDR1、2和3的氨基酸序列分别是TYAMS(SEQ ID NO：68)、EISSGGSYTYYSDTVTG(SEQ ID NO：69)和和KTLDYYALDY(SEQ ID NO：70)。在一些实施方式中，所述抗体的VL CDR1、2和3的氨基酸序列分别是RANKTISKYLA(SEQ ID NO：72)、SGSRLQS(SEQ ID NO：73)和QQHNEYPWT(SEQ ID NO：74)。
在一些实施方式中，所述抗体其VH的氨基酸序列为SEQ ID NO：67或与其具有至少70％序列同一性的序列，CDR1、2和3的氨基酸序列分别是TYAMS(SEQ ID NO：68)、EISSGGSYTYYSDTVTG(SEQ ID NO：69)和KTLDYYALDY(SEQ ID NO：70)。VL的氨基酸序列为SEQ ID NO：71或与其具有至少70％序列同一性的序列，CDR1、2和3的氨基酸序列分别是RANKTISKYLA(SEQ ID NO：72)、SGSRLQS(SEQ ID NO：73)和QQHNEYPWT(SEQ ID NO：74)。
在一些实施方式中，所述抗体其VH CDR1、2和3的氨基酸序列分别是TYAMS(SEQ ID NO：76)、EISSGGSHTFYSDTVTG(SEQ ID NO：77)和KTLDYYALDY(SEQ ID NO：78)。在一些实施方式中，所述抗体其VL CDR1、2和3的氨基酸序列分别是RASKSISKYLA(SEQ ID NO：80)、SGSRLQS(SEQ ID NO：81)和QQHNEYPWT(SEQ ID NO：82)。
在一些实施方式中，所述抗体其VH的氨基酸序列为SEQ ID NO：75或与其具有至少70％序列同一性的序列，CDR1、2和3的氨基酸序列分别是TYAMS(SEQ ID NO：76)、EISSGGSHTFYSDTVTG(SEQ ID NO：77)和KTLDYYALDY(SEQ ID NO：78)。VL的氨基酸序列为SEQ ID NO：79或与其具有至少70％序列同一性的序列，CDR1、2和3的氨基酸序列分别是RASKSISKYLA(SEQ ID NO：80)、SGSRLQS(SEQ ID NO：81)和QQHNEYPWT(SEQ ID NO：82)。
在一些实施方式中，所述抗体其VH CDR1、2和3的氨基酸序列分别是GYYML(SEQ ID NO：84)、RINPYNGATTYNQNFKD(SEQ ID NO：85)和WFGDFDF(SEQ ID NO：86)。在一些实施方式中，所述抗体其VL CDR1、2和3的氨基酸序列分别是RASQEISGYLS(SEQ ID NO：88)、ATSTLDS(SEQ ID NO：89)和LQYASYPFT(SEQ ID NO：90)。
在一些实施方式中，所述抗体其VH的氨基酸序列为SEQ ID NO：83或与其具有至少70％序列同一性的序列，CDR1、2和3的氨基酸序列分别是GYYML(SEQ ID NO：84)、RINPYNGATTYNQNFKD(SEQ ID NO：85)和WFGDFDF(SEQ ID NO：86)。VL的氨基酸序列为SEQ ID NO：87或与其具有至少70％序列同一性的序列，CDR1、2和3的氨基酸序列分别是RASQEISGYLS(SEQ ID NO：88)、ATSTLDS(SEQ ID NO：89)和LQYASYPFT(SEQ ID NO：90)。
在一些实施方式中，所述抗体为人源化抗体，其VH的氨基酸序列为SEQ ID NO：124、125、126、127中任一所示的重链可变区，CDR1、2和3的氨基酸序列分别是CDR1：SYNIY(SEQ ID NO：28)、CDR2：GVNPSNGNTNFNENFKS(SEQ ID NO：29)或GVNPSNGNTNFNENFKG(SEQ  ID NO：142)或GVNPSNGNTNFNENFQG(SEQ ID NO：128)和CDR3：GNYYGYEFAY(SEQ ID NO：30)。
在一些实施方式中，所述抗体为人源化抗体，其VL的氨基酸序列为SEQ ID NO：129、130中任一所示的轻链可变区，CDR1、2和3的氨基酸序列分别是KASQHVSTAVA(SEQ ID NO：32)、SPSYRYT(SEQ ID NO：33)和QQHYSTPWT(SEQ ID NO：34)。
在一些实施方式中，所述抗体为人源化抗体，其VH的氨基酸序列为SEQ ID NO：131、132、134、135、136任一所示的重链可变区，CDR1、2和3的氨基酸序列分别是CDR1：TYAMS(SEQ ID NO：76)、CDR2：EISSGGSHTFYSDTVTG(SEQ ID NO：77)或EISSGGSHTFYADTVKG(SEQ ID：133)或EISSGGSHTFYADTVTG(SEQ ID NO：143)、和CDR3：KTLDYYALDY(SEQ ID NO：78)。
在一些实施方式中，所述抗体为人源化抗体，其VL的氨基酸序列为SEQ ID NO：137、138、139、140、141中任一所示的轻链可变区，CDR1、2和3的氨基酸序列分别是RASKSISKYLA(SEQ ID NO：80)、SGSRLQS(SEQ ID NO：81)和QQHNEYPWT(SEQ ID NO：82)。
在一些实施方式中，非人源单克隆抗体可通过如下途径人源化：(1)同源替换，使用与非人源对应部分具有较大同源性的人FR进行替换；(2)表面重塑，对非人源CDR和FR表面氨基酸残基进行重塑，以使其类似于人抗体CDR的轮廓或者FR型式；(3)补偿变化，改编关键位置氨基酸残基，以补偿CDR移植；(4)定位保守，人源化单抗以FR保守序列为模板进行人源化，但保留非人源单抗可变区的关键氨基酸残基。
本文不仅包括完整的单克隆抗体，还包括具有免疫活性的抗体片段(抗原结合片段)，如Fab，Fab′，F(ab′)2，Fd，单链Fv或scFv，二硫键连接的Fv，V-NAR结构域，IgNar，胞内抗体，IgGΔCH2，小抗体，F(ab′)3，四抗体，三抗体、双特异性抗体，单结构域抗体，DVD-Ig，Fcab，mAb2，(scFv)2或scFv-Fc等。
在一些实施方式中，本公开的抗体不与替瑞利尤单抗竞争性结合TIGIT。在一些实施方式中，本公开的抗体结合的表位与替瑞利尤单抗不同。在一些实施方式中，上述抗体为例如7103-06、7103-07。
在一些实施方式中，本公开的抗体与替瑞利尤单抗竞争性结合TIGIT，且部分阻断替瑞利尤单抗结合。在一些实施方式中，本公开的抗体结合的表位与替瑞利尤单抗有重叠。在一些实施方式中，上述抗体为例如7103-01、7103-03、7103-04。
在一些实施方式中，本公开的抗体结合相同的表位。在一些实施方式中，抗体7103-01、7103-03、7103-04相互阻断彼此的TIGIT结合，结合相同的表 位。
在一些实施方式中，本公开的抗体结合人或其他灵长类动物的TIGIT，但不结合小鼠TIGIT。
单克隆抗体及其片段可用本领域技术人员熟知的各种方法来制得。例如，单克隆抗体可用杂交瘤方法(由Kohler等，Nature，256：495(1975)首先提出)制得，或用重组DNA方法(美国专利No.4,816,567)制得。单克隆抗体也可用例如Clackson等，Nature，352：624-628(1991)和Marks等，J.Mol.Biol.，222：581-597(1991)所述的技术从噬菌体抗体库中分离获得。
单克隆抗体或其片段的编码分子、含该分子的表达载体及宿主细胞
本文还提供了编码所述的抗TIGIT单克隆抗体或其片段的核酸分子。这些核酸分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。本文还公开了含有上述核酸分子的表达载体，例如为pcDNA3.4、pcDNA3.1等；本文还公开了被上述表达载体转化的宿主细胞，其可为例如CHO细胞、COS细胞等。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码所述的本文的单克隆抗体(或其片段，或其衍生物)的核酸序列。然后可将该序列引入本领域中已知的各种现有的分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本文蛋白序列中。
本文还涉及包含上述的编码序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞，例如CHO细胞、COS细胞等。通常用哺乳动物细胞系来作为表达真核细胞衍生多肽的宿主细胞。哺乳动物细胞在培养物中的繁殖是本领域熟知的。见《组织培养》，Academic Press，Kruse and Patterson编辑(1973)，该文纳入本文作为参考。较佳的哺乳动物细胞是许多可购得的无限增殖细胞系。这些无限增殖细胞系包括但不局限于，中国仓鼠卵巢(CHO)细胞、Vero细胞、Hela海拉细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞(如Hep G2)和其它许多细胞系。它们为蛋白质分子提供了翻译后修饰，包括正确 的折叠、正确的二硫键形成以及正确位点的糖基化。
本文还提供了可生产本文单克隆抗体的杂交瘤细胞系。在获得生产本文的单克隆抗体的杂交瘤，本领域人员可很方便地获知本文的抗体的结构(比如抗体的重链可变区和轻链可变区)，然后制备本文的单克隆抗体。
用表达载体转化宿主细胞的方法有很多种，所用的转化程序取决于待转化的宿主。将异源多核苷酸导入哺乳动物细胞中的方法是本领域所知的，其包括葡聚糖介导的转染、磷酸钙沉淀、Polybrene(1，5-二甲基-1，5-二氮十一亚甲基聚甲溴化物)介导转染、原生质体融合、电穿孔、脂质体介导转染以及将DNA直接显微注射到胞核中。在本文中，较佳的方法是电穿孔法或脂质体介导法等。例如可采用Invitrogen公司的脂质体法试剂盒来转染诸如COS、CHO细胞等宿主细胞。
在适合本文单克隆抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本文的单克隆抗体。
单克隆抗体的鉴定及纯化
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。单克隆抗体的结合亲和力例如可用Munson等，Anal.Biochem.，107：220(1980)的Scatchard分析来测定。
本文的单克隆抗体可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超声处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
偶联物、嵌合抗原受体及表达嵌合抗原受体的免疫细胞
本申请中还提供了包含本文抗体或其抗原结合片段的偶联物、嵌合抗原受体，以及表达所述嵌合抗原受体的免疫细胞。由于本申请抗体或其抗原结合片段对TIGIT的特异性，所述偶联物、嵌合抗原受体，以及表达所述嵌合抗原受体的免疫细胞可起到靶向作用于表达或过表达TIGIT的细胞或组织。并且，由于本申请的抗体或其抗原结合片段本身具有生物活性(例如具有免疫调节活性)，通过其偶联物、嵌合抗原受体及表达嵌合抗原受体的免疫细胞中的抗体 或抗原结合片段部分也可发挥相应的作用。
在一些实施方式中，抗体偶联物可包含：本文所述的抗体或其抗原结合片段；偶联部分，例如药物、毒素、细胞因子、放射性核素或酶；以及可选的，接头。在一些实施方式中，所述偶联通过酶促偶联、化学偶联、融合等方式实现。接头可以是促进偶联部分在细胞中的释放的“可切割接头”，例如酸不稳定接头、肽酶敏感性接头、光不稳定接头、二甲基接头，和含二硫化物的接头。
在一些实施方式中，与抗体连接的偶联部分可为例如生物活性剂，其可为增强和/或调节所希望的生物学作用的任何合成的、重组产生的或天然发生的物质。在一些实施方式中，所述生物活性剂可提供的作用包括但不限于：调节、刺激、和/或抑制生长信号；调节、刺激、和/或抑制抗凋亡信号；调节、刺激、和/或抑制凋亡或坏死信号；调节、刺激、和/或抑制ADCC级联；调节、刺激、和/或抑制CDC级联等。例如，其可为化疗剂、毒素、细胞因子、同位素、酶类等。
本申请中还提供了嵌合抗原受体(CAR)，其包含胞外结构域和胞内结构域，其中，所述胞外结构域包含本申请的抗体或其抗原结合片段。在一些实施方式中，本申请的CAR还包含连接胞外结构域和胞内结构域的跨膜结构域，例如CD28的跨膜部分。本申请的CAR可为第一代、第二代、第三代或第四代CAR。
在一些实施方式中，本申请CAR中的抗体或其抗原结合片段为scFv或VH sdAb，或由其组成。在一些实施方式中，CAR的胞内结构域包括模拟或近似通过天然抗原受体的信号、通过此类受体联合共刺激受体的信号，和/或通过单独共刺激受体的信号的那些。
在一些实施方式中，胞内结构域包括一个或多个共刺激结构域和活化结构域，例如选自下组的一种或多种：ITAM结构域、CD3ζ、CD28、4-1BB、OX40、CD27、ICOS或其组合，例如(CD28+CD3ζ)、(CD28+CD27+CD3ζ)、(CD28+OX40+CD3ζ)、(CD28+4-1BB+CD3ζ)、(CD28+CD27+OX40+CD3ζ)、(CD28+4-1BB+CD27+CD3ζ)、(CD28+4-1BB+OX40+CD3ζ)、(4-1BB+CD3ζ)、(4-1BB+OX40+CD3ζ)、(4-1BB+CD27+CD3ζ)、(CD27+CD3ζ)、(CD27+OX 40+CD3ζ)、(CD28Δ+CD3ζ)、(CD28Δ+CD27+CD3ζ)、(CD28Δ+OX40+CD3ζ)、(CD28Δ+4-1BB+CD3ζ)、(CD28Δ+4-1BB+OX40+CD3ζ)、(CD28Δ+CD27+OX40+CD3ζ)、(CD28Δ+4-1BB+CD27+CD3ζ))、(4-1BB+ICOS+CD3ζ)、(CD28+ICOS+CD3ζ)、(ICOS+CD3ζ)。
在一些实施方式中，CAR的胞外结构域还包含铰链区，例如所述铰链区源自IgG的铰链或CD8α/CD28胞外区，如选自：IgG4 FcΔEQ、IgG4 FcΔQ、(t-12AA+t-20AA)、mKate、phiLov、dsRed、Venus、eGFP、CH3 HA，(CD8α+t-20AA)、双重t-20 AA、(t-20AA+CD8α)、(CD8α+亮氨酸拉链Basep1)、(CD8α +亮氨酸拉链Acid1)、2D3、CD8α或IgG4 Fc。
本申请中还提供了一种经转化的免疫细胞，其表达本申请的嵌合抗原受体，例如所述免疫细胞选自T细胞，如αβT细胞、γδT细胞或NK T细胞或从多能细胞衍生的T细胞。免疫细胞可来自待接受治疗的个体或来自其他来源。可将经转化(可任选的扩增)的免疫细胞给予待治疗个体，例如用于过继疗法。
此外，本申请中还包括了用于转化免疫细胞的CAR构建体、表达载体、制备方法及其应用。
药物组合物
本文还提供了一种药物组合物，该组合物含有药学上有效量的本文单抗或其免疫偶联物以及药学上可接受的载体。
本文所用的术语“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。本文所用的“药学上可接受的载体”应当与本文的活性物质相容，即能与其共混而不会在通常情况下大幅度降低药物组合物的效果。这些载体是本领域普通技术人员所熟知的。在《雷明顿药物科学》(Remington′s Pharmaceutical Sciences，Mack Pub.Co.，N.J.1991年)中可找到关于药学上可接受的载体的充分讨论。
这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、佐剂、及其组合。另外，这些载体中还可能存在辅助性的物质，如润湿剂或乳化剂、pH缓冲物质等。
本文的组合物可通过口服以及静脉内、肌内或皮下等途径给药；优选的是口服或静脉内注射给药。本文的药物组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。
使用药物组合物时，是将安全有效量的单克隆抗体或免疫偶联物施用于哺乳动物，其中该安全有效量通常约0.1微克-5毫克/千克体重，而且在大多数情况下不超过约3毫克/千克体重，较佳地该剂量是约1-10微克/千克体重-约1毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围内的。
如本文所用，术语“单位剂型”是指为了服用方便，将本公开的组合物制备成单次服用所需的剂型，包括但不限于各种固体剂(如片剂)、液体剂、胶囊剂、缓释剂。
在本公开的另一优选实施方式中，所述组合物为单位剂型或多剂型，且其中活性物质的含量为0.01～2000mg/剂，优选0.1～1500mg/剂，更优选1～1000mg/剂。在本公开的另一个优选例中，每天施用1～6剂本公开的组合物， 优选施用1～3剂；最优选的，每天服用的剂量为1剂。
本文的单克隆抗体或免疫偶联物可有效阻断TIGIT与其配体CD155的结合，增强免疫细胞的细胞因子释放，提高免疫细胞活性、增强免疫应答，用于预防和/或治疗肿瘤、感染或感染性疾病。本文的活性物质可用于治疗多种类型的肿瘤，所述的肿瘤比如(包括但不限于)：腺癌、白血病、淋巴瘤、黑色素瘤、肉瘤；来自肾上腺、胆囊、骨、骨髓、脑、乳腺、胆管、胃肠道、心脏、肾脏、肝脏、肺、肌肉、卵巢、胰腺、甲状旁腺、阴茎、前列腺、皮肤、唾液腺、脾脏、睾丸、胸腺、甲状腺或子宫的肿瘤组织；中枢神经系统的肿瘤，如胶质细胞多样性瘤、或星细胞瘤；眼部肿瘤(例如基底细胞癌、鳞状细胞癌或黑色素瘤)、内分泌腺肿瘤、神经内分泌系统肿瘤、胃肠道胰腺内分泌系统肿瘤，生殖系统肿瘤或头颈部肿瘤。
检测试剂盒
本文还提供了一种检测TIGIT的试剂盒，它含有本文所述的抗TIGIT单克隆抗体或其活性片段、免疫偶联物。可采用本文的试剂盒来检测生物样品中是否存在TIGIT或其含量，该检测方法包括步骤：(a)将样品与本文试剂盒中的抗TIGIT单克隆抗体或其免疫偶联物接触；(b)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在TIGIT或定量检测所形成的抗原-抗体复合物的量以反映样品中TIGIT的含量。所述样品可为经过预处理或未经过预处理，例如可经过提取、纯化或浓缩等。
所述试剂盒含有容器以及位于容器内的本文的单克隆抗体、或者带有所述单克隆抗体的检测板，以及使用说明书。该试剂盒中还可含有检测所需的其它试剂，例如缓冲液、指示剂等。本领域技术人员可根据具体需要对试剂盒的内容物进行调整。
实施例
下面结合具体实施例，进一步阐述本公开。应理解，这些实施例仅用于说明本公开而不用于限制本公开的范围。本领域技术人员可对本公开做出适当的修改、变动，这些修改和变动都在本公开的范围之内。
下列实施例中未注明具体条件的实验方法，可采用本领域中的常规方法，例如参考《分子克隆实验指南》(第三版，纽约，冷泉港实验室出版社，New York：Cold Spring Harbor Laboratory Press，1989)或按照供应商所建议的条件。DNA的测序方法为本领域常规的方法，也可由商业公司提供测试。
除非另外说明，否则百分比和份数按重量计算。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与 所记载内容相似或均等的方法及材料皆可应用于本公开方法中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1.抗人TIGIT鼠单克隆抗体的产生
1.1免疫
将3只Balb/c小鼠(6～8周龄，体重18g左右，雌性，购自计划生育科学研究所实验动物经营部)以50μg重组人TIGIT(Human TIGIT His，Sino Biological，10917-H08H Accession#NP 776160.2.Met1-Phe138，C末端表达多组氨酸标签)在完全弗氏佐剂(complete Freund’s adjuvant CFA)存在下，按100μl蛋白和100μl CFA进行乳化，经由腹腔和皮下来进行多点免疫(每点注射量为50μl)。在不完全弗氏佐剂(incomplete Freund’s)存在下，同样按照100μl蛋白和100μl IFA进行乳化。以相同的免疫原hTIGIT在首次免疫后的两周、三周、四周、五周共对小鼠进行4次免疫追加。再一周后检测血清效价达到10W以上，用相同的25μg免疫原hTIGIT通过皮下注射进行冲刺免疫和准备进行骨髓瘤细胞融合。
1.2融合
使小鼠的脾细胞与淋巴细胞与小鼠骨髓瘤细胞(购自中国科学院细胞库，货号为TCM18)融合，具体步骤如下：
取2个T75Flask 24小时前传代的骨髓瘤细胞，用IMDM培养基洗一遍，重悬计数为2～4×10 7个。将冲刺免疫72小时后的小鼠眼球放血，拉颈处死，放入75％乙醇中消毒。无菌取脾，剥去周围的结缔组织，用IMDM培养基清洗。剪碎研磨脾细胞，吹散，经40μm的细胞过滤筛网过滤，离心1000rpm，5min，重悬，计数为1～2×10 8个。将两种细胞混匀，离心1000rpm，5min。弃尽细胞上清，用手轻击离心管底，使沉淀松散，于37℃水浴中预热。将预热至37℃的1ml PEG于60s内滴加到离心管中，边加边轻轻搅拌，约1min。于30s内加入1ml IMDM培养基，再于1min内加入3ml IMDM培养基，最后于1min内加入16ml IMDM培养基。静置10min，离心1000rpm，5min。弃去上清，用含20％FBS的IMDM培养基(含1×HAT(Sigma，H0262-10VL)和1×抗菌素溶液(双抗)(Gibco，15140122))重悬细胞，分装于25块96孔细胞培养板中，200μl/孔，5％CO 2、37℃孵育。
1.3杂交瘤筛选
融合后第5天～10天，根据细胞生长密度，全换液，培养基为含10％FBS的IMDM培养基(含1×HAT和1×双抗)，200μl/孔，37℃，5％CO 2孵育。融合 后第10～14天，根据细胞生长密度，进行ELISA检测。检测的阳性孔细胞进行第一次和第二次亚克隆。ELISA检测步骤如下：
TIGIT-Fc(Human TIGIT(Fc Tag)：Sino Biological，10917-H02H)包被：用包被液将TIGIT-Fc稀释至浓度为1μg/ml，混匀后用多孔移液器吸入96孔酶标板中，50μl/孔，用封板膜密封后置于4℃冰箱过夜。
酶标板封闭：250μl/孔PBS洗涤2次，拍干。再加入150μl/孔的封闭液，封板膜密封后置37℃，1.5小时。
一抗孵育：取杂交瘤上清100μl/孔加入酶标板，封板膜密封后置37℃，1小时。
二抗孵育：弃封闭液，250μl/孔PBST洗3遍，拍干。以结合液1∶3000稀释山羊抗鼠HRP二抗(Jackson Immuno Research，115-035-164)，100μl/孔加入酶标板，封板膜密封后置37℃，1小时。
TMB孵育：弃封闭液，250μl PBST洗3遍，拍干。TMB 50ul//孔加入酶标板，封板膜密封后置37℃，15-25分钟。
酶标仪检测：双波长450nm/655nm检测，结果以OD 450-OD 655计算。
1.4鼠单克隆抗体制备
共筛选出8株克隆，其信息如表1所示：
表1.克隆信息
编号 克隆 重链 轻链
7103-01 31E10-2-1 IgG1 kappa
7103-02 37C6-1-1 IgG1 kappa
7103-03 36H8-2-2 IgG1 kappa
7103-04 40E6-2-2 IgG1 kappa
7103-05 41D7-2-1 IgG1 kappa
7103-06 47A2-1-2 IgG1 kappa
7103-07 42C12-1-2 IgG1 kappa
7103-08 49F8-1-2 IgG2b kappa
培养所得细胞株，并扩增细胞。离心细胞培养液，收集上清，用Protein-A亲和层析柱从上清中纯化融合蛋白，跑胶分析，每个泳道分别上样2μg还原和非还原样品。电压150V，1小时。非还原的抗体的分子量在150KD左右，还原抗体的分子量分别在50KD和25KD左右，符合抗体的分子量特性(图1)。
1.5鼠单克隆抗体鉴定
1.5.1抗人TIGIT鼠单克隆抗体与重组人TIGIT抗原结合
通过ELISA，使用标准方法和步骤测试了8株TIGIT单克隆抗体与重组人 TIGIT抗原(Human TIGIT(His)，Sino Biological，10917-H08H)的结合，具体步骤如下：
TIGIT包被：用包被液将TIGIT稀释至浓度为1μg/ml，混匀后用多孔移液器吸入96孔酶标板中，50μl/孔，用封板膜密封后置于4℃冰箱过夜。
酶标板封闭：250μl/孔PBS洗涤2次，拍干。再加入150μl/孔的封闭液，封板膜密封后置37℃，1.5小时。
一抗孵育：梯度稀释抗体，100μl/孔加入酶标板，封板膜密封后置37℃，1小时。
二抗孵育：弃封闭液，250μl PBST洗3遍，拍干。以结合液1∶3000稀释山羊抗鼠HRP二抗(Jackson Immuno Research，115-035-164)，100μl/孔加入酶标板，封板膜密封后置37℃，1小时。
TMB孵育：弃封闭液，250μl PBST洗3遍，拍干。TMB 50μl/孔加入酶标板，封板膜密封后置37℃，15-25分钟。
酶标仪检测：双波长450nm/655nm检测，结果以OD 450-OD 655计算。
实验结果与分析：
ELISA试验结果显示(图2)：如表1中所示的8株抗人TIGIT鼠单克隆抗体(7103-01～7103-08)均能特异性结合人TIGIT，结合的亲和力EC50分别为：0.0647nM，0.0987nM，0.0674nM，0.0727nM，0.0571nM，0.0393nM，0.075nM和0.0372nM。
1.5.2抗人TIGIT鼠单克隆抗体对重组人TIGIT-CD155结合的抑制
为了评估抗人TIGIT鼠单克隆抗体与CD155(Human CD155(Fc Tag)：Sino Biological，10109-H02H)对重组人TIGIT(Human TIGIT(His)，Sino Biological，10917-H08H)的结合的竞争，进行ELISA检测，具体步骤如下：
TIGIT包被：用包被液将TIGIT稀释至浓度为0.8μg/ml，混匀后用多孔移液器吸入96孔酶标板中，50μl/孔，用封板膜密封后置于4℃冰箱过夜。
酶标板封闭：250μl/孔PBS洗涤2次，拍干。再加入150μl/孔的封闭液，封板膜密封后置37℃，1.5小时。
一抗孵育：250μl/孔PBS洗涤2次，拍干。分别稀释6μg/ml的CD155在结合液中，60μl/孔加入96孔稀释板中。再用结合液分别稀释筛选出的杂交瘤抗体，最高浓度30μg/ml，以3倍浓度稀释，60μl/孔加入上面的相应稀释板中，混匀后，再100μl/孔加入包被好的96孔酶标板中。封板膜密封后置37℃，1.5小时。
二抗孵育：弃封闭液，250μl PBST洗3遍，拍干。以结合液1∶3000稀释anti-human-Fc-AP(Jackson Immuno Research，109-055-098)，100μl/孔加入酶标 板，封板膜密封后置37℃，1小时。
PNPP孵育：弃封闭液，250μl PBST洗3遍，拍干。取1颗PNPP(SouthemBiotech，0201-01)于1ml 5×二乙醇胺底物，再加入4ml去离子水溶解，50μl/孔加入酶标板，封板膜密封后置37℃，15-25分钟。
酶标仪检测：双波长405nm/490nm检测，结果以OD 405-OD 490计算。
实验结果与分析：
实验结果(图3)显示了如表1所示的8株抗人TIGIT鼠单克隆抗体均能特异性阻断CD155与人hTIGIT的结合，其IC 50分别为：0.178nM，0.130nM，0.141nM，0.183nM，0.169nM，0.0607nM，0.159nM，0.0857nM。
1.5.3抗人TIGIT鼠单克隆抗体与重组食蟹猴TIGIT的结合
以如上同样的方法分别包被食蟹猴和小鼠的TIGIT抗原，梯度稀释抗人TIGIT鼠单克隆抗体，再加入抗鼠Fab段的二抗(Jackson Immuno Research，515-035-072)，TMB显色。ELISA检测8株鼠单克隆抗体与重组食蟹猴TIGIT的结合。
实验结果与分析：
结果如图4显示，8株抗人TIGIT鼠单克隆抗体均结合人TIGIT，而均不与小鼠的TIGIT结合。
8株中五株(编号为7103-01、7103-03、7103-04、7105-06、7103-07)的抗人TIGIT鼠单克隆抗体可以识别食蟹猴TIGIT，结合EC 50分别为：1.066nM，0.398nM，0.334nM，0.174nM，0.704nM。而有3株抗人TIGIT鼠单克隆抗体(编号7103-02、7103-05、7103-08)和食蟹猴TIGIT不结合。
1.5.4抗人TIGIT鼠单克隆抗体与CHO细胞膜表达的TIGIT的结合
将以上可以与食蟹猴TIGIT结合的纯化的抗人TIGIT鼠单克隆抗体与细胞膜上TIGIT结合，由流式细胞分析技术分析抗体结合强度，具体步骤如下：
稳定表达全长人TIGIT的CHO-K(CHO-K-TIGIT)细胞株构建如下：采用含有全长人TIGIT cDNA(NM_173799.2)的质粒(购自Sino Biological)把cDNA克隆到含有大鼠谷氨酰胺合成酶基因的哺乳细胞表达载体pcDNA3.1(Invitrogen)，然后用电转染(Bio-Rad，Gene Pulser Xcell)方法把上述构建好的质粒转染进CHO-K细胞，转染的细胞在OptiCHO培养基(Invitrogen)中培养24-48个小时后，把培养基换成筛选培养基。筛选培养基含有OptiCHO，5μg/ml重组人胰岛素和10μM氨基亚砜蛋氨酸(MSX)。细胞培养在37℃，8％CO 2的培养箱里。3个星期后，用抗TIGIT抗体对筛选培养的细胞进行流式分选(FACS)，得到表达在细胞膜上的人TIGIT的CHO-K单克隆细胞株。
将CHO-TIGIT计数并1000rpm 5min离心。用0.5％BSA PBS重悬，取5×10 5个细胞/孔加入96孔尖底板中。400g 5min离心，并弃去上清。梯度稀释抗TIGIT抗体，以100μl/孔加入96孔尖底板，4℃，30min。用0.5％BSAPBS以250μl/孔洗两次，将1∶100稀释Anti-mouse-IgG-PE(Jackson Immuno Research，115-116-071)以100μl/孔加入96孔尖底板，4℃，30min。用0.5％BSA PBS以250μl/孔洗两次，以200μl/孔加入0.5％BSA PBS。样品用Beckman CytoFLEX进行检测。
实验结果与分析
结果如图5所示，五株抗人TIGIT鼠单克隆抗体(7103-01、7103-03、7103-04、7105-06、7103-07)均结合细胞膜表面表达的TIGIT(CHO-TIGIT)，其结合CHO-TIGIT细胞膜上的EC 50分别为：0.499nM，0.347nM，0.440nM，0.256nM，0.226nM。
1.6重链和轻链可变区测序
1.6.1RNA提取及cDNA获得
取约1～2×10 5个杂交瘤细胞进行RNA提取(Sigma，GenElute TM Mammalian Total RNA Miniprep Kit，货号RTN70)。取约8μL的总RNA进行后续的反转录PCR，首先65℃，5min，以消除RNA的二级结构，再加2μL的5×gDNA wiper，42℃，2min，以去除基因组DNA的污染。最后再加2μL的10×RT Mix，2μL的HiscriptIII Enzyme Mix和1μL的随机六聚体(Random Hexamers，诺唯赞，R312)。轻弹管壁混匀，瞬时离心将液体甩到管底，最后用如下条件进行逆转录反应：25℃，5min；42℃，45min；85℃，5s。
1.6.2抗体可变区基因的扩增
将上述cDNA做为模板，用PCR方法以不同的引物组合扩增出杂交瘤抗体的VH和VL基因。本实验所用的引物主要参考文献von Boehmer，L.等，Sequencing and cloning of antigen-specific antibodies from mouse memory B cells.Nat Protoc，2016.11(10)：p.1908-1923.，其中重链分别用两组不同的引物primerHa(包括SEQ ID NO：1至SEQ ID NO：12)和primerHb来进行反应(SEQ ID NO：13和SEQ ID NO：14)，轻链分别用primerKb(包括SEQ ID NO：15至SEQ ID NO：24)和primerKc(SEQ ID NO：25和SEQ ID NO：26)来进行反应，反应条件：在94℃变性3分钟后，在94℃，30秒、55℃，30秒、72℃，35秒下各40个循环，在72℃下的循环末端孵育5分钟。
表2.用于扩增抗体可变区基因的引物
Figure PCTCN2022094422-appb-000001
Figure PCTCN2022094422-appb-000002
得到的PCR产物或直接进行测序或进行TA克隆测序，重链的测序引物为SEQ ID NO：14，轻链的测序引物为SEQ ID NO：26，经IgBlast比对，成功获得所有抗体的V区序列如下表所示：
表3. 8株TIGIT鼠单克隆抗体的V区序列。
Figure PCTCN2022094422-appb-000003
Figure PCTCN2022094422-appb-000004
Figure PCTCN2022094422-appb-000005
Figure PCTCN2022094422-appb-000006
下划线处为CDR。
实施例2.人鼠嵌合单克隆抗体的制备
2.1人鼠嵌合单克隆抗体表达质粒构建
为了进行抗体的重组表达，将杂交瘤抗体的V区构建到嵌合抗体表达载体(pcDNA3.1)上，即将V区序列连接到恒定区为人IgG1或者人λ/κ的表达载体上。首先根据不同的序列设计特异性的PCR引物，序列如下表所示。PCR反应条件如下：在94℃变性3分钟后，在94℃，30秒、55℃，30秒、72℃，35秒下各40个循环，在72℃下的循环末端孵育5分钟。以测序结果明确的PCR产物或者TA克隆菌液为模板，成功调取出待克隆的片段。
表4.用于嵌合抗体表达载体构建的引物序列
Figure PCTCN2022094422-appb-000007
Figure PCTCN2022094422-appb-000008
用上述PCR产物进行琼脂糖凝胶电泳，并回收目的条带，进行Xho I单酶切反应，把酶切的PCR片段克隆到同样酶切的表达载体中，将重组质粒转化进DH5a感受态细菌，用菌落PCR方法鉴定含有正确重组质粒的阳性菌落，提纯重组质粒，酶切和测序鉴定。
2.2人鼠嵌合单克隆抗体的表达
本实验所用的HEK293F细胞、培养基和转染试剂均购买于义翘神州，具体使用方法如下：转染前一天，取样计算细胞密度及活率，以2E6个细胞/mL的密度接种到新鲜的培养基中，置于37℃，5％CO 2，150-175rpm转速的恒温摇床培养箱中进行培养。转染当天，计算细胞活率，并将细胞调整至3E6个细胞/mL。转染液配制：以20mL的转染体积为例，用150mM的NaCl稀释20μg的DNA，总体积为500μL，温和混匀；用150mM的NaCl稀释100μL的sinofection转染试剂至总体积500μL，温和混匀；将稀释好的DNA和转染试剂同时单独静置5min后温和混匀，总体积1mL，之后静置10min，最后逐滴加入到细胞培养液中，混匀后继续培养。转染后的第3天后，每天检测细胞的活率，待到细胞活率为60％左右时，收获上清。
2.3人鼠嵌合单克隆抗体的纯化
将以上嵌合单克隆抗体(编号7103-01(hFc)、7103-03(hFc)、7103-04(hFc)、7103-06(hFc)、7103-07(hFc))进行哺乳动物细胞的表达纯化，将瞬转收获的细胞培养液4000rp，离心10min，弃去细胞沉淀。取适量蛋白A珠加入到上清中，室温孵育4h。离心弃去上清后，用PBS洗涤蛋白A珠两次，最后用pH＝3的枸橼酸洗脱液洗脱，最后用1M Tris调整pH为中性。
实验结果与分析：
纯化后的SDS-PAGE分析结果如图6所示。非还原的抗体的分子量在150KDa左右，还原抗体的分子量分别在55kDa和25KDa，符合抗体的分子量特性。
实施例3.人鼠嵌合单克隆抗体与TIGIT的结合
3.1用ELISA方法研究人鼠嵌合单克隆TIGIT抗体与hTIGIT的体外结合活性
为了确定本公开人鼠嵌合TIGIT单克隆抗体是否可以结合重组人TIGIT蛋白，进行体外测试。将替瑞利尤单抗(Tiragolumab)用作阳性对照。
用pH9.6的NaHCO3溶液稀释抗原hTIGIT(Human TIGIT(His)，Sino Biological，10917-H08H)至1μg/mL，每孔50μL加入到96孔酶标板中，4℃冰箱过夜。次日，PBS洗涤两遍之后，用3％的BSA封闭，每孔100μL，37℃孵育1.5h，用PBST洗涤4次之后，加入一抗稀释液后继续37℃孵育2h，继续用PBST洗涤4次后，加入一定比例稀释的抗人-Fc-AP抗体(Jackson Immuno Research，109-055-098)，37℃孵育1.5h，最后PBST洗涤4次后，加入显色液(对硝基苯磷酸盐，SouthernBiotech109-055-09)，37℃孵育10-15min后，酶标仪读数(波长405nm和490nm)。
实验结果与分析：
ELISA试验结果(图7)显示：5种抗TIGIT人鼠嵌合单克隆抗体(编号7103-01(hFc)、7103-03(hFc)、7103-04(hFc)、7103-06(hFc)、7103-07(hFc))能特异性结合人TIGIT，其结合的亲和力EC 50分别为0.499nM，0.380nM，0.554nM，0.554nM，0.310nM，而阳性对照替瑞利尤单抗的EC 50为1.194nM。
该结果表明重组表达的嵌合抗体结合抗原TIGIT的活性很好，与杂交瘤纯化的鼠源抗体相当。与Genetech的替瑞利尤单抗相比，本公开抗体对TIGIT的结合活性更佳，EC 50值仅为替瑞利尤单抗的1/4-1/2。
3.2用生物膜层光学干涉技术(BLI)研究人鼠嵌合TIGIT单克隆抗体与TIGIT的结合活性
用GATOR(ProbeLife)检测仪器以及HFC(LOT#2007065Tray 2)，Human antibody Capture探针来检测人鼠嵌合TIGIT单克隆抗体的解离常数(Kd)。将人鼠嵌合TIGIT单克隆抗体在结合缓冲液(Q buffer[PBS(10mM PH7.4)+0.02％Tween 20+0.2％BSA]中稀释至30nM。将重组人TIGIT蛋白在Q buffer中2倍梯度稀释至不同浓度，在40nM到10nM的范围内变化。通过将抗体捕获传感器置于上述连续稀释的抗原溶液中来开始进行动力学关联测定。打开Octet  Data Acquisition软件，选择New Kinetics Experiment-Basic Kinetics模式；
按照下面表格设置流程：
表5.用生物膜层光学干涉技术(BLI)研究TIGIT抗体结合活性的流程
过程 时间 浓度
基线1 30秒 Q缓冲液
加载 120秒 30nM抗体(在Q缓冲液中)
基线2 60秒 Q缓冲液
关联 120秒 40nM～10nM，用Q缓冲液2倍梯度稀释的抗原
解离 120秒 Q缓冲液
再生 25秒 Q缓冲液
表6.结合于人TIGIT的人鼠嵌合单克隆抗体的动力学分析结果
Figure PCTCN2022094422-appb-000009
实验结果显示所有抗体在Global拟合模式下的相关系数R2都大于0.95，符合系统适应性要求，结果可靠。
3.3用流式细胞术方法(flow cytometry)研究人鼠嵌合单克隆抗体与细胞膜上TIGIT蛋白的结合
为了确定本公开人鼠嵌合TIGIT单克隆抗体是否可以结合细胞膜上的TIGIT蛋白，进行体外测试。将替瑞利尤单抗(Tiragolumab)用作阳性对照。
首先配制FACS缓冲液，即1×PBS+0.5％BSA溶液。取所需数量的细胞，一个样品所需的细胞量约为1×10^5～5×10^5个，用FACS buffer将一抗以一定的比例稀释后重悬细胞，体积约为100μL，4℃冰箱孵育30min，用FACS缓冲液将细胞洗涤一次后，用FACS缓冲液稀释的FITC-抗人IgG(Abcam：6854)将细胞重悬后置于4℃冰箱孵育30min，最后，FACS缓冲液洗涤细胞后，用 200μL的FACS缓冲液重悬细胞，上机检测。
实验结果与分析：
结果如图8所示，五种TIGIT嵌合单克隆抗体(编号7103-01(hFc)、7103-03(hFc)、7103-04(hFc)、7103-06(hFc)、7103-07(hFc))均和CHO-TIGIT结合较好，结合CHO-TIGIT细胞膜上的EC 50分别为1.079nM，0.596nM，0.867nM，0.873nM，0.927nM，阳性对照替瑞利尤单抗为1.724nM。
该数据表明本公开的抗体不仅与固相抗原(ELISA法)结合能力很强，与细胞膜上的TIGIT结合活性也很高，EC 50数值为替瑞利尤单抗的1/3-1/2，表明本发明的抗体与细胞膜上TIGIT的结合显著优于替瑞利尤单抗与细胞膜表面TIGIT的结合。
实施例4.人鼠嵌合TIGIT单克隆抗体对重组人TIGIT-CD155结合的抑制
为了确定本公开人鼠嵌合TIGIT单克隆抗体是否可以抑制CD155与TIGIT的结合，进行体外测试。将替瑞利尤单抗(Tiragolumab)用作阳性对照。
用pH＝9.6的NaHCO 3溶液稀释抗原TIGIT(Human TIGIT(His)，Sino Biological，10917-H08H)至1μg/mL，每孔50μL加入到96孔酶标板中，4℃冰箱过夜。次日，PBS洗涤两遍之后，用3％的BSA封闭，每孔100μL，37℃孵育1.5h，用PBST洗涤4次。梯度稀释的一抗和CD155-mFC(6μg/mL)(Human CD155(Fc Tag)：Sino Biological，10109-H02H)等体积混匀后加入封闭洗涤后的酶标板中，37℃孵育2h，继续用PBST洗涤4次后，加入一定比例稀释的二抗HRP抗mFC(Jackson Immuno Research，115-035-164)，37℃孵育1.5h，最后PBST洗涤4次后，加入TMB显色液，37℃孵育10-15min后，酶标仪读数(波长450nm和655nm)。
实验结果与分析：
实验结果(图9)显示了5种嵌合TIGIT单克隆抗体(编号7103-01(hFc)、7103-03(hFc)、7103-04(hFc)、7103-06(hFc)、7103-07(hFc))能特异性阻断CD155与人hTIGIT的结合。IC 50分别为：4.029nM，1.455nM，3.298mM，2.029nM，1.698nM，阳性对照替瑞利尤单抗的IC 50为5.235nM。
由以上数据可得，本公开的抗体能够阻断TIGIT与其配体CD155的结合，且本公开的抗体阻断能力均优于替瑞利尤单抗，其中7103-03(hFc)、7103-06(hFc)和7103-07(hFc)的IC 50值为替瑞利尤单抗的1/3-1/2，阻断能力显著优于替瑞利尤单抗。
实施例5.人鼠嵌合TIGIT单克隆抗体结合表位分析
为了确定本公开人鼠嵌合TIGIT单克隆抗体与已公开的替瑞利尤单抗的 结合表位是否有交叉，进行了如下体外测试。
用pH 9.6的NaHCO 3溶液稀释抗原TIGIT(Human TIGIT(His)，Sino Biological，10917-H08H)至1μg/mL，每孔50μL加入到96孔酶标板中，4℃冰箱过夜。次日，PBS洗涤两遍之后，用3％的BSA封闭，每孔100μL，37℃孵育1.5h，用PBST洗涤4次。梯度稀释的本公开鼠源抗体和替瑞利尤单抗(终浓度0.83μg/mL，相当于替瑞利尤单抗与TIGIT-His结合的EC 80)等体积混匀后加入封闭洗涤后的酶标板中，37℃孵育2h，继续用PBST洗涤4次后，加入一定比例稀释的二抗HRP抗mFC(Jackson Immuno Research，115-035-164)，37℃孵育1.5h，最后PBST洗涤4次后，加入TMB显色液，37℃孵育10-15min后，酶标仪读数(波长450nm和655nm)。
实验结果与分析：
实验结果显示了本公开的抗体与替瑞利尤单抗结合TIGIT表位的基本情况。7103-06、7103-07与替瑞利尤单抗没有互相竞争，说明它们结合的表位与替瑞利尤单抗不同。而7103-01、7103-03、7103-04部分阻断替瑞利尤单抗与TIGIT的结合，说明7103-01、7103-03、7103-04结合的表位与替瑞利尤单抗有重叠，但是有所不同。接下来，我们采用同样的方法检测了7103-01、7103-03、7103-04之间相互竞争结合TIGIT的情况，结果显示它们之间互相阻断彼此结合抗原TIGIT，说明三者的结合表位是一致的。
按照结合表位，本发明的抗体可以分为2组：
A组：与替瑞利尤单抗没有互相竞争的抗体7103-06、7103-07，其结合的表位与替瑞利尤单抗不同；以及
B组：部分阻断替瑞利尤单抗与TIGIT结合的抗体7103-01、7103-03、7103-04，其结合的表位与替瑞利尤单抗有重叠，但是有所不同。并且，所述抗体7103-01、7103-03、7103-04相互阻断彼此的TIGIT结合，表明其结合相同的表位。
实施例6.人鼠嵌合TIGIT单克隆抗体体外增强PBMC的INFγ产生
T细胞激活后，淋巴细胞分泌细胞因子IFNγ。为了测试本公开人鼠嵌合TIGIT单克隆抗体作为T细胞活化的阳性调节剂功能，我们进行了以下实验，证明了本公开抗体由TIGIT信号阻断对PBMC细胞IFNγ产生的增强作用。
复苏市售冷冻的人外周血单核细胞PBMC(Oricells)，调整细胞密度至5×10 6个/mL，用RPMI 1640+10％FBS培养基稀释PHA至浓度6μg/mL，接着用该PHA稀释液将抗体稀释至抗体20μg/mL，加100μL的PHA和抗体稀释液至96孔板中，最后加100μL的细胞悬液至96孔板中，轻轻混匀后至37℃，5％CO 2培养箱中培养4天。4天后吸取上清100μL检测IFNγ的含量。
实验结果与分析：
图12显示，本公开的5株人鼠种嵌合TIGIT单克隆抗体(编号7103-01(hFc)、7103-03(hFc)、7103-04(hFc)、7103-06(hFc)、7103-07(hFc))可以促进PHA激活的PBMC产生IFNγ，其效果均优于替瑞利尤单抗，释放量增加百分比达到替瑞利尤单抗的42％-62％。
实施例7.人源化抗人TIGIT单克隆抗体设计
根据编号7103-01和7103-07的鼠源母本抗体序列，设计了人源化抗体序列，具体步骤如下：首先使用Discovery Studio和
Figure PCTCN2022094422-appb-000010
Antibody Modeling，采用同源建模方法构建可变区的三维分子模型。接下来，通过比对数据库已有抗体结构，分别对母本抗体可变区及CDR进行结构模拟。同时，选择分别与鼠源母本抗体VH和VL具有高度同源性的cDNA衍生的人种系(Germline)序列进行比对。重链VH选择同源度最高的IGHV1为人源化设计模板，设计序列。轻链VL选择IGKV1为人源化设计模板，设计序列。
结果与讨论
将鼠源母本抗体编号7103-01原始重链mVH序列设计为4个人源化序列：7103-01-huVH1(SEQ ID NO：124)、7103-01-huVH2(SEQ ID NO：125)、7103-01-huVH3(SEQ ID NO：126)和7103-01-huVH4(SEQ ID NO：127)；将抗体原始轻链mVL设计为2个人源化序列：7103-01-huVL1(SEQ ID：129)和7103-01-huVL2(SEQ ID：130)。
将鼠源母本抗体7103-07原始重链mVH序列设计为5个人源化序列：7103-07-huVH1(SEQ ID：131)、7103-07-huVH2(SEQ ID：132)、7103-07-huVH3(SEQ ID：134)、7103-07-huVH4(SEQ ID：135)、7103-07-huVH5(SEQ ID：136)；将原始轻链mVL设计为5个人源化序列：7103-07-huVL1(SEQ ID NO：137)、7103-07-huVL2(SEQ ID NO：138)、7103-07-huVL3(SEQ ID NO：139)、7103-07-huVL4(SEQ ID NO：140)、7103-07-huVL5(SEQ ID NO：141)。
具体序列如下表。
表7.人源化抗体的V区序列
Figure PCTCN2022094422-appb-000011
Figure PCTCN2022094422-appb-000012
Figure PCTCN2022094422-appb-000013
Figure PCTCN2022094422-appb-000014
表8.人源化抗体不同轻重链的组合方式以及对应编号：
人源化抗体来源 人源化抗体轻重链组合方式 组合编号
7103-01 huVH1VL1 P03486
7103-01 huVH2VL1 P03487
7103-01 huVH3VL1 P03488
7103-01 huVH4VL1 P03489
7103-01 huVH1VL2 P03490
7103-01 huVH2VL2 P03491
7103-01 huVH3VL2 P03492
7103-01 huVH4VL2 P03493
7103-07 huVH1VL1 P03476
7103-07 huVH1VL2 P03477
7103-07 huVH1VL3 P03478
7103-07 huVH2VL1 P03479
7103-07 huVH2VL2 P03480
7103-07 huVH2VL3 P03481
7103-07 huVH3VL1 P03482
7103-07 huVH3VL2 P03483
7103-07 huVH3VL3 P03484
7103-07 huVH4VL2 P04662
7103-07 huVH5VL2 P04663
7103-07 huVH4VL5 P04665
7103-07 huVH5VL4 P04666
7103-07 huVH5VL5 P04667
实施例8.人源化抗人TIGIT单克隆抗体鉴定
8.1表达载体的构建
根据上述人源化设计结果，将序列构建到pcDNA3.4载体上，经PCR、酶切、连接、转化、鉴定、测序、比对、抽提后得到质粒。
8.2各种组合的抗体的制备
根据人源化设计后的表达组合，运用ExpiCHO-S表达系统(Thermo Fisher，A29133)，将上述质粒按照要求进行在ExpiCHO-S细胞中为期7天的表达，并在表达第6天，通过HPLC测出表达量。最后经ProteinA亲和层析一步纯化，得到待检抗体蛋白。
8.3人源化单克隆抗体与TIGIT的结合
8.3.1用生物膜层光学干涉技术(BLI)研究人源化TIGIT单克隆抗体与 TIGIT的结合活性。
实验方法同实施例3.2
实验结果如下表：
表9.结合于人TIGIT的人源化单克隆抗体的动力学分析结果
表9.1以鼠源母本抗体7103-01设计的人源化抗体的动力学分析结果
Figure PCTCN2022094422-appb-000015
表9.2以鼠源母本抗体7103-07设计的人源化抗体的动力学分析结果
Figure PCTCN2022094422-appb-000016
实验结果显示所有抗体在Global拟合模式下的相关系数R 2都大于0.95，符合系统适应性要求，结果可靠。
8.3.2用ELISA方法研究人源化单克隆TIGIT抗体与hTIGIT的体外结合活性。
为了确定本公开人源化TIGIT单克隆抗体是否可以结合重组人TIGIT蛋白，进行体外测试。将鼠源抗体和替瑞利尤单抗(Tira)用作阳性对照。实验方法同实施例3.1
实验结果与分析：
ELISA试验结果(图13)显示：6株7103-01-抗人TIGIT人源化单克隆抗体(编号P03486、P03487、P03490、P03491、P03492、P03493)能特异性结合人TIGIT。结合的亲和力EC 50分别为0.511nM，0.532nM，0.501nM，0.471nM，0.463nM，0.444nM，对比替瑞利尤单抗的0.540nM，本发明的人源化单克隆抗体结合人TIGIT亲和力更高。
ELISA试验结果(图14A)显示：9株7103-07抗人TIGIT人源化单克隆抗体(编号P03476、P03477、P03478、P03479、P03480、P03481、P03482、P03483和P03484)能特异性结合人TIGIT。结合的亲和力EC 50分别为0.221nM，0.194nM，0.161nM，0.0858nM，0.252nM，0.122nM，0.182nM，0.212nM，0.187nM，对比人鼠嵌合抗体EC 50为0.206nM，以及Tira的EC 50为0.528nM，表明重组表达的人源化抗体结合抗原TIGIT的活性很好，与人鼠嵌合抗体相当，优于替瑞利尤单抗(Tira)。
用同种方法进行第二批次的实验，ELISA试验结果(图14B)显示：5株7103-07抗人TIGIT人源化单克隆抗体(编号P04662、P04663、P04664、P04665、P04666和P04667)能特异性结合人TIGIT。结合的亲和力EC 50分别为0.0419nM、0.0330nM、0.0913nM、0.0915nM、0.0836nM，对比人鼠嵌合抗体EC 50为0.0640nM，以及Tira的EC 50为0.1619nM，同样表明重组表达的人源化抗体结合抗原TIGIT的活性很好，与人鼠嵌合抗体相当，优于替瑞利尤单抗(Tira)。
8.3.3用流式细胞术方法(flow cytometry)研究人源化抗体与细胞膜上TIGIT蛋白的结合
为了确定本公开人源化TIGIT单克隆抗体是否可以结合细胞膜上的TIGIT蛋白，进行体外测试。将鼠源抗体和替瑞利尤单抗(Tira)用作阳性对照。
实验方法同实施例3.2
实验结果与分析：
如图15所示，3株7103-01抗人TIGIT人源化单克隆抗体(编号P03489、P03492、P03493)均和CHO-TIGIT结合较好，结合CHO-TIGIT细胞膜上的EC 50分别为：0.761nM，0.437nM，0.557nM，明显优于Tira的1.356nM。
如图16所示，8株7103-07抗人TIGIT人源化单克隆抗体(编号P03476、 P03477、P03478、P03479、P03480、P03481、P03482、P03483)均和CHO-TIGIT结合较好，结合CHO-TIGIT细胞膜上的EC 50分别为：1.518nM，1.738nM，2.582nM，0.963nM，2.193nM，2.824nM，2.376nM，2.607nM，对比人鼠嵌合单克隆抗体的1.347nM和Tira的3.844，本发明人源化抗体的亲和力更高。
本公开的人源化抗体不仅与固相抗原(ELISA法)结合能力很强，与细胞膜上的TIGIT结合活性也很高，并且同样优于Tira与细胞膜表面TIGIT的结合。
实验结果(图17A)显示了9株7103-07抗人TIGIT人源化单克隆抗体(编号P03476、P03477、P03478、P03479、P03480、P03481、P03482、P03483和P03484)能特异性阻断CD155与人TIGIT的结合。IC 50分别为：1.316nM，0.928nM，0.989nM，0.714nM，1.239nM，0.620nM，0.791nM，0.836nM，1.288nM，阻断能力与人鼠嵌合单克隆抗体IC 50值1.033nM相当或更优，且明显优于替瑞利尤单抗(IC 50值2.534nM)。
实验结果(图17B)显示了另一批次4株7103-07抗人TIGIT人源化单克隆抗体(编号P04662、P04663、P04666和P04667)能特异性阻断CD155与人TIGIT的结合，第一批次P03479用作对照。IC 50分别为：0.630nM，0.594nM，0.127nM，0.631nM，阻断能力与人鼠嵌合单克隆抗体IC 50值0.273nM相当，且优于替瑞利尤单抗(IC 50值0.775nM)。
实施例9.体外增强PBMC的INFγ产生
9.1体外增强PBMC的INFγ的产生
T细胞激活后，淋巴细胞分泌细胞因子IFNγ。为了测试本公开人源化TIGIT单克隆抗体作为T细胞活化的阳性调节剂功能，我们进行了以下实验，说明了本公开抗体对TIGIT信号阻断对PBMC细胞IFNγ产生的增强作用。
实验方法：复苏冷冻的人外周血单核细胞PBMC，调整细胞密度至5×10^6个/mL，用RPMI 1640+10％FBS培养基分别稀释抗体至抗体浓度为20μg/mL，PHA浓度6μg/mL，分别加50μL的PHA稀释液和50μL的抗体稀释液至96孔板中，最后加100μL的细胞悬液至96孔板中，轻轻混匀后至37℃，5％CO 2培养箱中培养4天。4天后吸取上清100μL检测IFNγ的含量。
实验结果与分析：
如图18显示，7株人源化TIGIT单克隆抗体(编号P03479、P03480、P03481、P03488、P03489、P03491和P03493)对PHA激活的PBMC释放IFNγ具有促进作用。加入人源化TIGIT抗体的IFNγ释放量比不加人源化TIGIT单克隆抗体的IFNγ释放量高出31％-81％，且效果均优于替瑞利尤单抗(16％)。
实施例10.体内抑制小鼠肿瘤生长的功能研究
由于本发明的抗体都不结合小鼠TIGIT，我们用人TIGIT基因敲入小鼠(C57BL/6)研究抗体的体内抗肿瘤效果。人TIGIT基因敲入小鼠饲养在SPF级别环境里。在本实施例中小鼠肿瘤模型为结肠癌MC38。
试验方法简述如下：小鼠结肠癌MC38肿瘤细胞来自上海南方模式生物科技股份有限公司。24只7～9周龄的人TIGIT基因敲入C57BL/6小鼠分成3组(4雄4雌/组)，用皮下接种的方法在小鼠腋下注射MC38细胞1×10 6个/只。在接种的当天(D0)，通过腹腔注射的方式分别给予各组小鼠PBS(对照组)、重组人IgG110mg/kg(对照组)和TIGIT人源化抗体P0347910mg/kg。每周给药2次，共4次。每次给药时测量瘤体积(即长径×短径 2/2)，小鼠称重。在D17结束试验。引颈脱臼处死小鼠，取瘤称重。
研究结果见图19。图19A显示各组每个小鼠的肿瘤体积与时间的关系，图19B显示在试验结束时(D17)各组每个小鼠的肿瘤重量。实验结果表明，TIGIT人源化抗体P03479能有效抑制小鼠肿瘤的生长(图19A和19B)，在实验结束时(D17)，P03479组小鼠肿瘤的平均重量显著小于hIgG1组(p＜0.05)。
在本公开提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本公开的上述讲授内容之后，本领域技术人员可以对本公开作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (18)

  1. 靶向T细胞免疫球蛋白和ITIM结构域蛋白(TIGIT)的单克隆抗体或其抗原结合片段，其与CD155竞争性结合TIGIT，且其与人TIGIT的结合亲和力EC 50为0.01～20nM。
  2. 靶向T细胞免疫球蛋白和ITIM结构域蛋白(TIGIT)的单克隆抗体或其抗原结合片段，其中，所述单克隆抗体具有选自下组的重链互补决定区(VH CDR)1～3和轻链互补决定区(VL CDR)1～3，或与其具有至少95％序列同一性的序列：
    选自下组的VH CDR1：SEQ ID NO：28、36、44、52、60、68、76、84；
    选自下组的VH CDR2：SEQ ID NO：29、37、45、53、61、69、77、85、128、133、142、143；
    选自下组的VH CDR3：SEQ ID NO：30、38、46、54、62、70、78、86；
    选自下组的VL CDR1：SEQ ID NO：32、40、48、56、64、72、80、88；
    选自下组的VL CDR2：SEQ ID NO：33、41、49、57、65、73、81、89；和
    选自下组的VL CDR3：SEQ ID NO：34、42、50、58、66、74、72、90；和/或
    氨基酸序列分别如下所述的VH CDR 1～3：SEQ ID NO：28～30；SEQ ID NO：36～38；SEQ ID NO：44～46；SEQ ID NO：52～54；SEQ ID NO：60～62；SEQ ID NO：68～70；SEQ ID NO：76～78；SEQ ID NO：84～86；SEQ ID NO：28、142、30；SEQ ID NO：28、128、30；SEQ ID NO：76、133、78；SEQ ID NO：76、143、78；以及氨基酸序列分别如下所述的VL CDR 1～3：SEQ ID NO：32～34；SEQ ID NO：40～42；SEQ ID NO：48～50；SEQ ID NO：56～58；SEQ ID NO：64～66；SEQ ID NO：72～74；SEQ ID NO：80～82；SEQ ID NO：88～90；和/或
    所述单克隆抗体具有选自下组的重链互补决定区(VH CDR)1～3和轻链互补决定区(VL CDR)1～3：
    (a)氨基酸序列分别如SEQ ID NO：28～30所述的VH CDR 1～3，以及氨基酸序列分别如SEQ ID NO：32～34所述的VL CDR 1～3；
    (b)氨基酸序列分别如SEQ ID NO：36～38所述的VH CDR 1～3，以及氨基酸序列分别如SEQ ID NO：40～42所述的VL CDR 1～3；
    (c)氨基酸序列分别如SEQ ID NO：44～46所述的VH CDR 1～3，以及氨 基酸序列分别如SEQ ID NO：48～50所述的VL CDR 1～3；
    (d)氨基酸序列分别如SEQ ID NO：52～54所述的VH CDR 1～3，以及氨基酸序列分别如SEQ ID NO：56～58所述的VL CDR 1～3；
    (e)氨基酸序列分别如SEQ ID NO：60～62所述的VH CDR 1～3，以及氨基酸序列分别如SEQ ID NO：64～66所述的VL CDR 1～3；
    (f)氨基酸序列分别如SEQ ID NO：68～70所述的VH CDR 1～3，以及氨基酸序列分别如SEQ ID NO：72～74所述的VL CDR 1～3；
    (g)氨基酸序列分别如SEQ ID NO：76～78所述的VH CDR 1～3，以及氨基酸序列分别如SEQ ID NO：80～82所述的VL CDR 1～3；
    (h)氨基酸序列分别如SEQ ID NO：84～86所述的VH CDR 1～3，以及氨基酸序列分别如SEQ ID NO：88～90所述的VL CDR 1～3；
    (i)氨基酸序列分别如SEQ ID NO：28、142、30所述的VH CDR 1～3，以及氨基酸序列分别如SEQ ID NO：32～34所述的VL CDR 1～3；
    (j)氨基酸序列分别如SEQ ID NO：28、128、30所述的VH CDR 1～3，以及氨基酸序列分别如SEQ ID NO：32～34所述的VL CDR 1～3；
    (k)氨基酸序列分别如SEQ ID NO：76、133、78所述的VH CDR 1～3，以及氨基酸序列分别如SEQ ID NO：80～82所述的VL CDR 1～3；或
    (l)氨基酸序列分别如SEQ ID NO：76、143、78所述的VH CDR 1～3，以及氨基酸序列分别如SEQ ID NO：80～82所述的VL CDR 1～3。
  3. 如权利要求1或2所述的单克隆抗体或其抗原结合片段，其中，所述单克隆抗体具有选自下组的重链可变区(VH)和轻链可变区(VL)序列：
    如SEQ ID NO：27、35、43、51、59、67、75和83中任一项所述的VH氨基酸序列，或与其具有至少70％序列同一性的序列；以及，如SEQ ID NO：31、39、47、55、63、71、79和87中任一项所述的VL氨基酸序列，或与其具有至少70％序列同一性的序列；
    例如，所述单克隆抗体具有选自下组的VH和VL序列：
    (A)如SEQ ID NO：27所述的VH氨基酸序列，以及如SEQ ID NO：31所述的VL氨基酸序列，或与其具有至少70％序列同一性的序列；
    (B)如SEQ ID NO：35所述的VH氨基酸序列，以及如SEQ ID NO：39所述的VL氨基酸序列，或与其具有至少70％序列同一性的序列；
    (C)如SEQ ID NO：43所述的VH氨基酸序列，以及如SEQ ID NO：47所述的VL氨基酸序列，或与其具有至少70％序列同一性的序列；
    (D)如SEQ ID NO：51所述的VH氨基酸序列，以及如SEQ ID NO：55所述的VL氨基酸序列，或与其具有至少70％序列同一性的序列；
    (E)如SEQ ID NO：59所述的VH氨基酸序列，以及如SEQ ID NO：63所述的VL氨基酸序列，或与其具有至少70％序列同一性的序列；
    (F)如SEQ ID NO：67所述的VH氨基酸序列，以及如SEQ ID NO：71所述的VL氨基酸序列，或与其具有至少70％序列同一性的序列；
    (G)如SEQ ID NO：75所述的VH氨基酸序列，以及如SEQ ID NO：79所述的VL氨基酸序列，或与其具有至少70％序列同一性的序列；或
    (H)如SEQ ID NO：83所述的VH氨基酸序列，以及如SEQ ID NO：87所述的VL氨基酸序列，或与其具有至少70％序列同一性的序列。
  4. 如权利要求1或2所述的单克隆抗体或其抗原结合片段，其中，所述单克隆抗体为经人源化的抗体，例如选自嵌合抗体、CDR移植抗体和表面重塑抗体。
  5. 如权利要求4所述的单克隆抗体或其抗原结合片段，其中，所述单克隆抗体是嵌合抗体，其中：
    其恒定区(C区)为人恒定区，例如其重链恒定区为人IgG(例如IgG1、IgG2、IgG3或IgG4)和/或其轻链恒定区为人κ或λ；
    其可变区(V区)为鼠可变区，例如，所述嵌合抗体还具有如权利要求2中所限定的VH和VL序列或如权利要求3所限定的VH CDR 1～3和VL CDR 1～3序列；或者
    所述嵌合抗体表达载体的构建中采用了选自SEQ ID NO：91～123中所述的任一对相应的成对引物。
  6. 如权利要求1或2所述的单克隆抗体或其抗原结合片段，其中，所述单克隆抗体为CDR移植抗体，其包括如权利要求3中所限定的VH CDR 1～3和VL CDR 1～3序列。
  7. 如权利要求1或2所述的单克隆抗体或其抗原结合片段，其中，所述单克隆抗体为表面重塑抗体，其中，所述单克隆抗体由权利要求2中所限定的单克隆抗体经VH和VL中一个或多个氨基酸残基的人源化设计和替代获得，所述氨基酸替代位于或不位于CDR中(例如SEQ ID NO：29的VH CDR 2被SEQ ID NO：128或SEQ ID NO：142的人源化VH CDR2替代；SEQ ID NO：77的VL CDR 2被SEQ ID NO：133或SEQ ID NO：143的人源化VL CDR2替代)；
    例如，所述单克隆抗体具有选自下组的VH和VL序列：
    如SEQ ID NO：124、125、126、127、131、132、134、135和136中任一 项所述的VH氨基酸序列，或与其具有至少70％序列同一性的序列；和/或，如SEQ ID NO：129、130、137、138、139、140和141中任一项所述的VL氨基酸序列，或与其具有至少70％序列同一性的序列；
    例如，所述单克隆抗体具有选自下组的VH和VL序列：
    (A′)如SEQ ID NO：124所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：129所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；
    (B′)如SEQ ID NO：125所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：129所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；
    (C′)如SEQ ID NO：126所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：129所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；
    (D′)如SEQ ID NO：127所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：129所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；
    (E′)如SEQ ID NO：124所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：130所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；
    (F′)如SEQ ID NO：125所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：130所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；
    (G′)如SEQ ID NO：126所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：130所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；
    (H′)如SEQ ID NO：127所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：130所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；
    (I′)如SEQ ID NO：131所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：137所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；
    (J′)如SEQ ID NO：131所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：138所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；
    (K′)如SEQ ID NO：131所述的VH氨基酸序列或与其具有至少70％序列 同一性的序列，以及如SEQ ID NO：139所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；
    (L′)如SEQ ID NO：132所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：137所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；
    (M′)如SEQ ID NO：132所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：138所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；
    (N′)如SEQ ID NO：132所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：139所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；
    (O′)如SEQ ID NO：134所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：137所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；
    (P′)如SEQ ID NO：134所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：138所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；或
    (Q′)如SEQ ID NO：134所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：139所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；
    (R′)如SEQ ID NO：135所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：138所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；
    (S′)如SEQ ID NO：136所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：138所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；
    (T′)如SEQ ID NO：135所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：141所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；
    (U′)如SEQ ID NO：136所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：140所述的VL氨基酸序列或与其具有至少70％序列同一性的序列；
    (V′)如SEQ ID NO：136所述的VH氨基酸序列或与其具有至少70％序列同一性的序列，以及如SEQ ID NO：141所述的VL氨基酸序列或与其具有至少70％序列同一性的序列。
  8. 如权利要求1或2所述的单克隆抗体或其抗原结合片段，其具有选自下组的一个或多个特征：
    (i)在体内或体外特异性结合人TIGIT，例如其通过ELISA测定的与人TIGIT的结合亲和力EC 50为0.001nM～100nM，0.01nM～50nM，0.1nM～10nM；
    (ii)竞争性抑制CD155与TIGIT的结合，例如，其通过ELISA测定的特异性阻断CD155与人TIGIT的IC 50为0.005μg/ml～1μg/ml，0.01μg/ml～0.9μg/ml；
    (iii)结合人或灵长类哺乳动物TIGIT，而不结合小鼠TIGIT；
    (iv)正向调节免疫细胞(例如T细胞和NK细胞)的活性，例如提高人外周血单核细胞(PBMC)(如淋巴细胞、T细胞)的促免疫反应细胞因子(例如IFNγ)产生、减少或逆转NK细胞耗竭；
    (v)减少或消除表达TIGIT的细胞，例如Treg细胞，降低免疫抑制作用；
    (vi)增强免疫应答；以及
    (vii)具有针对肿瘤、感染或感染性疾病的预防和/或治疗作用。
  9. 编码权利要求1～8中任一项所述的单克隆抗体或其抗原结合片段的核酸分子，或包含所述核酸分子的载体或宿主细胞。
  10. 一种免疫偶联物，其包含：
    (a)权利要求1～8中任一项所述的单克隆抗体或其抗原结合片段；
    (b)选自下组的偶联部分：药物、毒素、细胞因子、放射性核素或酶；和
    (c)可任选的，接头。
  11. 一种嵌合抗原受体，其包含胞外结构域和胞内结构域，其中，所述胞外结构域包含如权利要求1～8中任一项所述的单克隆抗体或其抗原结合片段。
  12. 如权利要求11所述的嵌合抗原受体，其中：
    所述抗体或其抗原结合片段包含scFv或VHsdAb，或由其组成；
    所述胞内结构域编码选自下组的一种或多种：ITAM结构域、CD3ζ、CD28、4-1BB、OX40、CD27、ICOS或其组合，例如(CD28+CD3ζ)、(CD28+CD27+CD3ζ)、(CD28+OX40+CD3ζ)、(CD28+4-1BB+CD3ζ)、(CD28+CD27+ OX40+CD3ζ)、(CD28+4-1BB+CD27+CD3ζ)、(CD28+4-1BB+OX40+CD3ζ)、(4-1BB+CD3ζ)、(4-1BB+OX40+CD3ζ)、(4-1BB+CD27+CD3ζ)、(CD27+CD3ζ)、(CD27+OX 40+CD3ζ)、(CD28Δ+CD3ζ)、(CD28Δ+CD27+CD3ζ)、(CD28Δ+OX40+CD3ζ)、(CD28Δ+4-1BB+CD3ζ)、(CD28Δ+4-1BB+OX40+CD3ζ)、(CD28Δ+CD27+OX40+CD3ζ)、(CD28Δ+4-1BB+CD27+CD3ζ))、(4-1BB+ICOS+CD3ζ)、(CD28+ICOS+CD3ζ)、(ICOS+CD3ζ)；和/或
    所述嵌合抗原受体的胞外结构域还包含铰链区，例如所述铰链区源自IgG的铰链或CD8α/CD28胞外区，如选自：IgG4 FcΔEQ、IgG4 FcΔQ、(t-12AA+t-20AA)、mKate、phiLov、dsRed、Venus、eGFP、CH3 HA，(CD8α+t-20AA)、双重t-20 AA、(t-20AA+CD8α)、(CD8α+亮氨酸拉链Basep 1)、(CD8α+亮氨酸拉链Acid1)、2D3、CD8α或IgG4 Fc；和/或
    所述嵌合抗原受体还包含连接胞外结构域和胞内结构域的跨膜结构域，例如源自T-细胞受体的α、β或ζ链，如CD28，CD3ε，CD45，CD4，CD5，CDS，CD9，CD 16，CD22，CD33，CD37，CD64，CD80，CD86，CD 134，CD137，CD 154的跨膜区。
  13. 一种核酸分子以及包含所述核苷酸分子的构建体或载体，其中，所述核酸分子包含权利要求11或12所述嵌合抗原受体的编码序列。
  14. 一种经转化的免疫细胞，其表达根据权利要求11或12所述的嵌合抗原受体，或采用权利要求13所述的核酸分子、构建体或载体转化，例如所述免疫细胞选自T细胞，如αβT细胞、γδT细胞或NK T细胞或从多能细胞衍生的T细胞。
  15. 一种组合物，其包含：(A)权利要求1～8中任一项所述的单克隆抗体或其抗原结合片段、如权利要求9所述的核酸分子、载体或宿主细胞或如权利要求10所述的免疫偶联物、如权利要求11或12所述的嵌合抗原受体、如权利要求13所述的核酸分子、构建体或载体、和/或如权利要求14所述的经转化的免疫细胞，以及(B)载剂；
    例如，所述组合物为：药物组合物，其还包含药学上可接受的载剂；检测试剂盒，其还包含检测TIGIT水平或其活性或其相关通路所需的试剂。
  16. 如权利要求1～8中任一项所述的单克隆抗体或其抗原结合片段、如权利要求9所述的核酸分子、载体或宿主细胞、如权利要求10所述的免疫偶联物、如权利要求11或12所述的嵌合抗原受体、如权利要求13所述的核酸分子、构建体或载体、如权利要求14所述的经转化的免疫细胞和/或如权利要求15所述的组合物在制备正向调节免疫细胞活性和/或提高免疫应答的产品中的应用，例如用于制备预防和/或治疗肿瘤、感染或感染性疾病的药物中的应用。
  17. 如权利要求1～8中任一项所述的单克隆抗体或其抗原结合片段、如权利要求9所述的核酸分子、载体或宿主细胞、如权利要求10所述的免疫偶联物、如权利要求11或12所述的嵌合抗原受体、如权利要求13所述的核酸分子、构建体或载体、如权利要求14所述的经转化的免疫细胞和/或如权利要求15所述的组合物在制备减少或消除表达TIGIT的细胞(如Treg细胞)的免疫抑制作用的产品中的应用。
  18. 如权利要求1～8中任一项所述的单克隆抗体或其抗原结合片段、如权利要求9所述的核酸分子、载体或宿主细胞、如权利要求10所述的免疫偶联物、如权利要求11或12所述的嵌合抗原受体、如权利要求13所述的核酸分子、构建体或载体、如权利要求14所述的经转化的免疫细胞和/或如权利要求15所述的组合物在制备检测TIGIT存在与否、其水平或活性的试剂盒中的应用。
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