WO2023138639A1 - 靶向tigit的抗原结合蛋白及其用途 - Google Patents

靶向tigit的抗原结合蛋白及其用途 Download PDF

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WO2023138639A1
WO2023138639A1 PCT/CN2023/073041 CN2023073041W WO2023138639A1 WO 2023138639 A1 WO2023138639 A1 WO 2023138639A1 CN 2023073041 W CN2023073041 W CN 2023073041W WO 2023138639 A1 WO2023138639 A1 WO 2023138639A1
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Prior art keywords
seq
binding protein
hcdr2
hcdr3
hcdr1
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PCT/CN2023/073041
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English (en)
French (fr)
Inventor
钟阵威
宁亚楠
邓奇
陈晓锐
李霄培
李萌
褚所
何晓文
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原启生物科技(上海)有限责任公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This application relates to the field of biomedicine, in particular to an antigen-binding protein targeting TIGIT and its application.
  • Immune checkpoint T cell immunoglobulin and immunoreceptor tyrosine inhibitory motif domain (TIGIT, also known as Vstm3, WUCAM) is a type I transmembrane protein.
  • TIGIT also known as Vstm3, WUCAM
  • CD155 also known as poliovirus receptor or PVR
  • CD112 also known as poliovirus receptor-related ligand 2, PVRL2, cohesin-2
  • TIGIT competes with the activating receptor CD226 to bind to the ligand CD155, transmits inhibitory signals, inhibits excessive cell activation and inhibits the secretion of pro-inflammatory cytokines (Manieri, Trends Immunol, 2017, 38(1):20-8).
  • TIGIT is mainly expressed on regulatory T cells (Treg), activated T cells and natural killer cells (NK) (YU, Nat Immunol, 2009, 10(1):48-57), which can directly inhibit the proliferation, killing, degranulation and cytokine secretion of T cells and NK cells, and can also inhibit T cell activity by promoting DC cells to secrete IL-10 (Nicholas, Trends Immunol.2017, 38(1) :20-28).
  • TIGIT expressed on Treg can maintain the immunosuppressive function of Treg (Manieri, Trends Immunol, 2017, 38(1):20-8).
  • TIGIT is significantly up-regulated in a variety of tumor cells, infiltrating lymphocytes and peripheral blood mononuclear cells, and its ligands CD155 and CD112 are also widely expressed in a variety of tumor cells, suggesting that TIGIT may play a key role in the process of tumor immune escape (Tassi, Cancer Res.2017,77:851-861.Kong,Clin Cancer Res,2016,22:3057-66. Immunol Immunother, 2009,58(9):1517-26.).
  • the known anti-TIGIT antibodies still have defects such as low specificity and limited lethality. Therefore, there is an urgent need to develop anti-TIGIT antigen-binding proteins with high affinity and specificity for TIGIT protein.
  • the present application provides an isolated antigen-binding protein, which can specifically bind to TIGIT protein.
  • the antigen-binding protein described in the present application has one or more of the following characteristics: (1) can specifically bind to TIGIT protein; (2) can have binding activity with both human TIGIT and cynomolgus monkey TIGIT protein; (3) can have a blocking effect on the interaction between cell surface TIGIT protein and CD155; and (4) can inhibit tumor growth and/or proliferation.
  • the antigen binding protein comprises an antibody or antigen binding fragment thereof.
  • the antigen-binding fragment comprises Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  • the antigen-binding fragment is VHH.
  • the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the antigen binding protein comprises at least one CDR derived from an antibody heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 132.
  • the antigen binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 127.
  • the antigen binding protein comprises HCDR3, and the HCDR3 comprises SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
  • the antigen binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 128.
  • the antigen binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.
  • the antigen binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1.
  • the antigen binding protein comprises HCDR1, HCDR2, and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:128, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:127.
  • the antigen binding protein comprises HCDR1, HCDR2, and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, and the HCDR3 comprises SEQ ID NO:4, SEQ ID NO:8 , SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20 amino acid sequence.
  • the antigen binding protein comprises HCDR1, HCDR2, and HCDR3, and the HCDR1, HCDR2, HCDR3 comprise any set of amino acid sequences selected from the group consisting of:
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:2
  • HCDR3 SEQ ID NO:4;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:4;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:8;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:9;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:10
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:11;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:12
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:13
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:14;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:15;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:16;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:17
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:18;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:19;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:20;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:5
  • HCDR3 SEQ ID NO:18;
  • HCDR1 SEQ ID NO: 1
  • HCDR2 SEQ ID NO: 6
  • HCDR3 SEQ ID NO: 18;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:7
  • HCDR3 SEQ ID NO:18.
  • the antigen binding protein comprises H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 129.
  • said H-FR1 of said antigen binding protein comprises SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID The amino acid sequence shown in any one of NO:31 and SEQ ID NO:32.
  • the antigen binding protein comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:33.
  • the antigen binding protein comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the amino acid sequence of the H-FR3 SEQ ID NO: 130.
  • the H-FR3 of the antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37.
  • the antigen binding protein comprises H-FR4, the N-terminus of the H-FR4 is connected to the
  • H-FR4 comprises the amino acid sequence shown in SEQ ID NO:131.
  • said H-FR4 of said antigen binding protein comprises SEQ ID NO: 38 or SEQ ID The amino acid sequence shown in any one of NO:39.
  • the antigen binding protein comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:132.
  • said VH of said antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO: 40-67.
  • the antigen binding protein is a VHH
  • the VHH comprises the amino acid sequence shown in any one of SEQ ID NOs: 40-67.
  • the antigen binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:70.
  • the antigen binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76 and SEQ ID NO:77.
  • the antigen binding protein comprises HCDR1
  • the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81 and SEQ ID NO:82.
  • the antigen binding protein comprises HCDR1, HCDR2, and HCDR3, the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81 and SEQ ID NO:82, and the HCDR2 comprises SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SE The amino acid sequence shown in any one of Q ID NO:74, SEQ ID NO:75, SEQ ID NO:76 and SEQ ID NO:77, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:70.
  • the antigen binding protein comprises HCDR1, HCDR2, and HCDR3, and the HCDR1, HCDR2, HCDR3 comprise any set of amino acid sequences selected from the group consisting of:
  • HCDR1 SEQ ID NO:78
  • HCDR2 SEQ ID NO:71
  • HCDR3 SEQ ID NO:68
  • HCDR1 SEQ ID NO:78
  • HCDR2 SEQ ID NO:72
  • HCDR3 SEQ ID NO:68
  • HCDR1 SEQ ID NO:79
  • HCDR2 SEQ ID NO:72
  • HCDR3 SEQ ID NO:68
  • HCDR1 SEQ ID NO:78
  • HCDR2 SEQ ID NO:73
  • HCDR3 SEQ ID NO:68
  • HCDR1 SEQ ID NO:80
  • HCDR2 SEQ ID NO:73
  • HCDR3 SEQ ID NO:68
  • HCDR1 SEQ ID NO:78
  • HCDR2 SEQ ID NO:71
  • HCDR3 SEQ ID NO:68
  • HCDR1 SEQ ID NO:78
  • HCDR2 SEQ ID NO:74
  • HCDR3 SEQ ID NO:68
  • HCDR1 SEQ ID NO:80
  • HCDR2 SEQ ID NO:75
  • HCDR3 SEQ ID NO:68
  • HCDR1 SEQ ID NO:81
  • HCDR2 SEQ ID NO:76
  • HCDR3 SEQ ID NO:69
  • HCDR1 SEQ ID NO:82
  • HCDR2 SEQ ID NO:77
  • HCDR3 SEQ ID NO:70.
  • the antigen binding protein comprises H-FR1
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1
  • the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 133.
  • said H-FR1 of said antigen binding protein comprises SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID The amino acid sequence shown in any one of NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:99.
  • the antigen binding protein comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:134.
  • the H-FR2 of the antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:100, SEQ ID NO:101 and SEQ ID NO:102.
  • the antigen binding protein comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109 .
  • the antigen binding protein comprises H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:38.
  • the antigen binding protein comprises a VH
  • the VH comprises the amino acid sequence shown in any one of SEQ ID NOs: 110-126.
  • the antigen binding protein is a VHH
  • the VHH comprises the amino acid sequence shown in any one of SEQ ID NOs: 110-126.
  • the antigen binding protein comprises an antibody heavy chain constant region derived from IgG.
  • the antigen binding protein comprises an antibody heavy chain constant region derived from human IgG.
  • the antigen binding protein comprises an antibody heavy chain constant region derived from human IgGl.
  • the present application also provides a polypeptide comprising the antigen-binding protein described in the present application.
  • the present application also provides one or more isolated nucleic acid molecules encoding the antigen binding proteins described herein.
  • the present application also provides a vector comprising the nucleic acid molecule.
  • the present application also provides a cell comprising the vector.
  • the present application also provides a method for preparing the antigen-binding protein, the method comprising culturing the cells under conditions that allow the expression of the antigen-binding protein.
  • the present application also provides a pharmaceutical composition, which comprises the antigen-binding protein, and optionally a pharmaceutically acceptable carrier.
  • the present application also provides a pharmaceutical combination, which comprises the antigen-binding protein and an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor comprises an antigen binding protein targeting PD-L1.
  • the antigen binding protein targeting PD-L1 in the pharmaceutical combination comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:135, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:136, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:137.
  • the antigen binding protein targeting PD-L1 in the pharmaceutical combination comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:139, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:140, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:141.
  • the antigen binding protein targeting PD-L1 in the pharmaceutical combination comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:138.
  • the antigen binding protein targeting PD-L1 in the pharmaceutical combination comprises VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:142.
  • the present application also provides the use of the antigen-binding protein, the polypeptide, the nucleic acid molecule, the carrier, the cell, the pharmaceutical composition, and/or the pharmaceutical combination in the preparation of medicines for preventing, treating and/or alleviating diseases and/or conditions.
  • the diseases and/or disorders include diseases and/or disorders associated with aberrant expression of TIGIT.
  • the disease and/or condition comprises a tumor.
  • the tumor comprises a solid tumor.
  • the tumor comprises a non-solid tumor.
  • the tumor comprises an immune checkpoint sensitive tumor.
  • the tumor comprises colon cancer, melanoma, non-small cell lung cancer, renal cell carcinoma, and/or hepatocellular carcinoma.
  • the present application also provides a method for preventing, treating and/or alleviating diseases and/or disorders, the method comprising administering the antigen-binding protein, the polypeptide, the nucleic acid molecule, the carrier, the cell, the pharmaceutical composition, and/or the pharmaceutical combination to a subject in need.
  • the diseases and/or disorders include diseases and/or disorders associated with aberrant expression of TIGIT.
  • the disease and/or condition comprises a tumor.
  • the tumor comprises a solid tumor.
  • the tumor comprises a non-solid tumor.
  • the tumor comprises an immune checkpoint sensitive tumor.
  • the tumor comprises colon cancer, melanoma, non-small cell lung cancer, renal cell carcinoma, and/or hepatocellular carcinoma.
  • the present application also provides a method for blocking the interaction between TIGIT protein and CD155, the method comprising administering the antigen-binding protein, the polypeptide, the nucleic acid molecule, the carrier, the cell, the pharmaceutical composition, and/or the pharmaceutical combination to a subject in need.
  • the methods are non-diagnostic and non-therapeutic methods.
  • the method is an in vitro method and/or an ex vivo method.
  • Figure 1 shows the flow cytometric detection of the binding activity of the antigen-binding protein described in this application to TIGIT on the cell surface.
  • Figure 2 shows flow cytometric detection of the blocking effect of the antigen-binding protein described in this application on the interaction between TIGIT and CD155 on the cell surface.
  • Figures 3A-3AA show the results of affinity assays for antigen-binding proteins with blocking activity.
  • Figure 4A shows the results of the mouse tumor volume inhibition of the antigen-binding protein described in the present application
  • Figure 4B shows the results of the mouse tumor weight inhibition of the antigen-binding protein described in the application.
  • Figures 5A-5C show the in vivo efficacy testing of the combination of antigen binding protein and PD-L1 antibody described in this application.
  • Figure 6A shows the EC50 assay results of the antigen-binding proteins described in the present application
  • Figure 6B shows the results of identification of the aggregation degree of the antigen-binding proteins described in the present application.
  • Figure 7A shows the affinity detection results of the modified antigen-binding protein and human TIGIT
  • Figure 7B shows the affinity detection results of the modified antigen-binding protein and cynomolgus TIGIT
  • Figure 7C- Figure 7O shows the results of identification of the aggregation degree of the antigen-binding protein described in this application.
  • Figure 8A shows the affinity test results of the antigen-binding protein described in the present application and human TIGIT
  • Figure 8B shows the affinity test results of the antigen-binding protein described in the present application and cynomolgus monkey TIGIT
  • Figure 8C shows the ELISA verification of the binding activity of the antigen-binding protein described in the present application at different temperatures
  • Figure 8D shows the verification of the binding activity of the antigen-binding protein described in the present application by SEC;
  • 9A-9B show the analysis of the binding activity of the antigen-binding protein described in the present application to TIGIT on PBMCs of cynomolgus monkeys after activation.
  • FIGS 10A-10D show the results of antigen-binding epitope verification of the antigen-binding protein described in this application.
  • Figure 11 shows the detection of blocking activity of the antigen binding proteins described in the present application.
  • Figure 12 shows the detection of ADCC activity of the antigen binding protein described in this application.
  • Figure 13 shows the results of mouse tumor volume inhibition of the antigen binding proteins described in the present application.
  • isolated antigen-binding protein generally refers to a protein having antigen-binding ability that is removed from its naturally occurring state.
  • Said “isolated antigen binding protein” may comprise an antigen-binding moiety and optionally a framework or framework portion which permits the antigen-binding moiety to adopt a conformation which facilitates its binding to antigen.
  • Antigen binding proteins may comprise, for example, antibody-derived protein framework regions (FR) or alternative or artificial framework regions with grafted variable regions (CDR) or CDR derivatives.
  • the antigen binding protein may comprise an antibody or an antigen binding fragment thereof.
  • the antigen binding protein can bind a TIGIT protein.
  • the antigen binding protein can compete with a reference antibody for binding to TIGIT protein.
  • the antigen binding protein may comprise an antibody heavy chain variable region VH.
  • the antigen binding protein can comprise derived from At least one CDR in the heavy chain variable region VH.
  • the VH may comprise HCDR3, HCDR2 and/or HCDR1.
  • the VH may include a framework region H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1.
  • the VH may comprise a framework region H-FR2 located between the HCDR1 and the HCDR2.
  • the VH may comprise a framework region H-FR3 located between the HCDR2 and the HCDR3.
  • the VH may include a framework region H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3.
  • the antigen binding protein can be VHH.
  • the antigen binding protein may comprise an antibody heavy chain constant region, which may be derived from IgG.
  • the antibody heavy chain constant region may be derived from human IgG.
  • the antibody heavy chain constant region may be derived from human IgG1.
  • antibody as used includes whole antibodies and binding fragments thereof. Typically, a fragment competes with the intact antibody from which it is derived for specific binding to the antigen.
  • antibodies or binding fragments thereof can be chemically conjugated to other proteins, or expressed as fusion proteins with other proteins.
  • the antibodies can be monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the binding protein of the antibody or binding fragment thereof can include TIGIT.
  • the antibody or binding fragment thereof can be specific for TIGIT.
  • antigen-binding fragment refers to a portion of an intact antibody and refers to the antigenically determining variable region of an intact antibody.
  • the antigen-binding fragments may include Fab, Fab', F(ab')2, Fv fragments and single-chain Fv fragments, tandem Fv fragments, VHH, bispecific antibodies.
  • the antigen-binding fragment can be a VHH.
  • the antigen-binding fragment can bind TIGIT.
  • the antigen-binding fragment can be specific for TIGIT.
  • VHH generally refers to an antibody comprising the variable antigen-binding domain of a heavy chain antibody.
  • VHHs may also be referred to as Nanobodies (Nb) and/or Single Domain Antibodies.
  • Nb Nanobodies
  • the VHH can bind TIGIT.
  • the VHH can be specific for TIGIT.
  • the antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • the term "heavy chain constant region” consists of three domains CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region.
  • the term "light chain constant region” consists of one domain, CL.
  • the VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors.
  • TIGIT protein or “TIGIT antigen” are used interchangeably and include any functionally active fragments, variants and homologues of TIGIT that are naturally expressed by cells or expressed on cells transfected with the TIGIT gene.
  • TIGIT may be human TIGIT, whose accession number in UniProt/Swiss-Prot is Q495A1.
  • TIGIT can be a functionally active fragment of human TIGIT.
  • such "functionally active fragments” may include fragments that retain at least one endogenous function of a naturally occurring protein (eg, binding to an antigen binding protein described herein).
  • the "functionally active fragment” may include a domain that binds to the antigen-binding protein of the present application.
  • TIGIT can be expressed on the surface of immune cells.
  • it can be expressed on the surface of regulatory T cells (Treg).
  • a functionally active fragment refers to a polypeptide having substantially the same amino acid sequence or encoded by a substantially identical nucleotide sequence as the naturally occurring sequence and capable of possessing one or more activities of the naturally occurring sequence.
  • a functionally active fragment of any given sequence is one in which a particular sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially retains at least one endogenous function.
  • Sequences encoding functionally active fragments can be obtained by addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in naturally occurring proteins and/or polynucleotides, as long as the original functional activity is maintained.
  • the term “derivative” generally refers to the polypeptide or polynucleotide of the present application including any substitution, variation, modification, substitution, deletion and/or addition of one (or more) amino acid residues from/to the sequence, as long as the resulting polypeptide or polynucleotide substantially retains at least one endogenous function thereof.
  • analogue generally refers to a polypeptide or polynucleotide, including any mimetic of the polypeptide or polynucleotide, that is, a chemical compound possessing at least one endogenous function of the polypeptide or polynucleotide simulated by the mimetic.
  • amino acid substitutions such as at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 20) amino acid substitutions can be made as long as the modified sequence substantially retains the desired activity or ability.
  • Amino acid substitutions may involve the use of non-naturally occurring analogs.
  • homologue generally refers to an amino acid sequence or nucleotide sequence having a certain homology to a naturally occurring sequence.
  • the term “homology” may be equated with sequence "identity”.
  • homologous sequences may include amino acid sequences that may be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence.
  • a homologue will comprise the same active site, etc., as the subject amino acid sequence.
  • Homology can be considered in terms of similarity (ie, amino acid residues having similar chemical properties/functions), or can be expressed in terms of sequence identity.
  • a reference to a sequence having a percentage identity to any of the SEQ ID NOs of an amino acid sequence or a nucleotide sequence refers to a sequence having said percentage identity over the entire length of the referenced SEQ ID NO.
  • sequence alignment can be performed by various means known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software and the like. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment across the full-length sequences being compared.
  • Proteins or polypeptides for use in this application may also have deletions, insertions or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins.
  • Deliberate amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphiphilic properties of the residues, so long as endogenous function is preserved.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids containing uncharged polar headgroups with similar hydrophilicity values include asparagine, glutamine, serine, threonine, and tyrosine.
  • IgG refers to a polypeptide belonging to the class of antibodies substantially encoded by the recognized immunoglobulin gamma genes. In humans, this class includes IgG1, IgG2, IgG3 and IgG4. In mice, this class includes IgGl, IgG2a, IgG2b, and IgG3.
  • polypeptide polypeptide
  • peptide protein
  • protein polymer of amino acid residues.
  • the term can be used to refer to amino acid polymers in which one or more amino acid residues are synthetic chemical mimics of the corresponding natural amino acid, as well as to natural amino acid polymers, those containing modified residues, and non-natural amino acid polymers.
  • said polypeptide may comprise said antigen binding protein.
  • nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or analogs thereof of any length isolated from their natural environment or artificially synthesized.
  • the term "cell” generally refers to an individual cell, cell line or cell culture that can or has contained a plasmid or vector comprising a nucleic acid molecule described herein, or is capable of expressing a polypeptide described herein or an antigen binding protein described herein.
  • the cells may include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny cells may not necessarily be completely identical in morphology or genome to the original parent cells, but it is sufficient to be able to express the polypeptide or antigen-binding protein described in this application.
  • the cells can be obtained by transfecting cells in vitro with the vectors described in this application.
  • the cells can be prokaryotic cells (such as Escherichia coli) or eukaryotic cells (such as yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells or myeloma cells).
  • prokaryotic cells such as Escherichia coli
  • eukaryotic cells such as yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells or myeloma cells.
  • the term "pharmaceutical composition” generally refers to a chemical or biological composition suitable for administration to a mammalian subject.
  • the pharmaceutical composition may include the antigen binding protein, the polypeptide, the nucleic acid molecule, the carrier and/or the cell, and optionally a pharmaceutically acceptable carrier.
  • the term "pharmaceutical combination” generally refers to a combination with at least two active ingredients. Often, different active ingredients in a pharmaceutical combination can be used for joint administration. For example, in the pharmaceutical combination, the two active ingredients can be placed in admixture or separately. For example, when administering, the pharmaceutical combination may be administered together or sequentially.
  • the application provides an isolated antigen-binding protein, which has one or more of the following properties: (1) can specifically bind TIGIT protein; (2) can have binding activity with both human TIGIT and cynomolgus monkey TIGIT protein; (3) can have a blocking effect on the interaction between cell surface TIGIT protein and CD155; and (4) can inhibit tumor growth and/or proliferation.
  • the binding activity of the isolated antigen-binding protein to TIGIT protein can be detected by flow cytometry fluorescence sorting technique (FACS).
  • the antigen binding protein comprises an antibody or antigen binding fragment thereof.
  • the antigen-binding fragment comprises Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  • the antigen-binding fragment is VHH.
  • the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the CDR of an antibody also known as the complementarity determining region, is part of the variable region.
  • the amino acid residues in this region may make contacts with the antigen or antigenic epitope.
  • Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, comprehensive consideration of Kabat/Chothia, etc. These numbering systems are known in the art, see, for example, http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region according to the sequence and structure of the antibody. There may be differences in the CDR regions using different coding systems.
  • the CDR covers the CDR sequence divided according to any CDR division method; it also covers its variants, and the variant includes the amino acid sequence of the CDR being substituted, deleted and/or added with one or more amino acids.
  • the variant includes the amino acid sequence of the CDR being substituted, deleted and/or added with one or more amino acids.
  • the isolated antigen binding proteins described herein are defined by the Kabat coding system.
  • the antigen binding protein may comprise at least one CDR derived from the variable region VH of the heavy chain of an antibody.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 132.
  • the VH can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 40-67.
  • the antigen binding protein may comprise HCDR3.
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 127.
  • the antigen binding protein may include HCDR3.
  • the HCDR3 comprises SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:1 8.
  • the antigen binding protein may comprise HCDR2.
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 128.
  • the antigen binding protein may comprise HCDR2.
  • the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.
  • the antigen binding protein may comprise HCDR1.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:1.
  • the antigen binding protein may comprise HCDR1, HCDR2, and HCDR3.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:12
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:127.
  • the antigen binding protein may comprise HCDR1, HCDR2, and HCDR3.
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1
  • the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7
  • the HCDR3 comprises SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:
  • the antigen-binding protein may comprise HCDR1, HCDR2, and HCDR3, and the HCDR1, HCDR2, and HCDR3 may comprise any amino acid sequence selected from the following group:
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:2
  • HCDR3 SEQ ID NO:4;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:4;
  • HCDR1 SEQ ID NO: 1
  • HCDR2 SEQ ID NO: 3
  • HCDR3 SEQ ID NO: 8;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:9;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:10
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:11;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:12
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:13
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:14;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:15;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:16;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:17
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:18;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:19;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:3
  • HCDR3 SEQ ID NO:20;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:5
  • HCDR3 SEQ ID NO:18;
  • HCDR1 SEQ ID NO: 1
  • HCDR2 SEQ ID NO: 6
  • HCDR3 SEQ ID NO: 18;
  • HCDR1 SEQ ID NO:1
  • HCDR2 SEQ ID NO:7
  • HCDR3 SEQ ID NO:18.
  • the antigen binding protein may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:70.
  • the antigen binding protein may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76 and SEQ ID NO:77.
  • the antigen binding protein may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81 and SEQ ID NO:82.
  • the antigen binding protein may comprise HCDR1, HCDR2, and HCDR3.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81 and SEQ ID NO:82
  • the HCDR2 may comprise SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO :75
  • the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:70.
  • the antigen binding protein may comprise HCDR1, HCDR2, and HCDR3, and the HCDR1, HCDR2 and HCDR3 may comprise any set of amino acid sequences selected from the following group:
  • HCDR1 SEQ ID NO:78
  • HCDR2 SEQ ID NO:71
  • HCDR3 SEQ ID NO:68
  • HCDR1 SEQ ID NO:78
  • HCDR2 SEQ ID NO:72
  • HCDR3 SEQ ID NO:68
  • HCDR1 SEQ ID NO:79
  • HCDR2 SEQ ID NO:72
  • HCDR3 SEQ ID NO:68
  • HCDR1 SEQ ID NO:78
  • HCDR2 SEQ ID NO:73
  • HCDR3 SEQ ID NO:68
  • HCDR1 SEQ ID NO:80
  • HCDR2 SEQ ID NO:73
  • HCDR3 SEQ ID NO:68
  • HCDR1 SEQ ID NO:78
  • HCDR2 SEQ ID NO:71
  • HCDR3 SEQ ID NO:68
  • HCDR1 SEQ ID NO:78
  • HCDR2 SEQ ID NO:74
  • HCDR3 SEQ ID NO:68
  • HCDR1 SEQ ID NO:80
  • HCDR2 SEQ ID NO:75
  • HCDR3 SEQ ID NO:68
  • HCDR1 SEQ ID NO:81
  • HCDR2 SEQ ID NO:76
  • HCDR3 SEQ ID NO:69
  • HCDR1 SEQ ID NO:82
  • HCDR2 SEQ ID NO:77
  • HCDR3 SEQ ID NO:70.
  • the antibody framework region FR refers to the part of the antibody variable region that exists between the more divergent (ie hypervariable) CDRs.
  • Such framework regions are typically referred to as frameworks 1 to 4 (FR1, FR2, FR3 and FR4) and provide a framework for representing the CDRs in three-dimensional space to form an antigen-binding surface.
  • the antigen-binding protein may comprise H-FR1, and the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 129.
  • the H-FR1 of the antigen binding protein may comprise SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID The amino acid sequence shown in any one of NO:31 and SEQ ID NO:32.
  • the antigen binding protein may comprise H-FR2, and the H-FR2 is located between the HCDR1 and the HCDR2.
  • the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:33.
  • the antigen binding protein comprises H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the amino acid sequence of the H-FR3 SEQ ID NO: 130.
  • the H-FR3 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37.
  • the antigen-binding protein may comprise H-FR4, and the N-terminal of the H-FR4 is connected to the C-terminal of the HCDR3.
  • the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:131.
  • the H-FR4 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:38 or SEQ ID NO:39.
  • the antigen binding protein may comprise H-FR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:133.
  • the H-FR1 of the antigen binding protein may comprise SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID The amino acid sequence shown in any one of NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:99.
  • the antigen binding protein may comprise H-FR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:134.
  • the H-FR2 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:100, SEQ ID NO:101 and SEQ ID NO:102.
  • the antigen binding protein may comprise H-FR3, and the H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109.
  • the antigen binding protein may comprise H-FR4, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:38.
  • the antigen-binding protein may comprise an antibody heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:132.
  • the VH of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO: 40-67.
  • the antigen binding protein can be VHH, and the VHH can comprise the amino acid sequence shown in any one of SEQ ID NO:40-67.
  • the antigen binding protein may comprise VH, and the VH may comprise the amino acid sequence shown in any one of SEQ ID NO: 110-126.
  • the antigen binding protein can be VHH, and the VHH can comprise the amino acid sequence shown in any one of SEQ ID NO: 1110-126.
  • said isolated antigen binding protein may comprise a heavy chain constant region.
  • the heavy chain constant region refers to the A region comprising at least three heavy chain constant domains, CH1, CH2, and CH3.
  • Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha.
  • Non-limiting exemplary heavy chain constant regions also include ⁇ and ⁇ .
  • Each heavy chain constant region corresponds to an antibody isotype.
  • an antibody comprising a ⁇ constant region is an IgG antibody
  • an antibody comprising a ⁇ constant region is an IgD antibody
  • an antibody comprising an ⁇ constant region is an IgA antibody.
  • an antibody containing a mu constant region is an IgM antibody
  • an antibody containing an epsilon constant region is an IgE antibody.
  • IgG antibodies include, but are not limited to, IgG1 (comprising the ⁇ 1 constant region), IgG2 (comprising the ⁇ 2 constant region), IgG3 (comprising the ⁇ 3 constant region), and IgG4 (comprising the ⁇ 4 constant region) antibodies
  • IgA antibodies include, but are not limited to, IgA1 (comprising the ⁇ 1 constant region) and IgA2 (comprising the ⁇ 2 constant region) antibodies
  • IgM include, but are not limited to, IgM1 and IgM2.
  • the antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from IgG.
  • the antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from human IgG.
  • the antigen-binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from human IgG1.
  • the present application also provides one or more nucleic acid molecules, which can be nucleotides, deoxynucleotides and/ribonucleotides in isolated form of any length, and can encode the isolated antigen-binding protein.
  • the present application also provides a carrier, which may include the nucleic acid molecule.
  • the vector can transform, transduce or transfect host cells, so that the genetic material elements carried by it can be expressed in the host cells.
  • vectors can include promoters, transcripts, enhancers, replicons, selection elements, and reporter genes.
  • a carrier may include components that facilitate entry into cells.
  • the 5' and 3' ends of the nucleic acid molecule may also contain long terminal repeats.
  • the present application also provides cells, which may include the isolated antigen-binding protein, the chimeric antigen receptor, the nucleic acid molecule and/or the carrier.
  • the cells may include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny may not necessarily be completely identical (either in the morphology of the total DNA complement or in the genome) to the original parent cell.
  • the present application also provides a pharmaceutical composition, which may include the isolated antigen-binding protein, the polypeptide, the nucleic acid molecule, the carrier and/or the cell, and optionally a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition may also comprise one or more (pharmaceutically effective) carriers, stabilizing Suitable formulations of agents, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives.
  • the acceptable ingredients of the compositions are preferably nontoxic to recipients at the dosages and concentrations employed.
  • Pharmaceutical compositions of the present invention may include liquid, frozen and lyophilized compositions.
  • the pharmaceutically acceptable adjuvants can include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents that are compatible with pharmaceutical administration, are generally safe, nontoxic, and are neither biologically nor otherwise undesirable.
  • the pharmaceutical composition may comprise parenteral, transdermal, intracavity, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue.
  • the pharmaceutical composition can be administered to a patient or subject by infusion or injection.
  • the administration of the pharmaceutical composition can be performed by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • a pharmaceutical combination as described herein refers to a product resulting from the admixture or combination of more than one active ingredient and includes both fixed and non-fixed combinations of active ingredients.
  • the term "fixed combination” means that the active ingredients, eg, an antigen binding protein described herein, and one or more combination partners are both administered to a patient simultaneously in the form of a single entity or dosage.
  • the term “non-fixed combination” means that the active ingredients, such as a compound of the present invention, and one or more combination partners are administered to a patient as separate entities simultaneously, concurrently or sequentially (with no particular time limit), wherein such administration provides therapeutically effective levels of the two or more active ingredients in the patient.
  • the pharmaceutical combination may comprise the antigen-binding protein described in the present application, and an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor comprises an antigen binding protein targeting PD-L1.
  • the antigen binding protein targeting PD-L1 may comprise HCDR1, HCDR2 and HCDR3.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:135
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:136
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:137.
  • the antigen binding protein targeting PD-L1 may comprise LCDR1, LCDR2 and LCDR3.
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:139
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:140
  • the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:141.
  • the antigen-binding protein targeting PD-L1 in the pharmaceutical combination may comprise VH.
  • the VH can comprise the amino acid sequence shown in SEQ ID NO: 138.
  • the antigen-binding protein targeting PD-L1 in the pharmaceutical combination may comprise VL.
  • the VL can comprise the amino acid sequence shown in SEQ ID NO: 142.
  • the antigen-binding protein targeting PD-L1 in the pharmaceutical combination may comprise VH and VL.
  • the VH may comprise the amino acid sequence set forth in SEQ ID NO:138
  • the VL may comprise the amino acid sequence set forth in SEQ ID NO:142.
  • the present application also provides a method for preparing the isolated antigen-binding protein.
  • the method may comprise culturing the cell under conditions such that the antigen binding protein is expressed.
  • the present application also provides the use of the isolated antigen-binding protein, the polypeptide, the nucleic acid molecule, the carrier, the cell, the pharmaceutical composition and/or the pharmaceutical combination in the preparation of medicines, and the medicines can be used to prevent, alleviate and/or treat diseases and/or conditions.
  • the present application also provides a method for preventing, alleviating and/or treating a disease and/or a disease, and the method may include administering the isolated antigen-binding protein, the polypeptide, the nucleic acid molecule, the carrier, the cell, the pharmaceutical composition and/or the pharmaceutical combination to a subject.
  • the present application also provides the isolated antigen-binding protein, the polypeptide, the nucleic acid molecule, the carrier, the cell, the pharmaceutical composition and/or the pharmaceutical combination, which are used for preventing, alleviating and/or treating diseases and/or conditions.
  • the diseases and/or disorders include diseases and/or disorders associated with abnormal expression of TIGIT.
  • the diseases and/or conditions include tumors.
  • the tumor includes solid tumors.
  • tumors include non-solid tumors.
  • the tumor comprises an immune checkpoint sensitive tumor.
  • the tumor comprises colon cancer, melanoma, non-small cell lung cancer, renal cell carcinoma, and/or hepatocellular carcinoma.
  • the present application also provides a method for blocking the interaction between TIGIT protein and CD155, the method comprising administering the antigen-binding protein, the polypeptide, the nucleic acid molecule, the carrier, the cell, the pharmaceutical composition, and/or the pharmaceutical combination to a subject in need.
  • the methods are non-diagnostic and non-therapeutic methods.
  • the method is an in vitro method and/or an ex vivo method.
  • VHH variable region
  • CALL002 5'-GGTACGTGCTGTTGAACTGTTCC-3'; (SEQ ID NO: 144)
  • VHH-Back 5'-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3'; (SEQ ID NO: 145)
  • VHH-For 5'-CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3'. (SEQ ID NO: 146)
  • the target fragment was recovered from the gel and cloned into the phage display vector pMES4 (GenBank GQ907248) using restriction enzymes (from Thermo) PstI and Eco91I. After the plasmid was desalted, it was electroporated into electroporation-competent Escherichia coli TG1 to construct the phage display nanobody library NanoTIGI, and evaluate the diversity of the library.
  • the library size was calculated to be 2 ⁇ 10 9 by gradient dilution, and then 22 single clones were picked for sequencing. The mutation rate was 81.8% (18/22) and the diversity was 1.6E9.
  • TIGIT-His protein TIT-H52H5-100ug, ACRO
  • the VHH sequence of TIGI7 is shown in SEQ ID NO:40
  • the VHH sequence of TIGI36 is shown in SEQ ID NO:41
  • the VHH sequence of TIGI40 is shown in SEQ ID NO:42
  • the VHH sequence of TIGI41 is shown in SEQ ID NO:43
  • the VHH sequence of TIGI42 is shown in SEQ ID NO:44
  • the VHH sequence of TIGI53 is shown in SEQ ID NO :45
  • the VHH sequence of TIGI44 is shown in SEQ ID NO:46
  • the VHH sequence of TIGI56 is shown in SEQ ID NO:47
  • the VHH sequence of TIGI60 is shown in SEQ ID NO:48
  • the VHH sequence of TIGI63 is shown in SEQ ID NO:49
  • the VHH sequence of TIGI64 is shown in SEQ ID NO:50
  • the VHH sequence of TIGI35 is shown in SEQ ID As shown in NO:110
  • the VHH sequence of TIGI37 is
  • the VHH sequence of TIGI51 is shown in SEQ ID NO:116
  • the VHH sequence of TIGI51 is shown in SEQ ID NO:117
  • the VHH sequence of TIGI52 is shown in SEQ ID NO:118
  • the VHH sequence of TIGI54 is shown in SEQ ID NO:119
  • the VHH sequence of TIGI57 is shown in SEQ ID NO:120
  • the VHH sequence of TIGI58 is shown in SEQ ID NO:1 21
  • the VHH sequence of TIGI59 is shown in SEQ ID NO:122
  • the VHH sequence of TIGI61 is shown in SEQ ID NO:123
  • the VHH sequence of TIGI65 is shown in SEQ ID NO:124
  • the VHH sequence of TIGI33 is shown in SEQ ID NO:125
  • the VHH sequence of TIGI43 is shown in SEQ ID NO:126.
  • nucleotide sequences of 65 nanobodies obtained by sequence analysis were subcloned into eukaryotic expression vector pcDNA3.4, then transfected into EXPI293, cultured and expressed for 5 days to prepare bivalent antibodies. The supernatant was collected and purified by protein A (GenScript, catalog number: L00695-80).
  • a 384-well plate was coated with human TIGIT-Fc fusion protein and 5% Milk, and left at 4°C overnight. The next day, 5% Milk was added to block for 2 hours.
  • the purified antibody was formulated to 100 nM, diluted 3 times, and 11 gradients in total. After blocking, wash 3 times, add diluted bivalent antibody, incubate with shaking at room temperature for 1 hour, add anti-human IgG Fc-HRP after washing, incubate with shaking at room temperature for 1 hour, add TMB chromogenic solution after washing, add 2M phosphoric acid after 3-5 minutes to stop the color reaction, and read OD450.
  • BD Streptavidin- APC conjugate
  • TIGIT nanobody with blocking activity Dilute the TIGIT nanobody with blocking activity to 100nM, and add the antibody into a 384-well plate.
  • Dilute human TIGIT-His antigen Beijing Baipu Saisi Biotechnology Co., Ltd., TIT-H52H5-100ug
  • the AHC probe (Sartorius, Cat. No.: 18-5060) was used to set the binding time to 180 sec, the dissociation time to 300 sec, the baseline to 60 sec, and three regeneration cycles of 5 sec each. Then use software to fit the binding-dissociation curve and calculate the affinity of the antibody. The results are shown in Figure 3A- Figure 3AA, and the affinity of most nanobodies is at the nM level.
  • mice During the period of administration and observation, the body weight and tumor volume of the mice were measured 3 times a week, and the measured values were recorded. The results are shown in Figure 4A, and the tumor volume inhibition rate (TGI TV %) was calculated. At the end of the experiment, the animals were euthanized, the tumors were stripped, weighed, and photographed, and the tumor weight inhibition rate (TGI TW %) was calculated, and the experimental results were shown in Figure 4B.
  • TIGI7, TIGI37, TIGI39, TIGI46, TIGI48 and Tiragolumab all had tumor-inhibiting effects, and their tumor weight inhibition rates were 61.37%, 47.06%, 55.69%, 50.98%, 54.51% and 64.51%, respectively; their tumor volume inhibition rates were 62.98%, 53.63%, 59.80%, 52. 94%, 60.86%, 67.23%.
  • the TIGIT humanized mouse Balb/c and CT26 colon cancer models were used to study the anti-tumor effect of TIGI7 and YN035 (the sequence of VH is shown in SEQ ID NO: 138, and the sequence of VL is shown in SEQ ID NO: 142) in vivo.
  • Humanized mice aged 6-8 weeks were purchased from Jizui Pharmacy, and were inoculated with CT26 cells after one week of feeding, with a cell number of 1E6.
  • One week later, when the tumor volume grew to 80-100 mm 3 the patients were divided into random groups for administration.
  • the route of administration for all groups was intraperitoneal injection, administered once every three days, for 6 consecutive administrations, and the experiment ended about 11 days after the last administration.
  • the body weight and tumor volume of the mice were measured 3 times a week, and the measured values were recorded, and the tumor volume inhibition rate (TGI TV %) was calculated.
  • TGI TV tumor volume inhibition rate
  • a single mouse was euthanized, and the median survival period of each group was calculated.
  • the experimental results are shown in Figure 5A-5C.
  • the tumor inhibition rate of YN035 (PD-L1 monoclonal antibody) was 55.97%
  • the tumor inhibition rate of TIGI7 single drug was 59.59%
  • the tumor inhibition rate of combined drug was 62.39%.
  • the median survival time was 29.5 days, 32 days and 32.5 days respectively. Therefore, it was judged that YN035 and TIGI7 had a synergistic antitumor effect.
  • TIGI7 was selected for humanization of its framework regions. To put it simply, the human antibody DP-47 sequence was obtained from the literature, DP-47 and TIGI7 were numbered on the IMGT website (http://www.imgt.org/), the framework region and CDR were determined, and the amino acids on the TIGI7 framework were replaced with the corresponding amino acids on the DP-47 framework region. Since FR2 has an important role for Nanobody stability, it was left unchanged.
  • VHH sequence of HTIGI7 is shown in SEQ ID NO: 51
  • the VHH sequence of HTIGI7.1 is shown in SEQ ID NO: 52
  • the VHH sequence of HTIGI7.2 is shown in SEQ ID NO: 53
  • the VHH sequence of HTIGI7.3 is shown in SEQ ID NO: 54, HT.
  • VHH sequence of IGI7.4 is shown in SEQ ID NO: 55
  • HTIGI7.5 VHH sequences such as SEQ ID NO: 56
  • HTIGI7.6 VHH sequences such as SEQ ID NO: 57
  • HTIGI7.7 VHH sequences are shown in SEQ ID NO: 58
  • HTIGI HTIGI
  • the VHH sequence of 7.8 is shown in SEQ ID NO: 59
  • the VHH sequence of HTIGI7.9 is shown in SEQ ID NO: 60
  • the VHH sequence of HTIGI7.10 is shown in SEQ ID NO: 61
  • the VHH sequence of HTIGI7.11 is shown in SEQ ID NO: 62.
  • VHH sequence of .12 is shown in SEQ ID NO: 63
  • VHH sequence of HTIGI7.13 is shown in SEQ ID NO: 64.
  • the humanized sequence cloning is cloned into the expression carrier PCDNA3.4, and antibody protein is generated by instant transfection of Expi293, and purified by Protein A.
  • Normal cultured 293T-hTIGIT cells were digested and centrifuged, resuspended with PBS containing 0.1% BSA, and 4E4 cells were added to each well of a 96-well pointed-bottom plate, and the volume was adjusted to 30 ⁇ l.
  • the corresponding antibody was diluted to 100 nM, and then diluted 7 gradients by 3 times, and no antibody was added to the last gradient. Take 30 ⁇ l of antibody solution, mix it with the cell suspension, and incubate at 4°C for 1 hour.
  • the cells were washed twice with PBS containing 0.1% BSA, centrifuged at 500 g for 5 min each time, and the liquid in the wells was dried as much as possible.
  • Anti-human IgG Fc-650 secondary antibody (abcam, product number: ab98593) was diluted 1:200 times with PBS containing 0.1% BSA, added 30 ⁇ l to each well, mixed with cells, and incubated at 4°C for 30 min. Wash the cells 3 times with PBS containing 0.1% BSA, centrifuge at 500 g for 5 min each time, and finally resuspend the cells in 30 ⁇ l PBS containing 0.1% BSA, put them on the machine, and use Graphpad to make a four-parameter fitting curve after reading the data, and calculate EC50, the result is shown in Figure 6A. Stability identification was performed by running HPLC-SEC to analyze the degree of antibody aggregation. The results are shown in Figure 6B. The humanized TIGI7 binding activity decreased, but SEC showed that the nanobody had no aggregation and had good druggability.
  • the hTIGI7 affinity matured phage display antibody library was constructed. To put it simply, the nucleotide sequence of hTIGI7 was cloned into the HP153 vector, and then transformed into Escherichia coli CJ236, the phage was cultured, and the single-stranded DNA containing uracil was extracted. In vitro, artificially synthesized mutant primers were mixed with single-stranded DNA to prepare CCC-dsDNA.
  • CCC-dsDNA was desalted and electrotransformed into M13KO7 pre-infected TG1, then scaled up overnight, and the phages were collected the next day to screen TIGIT nanobodies with human-monkey cross-activity.
  • the screening process is similar to Example 1.2, the antigen used in the first round is 100nM cynomolgus TIGIT, the antigen used in the second round was 100nM cynomolgus TIGIT, the third round was 10nM human TIGIT, the fourth round was 1nM Cynomolgus TIGIT, the fifth round was 0.01nM human TIGIT, and the number of washings was 50 times.
  • positive sequences that can simultaneously bind to human and monkey TIGIT were selected by phage monoclonal ELISA. A total of 13 nanobodies were obtained after sequencing.
  • Digest and centrifuge normally cultured 293T-human TIGIT and 293T-cynomolgus TIGIT cells resuspend in PBS containing 0.1% BSA, take a 96-well tip-bottom plate, add 4E4 cells to each well, 30 ⁇ l per well.
  • the corresponding antibody hTIGI7.1-hTIGI7.13 was diluted to 200nM, and then 11 gradients were diluted by 3 times, and no antibody was added to the last gradient. Take 30 ⁇ l of antibody solution, mix it with the cell suspension, and incubate at 4°C for 1 hour.
  • the cells were washed twice with PBS containing 0.1% BSA, centrifuged at 500 g for 5 min each time, and the liquid in the wells was shaken dry.
  • Anti-human IgG Fc-650 secondary antibody (abcam, product number: ab98593) was diluted 1:200 times with PBS containing 0.1% BSA, added 30 ⁇ l to each well, mixed with cells, and incubated at 4°C for 30 min. Wash the cells 3 times with PBS containing 0.1% BSA, 500g each time, centrifuge for 5 minutes, and finally resuspend the cells in 30 ⁇ l PBS containing 0.1% BSA, and put them on the machine. After reading the data, use Graphpad to make a four-parameter fitting curve and calculate EC50.
  • hTIGI7.11E VHH sequence is SEQ ID NO: 65
  • hTIGI7.11T VHH sequence is SEQ ID NO: 66
  • hTIGI7 .11A the VHH sequence is SEQ ID NO:67.
  • the mutated sequence was subcloned into the eukaryotic expression vector pcDNA3.4, then transiently transfected into EXPI293 for expression, and purified with protein A.
  • the purified antibody was verified by flow cytometry with 293T-human TIGIT and 293T-cynomolgus TIGIT, as shown in Figure 8A and 8B, it was found that the mutated antibody did not change its affinity.
  • the three antibodies were diluted to 100nM and added to 384-well plate.
  • a total of 8 AHC probes (Sartorius, catalog number: 18-5060) were used, and each probe was used as a group, a total of 8 groups.
  • the 8 probes were first loaded with 100nM human TIGIT, and then the antibodies were run sequentially.
  • the first antibody of the first group is hTIGI7.11E, the second antibody is hTIGI7.11E; the first antibody of the second group is hTIGI7.11E, the second antibody Tiragoluamab; the first antibody of the third group is Tiragoluamab, the second antibody Tiragoluamab;
  • the first antibody of the sixth group is hTIGI7.11E, and the second antibody is 2A3; the first antibody of the seventh group is 2A3, and the second antibody is 2A3; the first antibody of the eighth group is 2A3, and the second antibody is hTIGI7.11E.
  • the time of Baseline is 60sec, and the time of combination is 300sec.
  • the target cells are: 293T-human TIGIT cells, which are 293T cell lines that can stably express human TIGIT and luciferase luciferase established in the laboratory; target cells are counted and centrifuged and then resuspended in "DMEM+10% FBS" medium, with a cell density of 2E5/ml, 50 ⁇ l/well, that is, 5000 target cells per well; a frozen PBMC, 8E7cells, was shipped from the company's warehouse; washed 3 times with PBS, 500g/5min, 400g/5min, 300g/5min, centrifuge at room temperature.
  • the antibodies were: hTIGIT7.11E, IgG1-FC, Tiragolumab, the initial working concentration of the antibody was 100nM, 8-fold serial dilution, a total of 7 concentration gradients were formed, 50 ⁇ l/well Add target cells, effector cells and antibodies together in a 96-well cell culture plate with an opaque white background, and set a separate well for target cells as a control.
  • cleavage percentage (%) (single target well-experimental well)/target single well ⁇ 100.
  • CT26 cells were inoculated subcutaneously on the right side of Balb/c-hTIGIT humanized mice at a concentration of 1 ⁇ 10 6 cells/0.1 mL.
  • the route of administration in all groups was intraperitoneal injection, administered once every three days, and administered continuously for 6 times.
  • the experiment was terminated 11 days after the last administration.
  • the body weight and tumor volume of the mice were measured 3 times a week, and the measured values were recorded, and the tumor volume inhibition rate (TGITV%) was calculated.
  • TGITV% tumor volume inhibition rate
  • the animals were euthanized. The results are shown in Figure 13. On the 17th day, compared with IgG1-Fc in the control group, both medium and high concentrations of hTIGI7.11E could significantly inhibit tumor growth.

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Abstract

提供一种分离的抗原结合蛋白，其具有下述性质中的一种或多种：(1)能够特异性结合TIGIT蛋白；(2)能够与人TIGIT和食蟹猴TIGIT蛋白均有结合活性；(3)能够对细胞表面TIGIT蛋白与CD155的相互作用具有阻断效果；以及(4)能够抑制肿瘤的生长和/或增殖。还提供了所述分离的抗原结合蛋白的制备方法及用途。

Description

靶向TIGIT的抗原结合蛋白及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种靶向TIGIT的抗原结合蛋白及其应用。
背景技术
免疫检查点T细胞免疫球蛋白和免疫受体酪氨酸抑制性基序结构域(TIGIT，又名Vstm3，WUCAM)是I型跨膜蛋白。TIGIT的配体有两种，CD155(也称为脊髓灰质炎病毒受体或PVR)和CD112(也称为脊髓灰质炎病毒受体相关配体2、PVRL2、粘连蛋白-2)。
TIGIT与激活性受体CD226竞争性结合配体CD155，传递抑制信号，抑制细胞过度活化并抑制促炎性细胞因子分泌(Manieri,Trends Immunol,2017,38(1):20-8)。TIGIT主要在调节性T细胞(Treg)、活化的T细胞和自然杀伤细胞(NK)等细胞上表达(YU,Nat Immunol,2009,10(1):48-57)，可以直接抑制T细胞和NK细胞的增殖、杀伤、脱颗粒及细胞因子分泌，也可以通过促进DC细胞分泌IL-10抑制T细胞活性(Nicholas,Trends Immunol.2017,38(1):20-28)。此外，表达在Treg上的TIGIT可以维持Treg的免疫抑制功能(Manieri,Trends Immunol,2017,38(1):20-8)。TIGIT在多种肿瘤细胞、浸润淋巴细胞和外周血单核细胞中显著上调，其配体CD155和CD112也在多种肿瘤细胞广泛表达，提示TIGIT在肿瘤免疫逃逸过程中可能起着关键作用(Tassi,Cancer Res.2017,77:851-861.Kong,Clin Cancer Res,2016,22:3057-66.Casado,Cancer Immunol Immunother,2009,58(9):1517-26.)。
目前，已知的抗TIGIT抗体还存在特异性不强，杀伤力有限等缺陷。因此，亟需开发对TIGIT蛋白亲和力高、特异性强的抗TIGIT抗原结合蛋白。
发明内容
本申请提供了一种分离的抗原结合蛋白，其能够特异性结合TIGIT蛋白。本申请所述抗原结合蛋白具有以下一种或多种特点：(1)能够特异性结合TIGIT蛋白；(2)能够与人TIGIT和食蟹猴TIGIT蛋白均有结合活性；(3)能够对细胞表面TIGIT蛋白与CD155的相互作用具有阻断效果；以及(4)能够抑制肿瘤的生长和/或增殖。
在某些实施方式中，所述抗原结合蛋白包括抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
在某些实施方式中，所述抗原结合片段为VHH。
在某些实施方式中，抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
在某些实施方式中，所述抗原结合蛋白包含源自抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:132所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包括HCDR3，且所述HCDR3包含SEQ ID NO:127所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包括HCDR3，且所述HCDR3包含SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包含HCDR2，且所述HCDR2包含SEQ ID NO:128所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包含HCDR2，且所述HCDR2包含SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:6和SEQ ID NO:7中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包含HCDR1，且所述HCDR1包含SEQ ID NO:1所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包含HCDR1，HCDR2，和HCDR3，所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:128所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:127所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包含HCDR1，HCDR2，和HCDR3，所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2、SEQ ID NO:3和SEQ ID NO:5、SEQ ID NO:6和SEQ ID NO:7中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包含HCDR1，HCDR2，和HCDR3，且所述HCDR1、HCDR2、HCDR3包含选自下组的任意一组氨基酸序列：
(1)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:2；和HCDR3：SEQ ID NO:4；
(2)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:4；
(3)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:8；
(4)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:9；
(5)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:10；
(6)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:11；
(7)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:12；
(8)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:13；
(9)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:14；
(10)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:15；
(11)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:16；
(12)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:17；
(13)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:18；
(14)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:19；
(15)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:20；
(16)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:5；HCDR3：SEQ ID NO:18；
(17)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:6；HCDR3：SEQ ID NO:18；以及
(18)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:7；HCDR3：SEQ ID NO:18。
在某些实施方式中，所述抗原结合蛋白包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:129所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白的所述H-FR1包含SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31和SEQ ID NO:32中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:33所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3SEQ ID NO:130所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白的所述H-FR3包含SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36和SEQ ID NO:37中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包含H-FR4，所述H-FR4的N末端与所述
HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:131所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白的所述H-FR4包含SEQ ID NO:38或SEQ ID  NO:39中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包括抗体重链可变区VH，且所述VH包含SEQ ID NO:132所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白的所述VH包含SEQ ID NO:40-67中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白为VHH，且所述VHH包含SEQ ID NO:40-67中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包括HCDR3，且所述HCDR3包含SEQ ID NO:68、SEQ ID NO:69和SEQ ID NO:70中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包含HCDR2，且所述HCDR2包含SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76和SEQ ID NO:77中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包含HCDR1，且所述HCDR1包含SEQ ID NO:78、SEQ ID NO:79、SEQ ID NO:80、SEQ ID NO:81和SEQ ID NO:82中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包含HCDR1，HCDR2，和HCDR3，所述HCDR1包含SEQ ID NO:78、SEQ ID NO:79、SEQ ID NO:80、SEQ ID NO:81和SEQ ID NO:82中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76和SEQ ID NO:77中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:68、SEQ ID NO:69和SEQ ID NO:70中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包含HCDR1，HCDR2，和HCDR3，且所述HCDR1、HCDR2、HCDR3包含选自下组的任意一组氨基酸序列：
(1)HCDR1：SEQ ID NO:78；HCDR2：SEQ ID NO:71；和HCDR3：SEQ ID NO:68；
(2)HCDR1：SEQ ID NO:78；HCDR2：SEQ ID NO:72；和HCDR3：SEQ ID NO:68；
(3)HCDR1：SEQ ID NO:79；HCDR2：SEQ ID NO:72；和HCDR3：SEQ ID NO:68；
(4)HCDR1：SEQ ID NO:78；HCDR2：SEQ ID NO:73；和HCDR3：SEQ ID NO:68；
(5)HCDR1：SEQ ID NO:80；HCDR2：SEQ ID NO:73；和HCDR3：SEQ ID NO:68；
(6)HCDR1：SEQ ID NO:78；HCDR2：SEQ ID NO:71；和HCDR3：SEQ ID NO:68；
(7)HCDR1：SEQ ID NO:78；HCDR2：SEQ ID NO:74；和HCDR3：SEQ ID NO:68；
(8)HCDR1：SEQ ID NO:80；HCDR2：SEQ ID NO:75；和HCDR3：SEQ ID NO:68；
(9)HCDR1：SEQ ID NO:81；HCDR2：SEQ ID NO:76；和HCDR3：SEQ ID NO:69；以及
(10)HCDR1：SEQ ID NO:82；HCDR2：SEQ ID NO:77；和HCDR3：SEQ ID NO:70。
在某些实施方式中，所述抗原结合蛋白包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:133所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白的所述H-FR1包含SEQ ID NO:83、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91、SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、SEQ ID NO:97、SEQ ID NO:98和SEQ ID NO:99中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:134所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白的所述H-FR2包含SEQ ID NO:100、SEQ ID NO:101和SEQ ID NO:102中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:103SEQ ID NO:104、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:108和SEQ ID NO:109中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:38所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包含VH，且所述VH包含SEQ ID NO:110-126中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白为VHH，且所述VHH包含SEQ ID NO:110-126中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包含抗体重链恒定区，所述抗体重链恒定区源自IgG。
在某些实施方式中，所述抗原结合蛋白包含抗体重链恒定区，所述抗体重链恒定区源自人IgG。
在某些实施方式中，所述抗原结合蛋白包含抗体重链恒定区，所述抗体重链恒定区源自人IgG1。
另一方面，本申请还提供了一种多肽，其包含本申请所述的抗原结合蛋白。
另一方面，本申请还提供了一种或多种分离的核酸分子，其编码本申请所述的抗原结合蛋白。
另一方面，本申请还提供了一种载体，其包含所述的核酸分子。
另一方面，本申请还提供了一种细胞，其包含所述的载体。
另一方面，本申请还提供了一种制备所述的抗原结合蛋白的方法，所述方法包括在使得所述抗原结合蛋白表达的条件下，培养所述的细胞。
另一方面，本申请还提供了一种药物组合物，其包含所述的抗原结合蛋白，以及任选地药学上可接受的载体。
另一方面，本申请还提供了一种药物组合，其包含所述的抗原结合蛋白，以及免疫检查点抑制剂。
在某些实施方式中，所述免疫检查点抑制剂包括靶向PD-L1的抗原结合蛋白。
在某些实施方式中，所述药物组合中所述靶向PD-L1的抗原结合蛋白包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:135所示的氨基酸序列，所述HCDR2包含SEQ ID NO:136所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:137所示的氨基酸序列。
在某些实施方式中，所述药物组合中所述靶向PD-L1的抗原结合蛋白包含LCDR1，LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:139所示的氨基酸序列，所述LCDR2包含SEQ ID NO:140所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:141所示的氨基酸序列。
在某些实施方式中，所述药物组合中所述靶向PD-L1的抗原结合蛋白包含VH，且所述VH包含SEQ ID NO:138所示的氨基酸序列。
在某些实施方式中，所述药物组合中所述靶向PD-L1的抗原结合蛋白包含VL，且所述VL包含SEQ ID NO:142所示的氨基酸序列。
另一方面，本申请还提供了所述的抗原结合蛋白，所述的多肽，所述的核酸分子，所述的载体，所述的细胞，所述的药物组合物，和/或所述的药物组合在制备药物中的用途，所述药物用于预防、治疗和/或缓解疾病和/或病症。
在某些实施方式中，所述疾病和/或病症包括与TIGIT异常表达相关的疾病和/或病症。
在某些实施方式中，所述疾病和/或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤。
在某些实施方式中，肿瘤包括非实体瘤。
在某些实施方式中，所述肿瘤包括对免疫检查点敏感的肿瘤。
在某些实施方式中，所述肿瘤包括结肠癌、黑色素瘤，非小细胞肺癌，肾细胞癌和/或肝细胞癌。
另一方面，本申请还提供了一种预防、治疗和/或缓解疾病和/或病症的方法，所述方法包括向有需要的受试者施用所述的抗原结合蛋白，所述的多肽，所述的核酸分子，所述的载体，所述的细胞，所述的药物组合物，和/或所述的药物组合。
在某些实施方式中，所述疾病和/或病症包括与TIGIT异常表达相关的疾病和/或病症。
在某些实施方式中，所述疾病和/或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤。
在某些实施方式中，肿瘤包括非实体瘤。
在某些实施方式中，所述肿瘤包括对免疫检查点敏感的肿瘤。
在某些实施方式中，所述肿瘤包括结肠癌、黑色素瘤，非小细胞肺癌，肾细胞癌和/或肝细胞癌。
另一方面，本申请还提供了一种阻断TIGIT蛋白和CD155相互作用的方法，所述方法包括向有需要的受试者施用所述的抗原结合蛋白，所述的多肽，所述的核酸分子，所述的载体，所述的细胞，所述的药物组合物，和/或所述的药物组合。
在某些实施方式中，所述方法为非诊断和非治疗目的的方法。
在某些实施方式中，所述方法为体外方法和/或离体方法。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是流式检测本申请所述抗原结合蛋白与细胞表面TIGIT的结合活性。
图2显示的是流式检测本申请所述抗原结合蛋白对细胞表面TIGIT与CD155相互作用的阻断效果。
图3A-图3AA显示的是具有阻断活性的抗原结合蛋白的亲和力检测结果。
图4A显示的是本申请所述的抗原结合蛋白的小鼠肿瘤体积抑制结果；图4B显示的是本申请所述的抗原结合蛋白的小鼠瘤重抑制结果。
图5A-5C显示的是本申请所述的抗原结合蛋白与PD-L1抗体联合用药的体内药效检测。
图6A显示的是本申请所述抗原结合蛋白的EC50测定结果；图6B显示的是本申请所述的抗原结合蛋白的聚集程度鉴定结果。
图7A显示的是改造后的抗原结合蛋白与人TIGIT的亲和力检测结果；图7B显示的是改造后的抗原结合蛋白与食蟹猴TIGIT的亲和力检测结果；图7C-图7O显示的是本申请所述的抗原结合蛋白的聚集程度鉴定结果。
图8A显示的是本申请所述抗原结合蛋白与人TIGIT的亲和力检测结果；图8B显示的是本申请所述抗原结合蛋白与食蟹猴TIGIT的亲和力检测结果；图8C显示的是不同温度下ELISA验证本申请所述的抗原结合蛋白的结合活性；图8D显示的是SEC验证本申请所述的抗原结合蛋白的结合活性；图8E显示的是流式验证本申请所述的抗原结合蛋白的结合活性。
图9A-图9B显示的是本申请所述的抗原结合蛋白与激活后食蟹猴PBMC上TIGIT的结合活性分析。
图10A-图10D显示的是本申请所述抗原结合蛋白的抗原结合表位验证结果。
图11显示的是本申请所述抗原结合蛋白的阻断活性检测。
图12显示的是本申请所述抗原结合蛋白的ADCC活性检测。
图13显示的是本申请所述抗原结合蛋白的小鼠肿瘤体积抑制结果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“分离的抗原结合蛋白”通常是指脱离了其天然存在状态的具有抗原结合能力的蛋白。所述“分离的抗原结合蛋白”可以包含结合抗原的部分和任选的，允许抗原结合部分采用促进其结合抗原的构象的框架或构架部分。抗原结合蛋白可以包含例如抗体来源的蛋白框架区(FR)或具有移植的可变区(CDR)或CDR衍生物的备选蛋白框架区或人工框架区。例如，所述抗原结合蛋白可以包括抗体或其抗原结合片段。例如，所述抗原结合蛋白可以结合TIGIT蛋白。例如，所述抗原结合蛋白可以与参比抗体竞争结TIGIT蛋白。例如，所述抗原结合蛋白可以包括抗体重链可变区VH。例如，所述抗原结合蛋白可以包括源自抗 体重链可变区VH中的至少一个CDR。例如，所述VH可以包含HCDR3、HCDR2和/或HCDR1。例如，所述VH可以包括框架区H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连。例如，所述VH可以包括框架区H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间。例如，所述VH可以包括框架区H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间。例如，所述VH可以包括框架区H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连。例如，所述抗原结合蛋白可以为VHH。例如，所述的抗原结合蛋白可以包括抗体重链恒定区，所述抗体重链恒定区可以源自IgG。例如，所述抗体重链恒定区可以源自人IgG。例如，所述抗体重链恒定区可以源自人IgG1。
使用的术语“抗体”包括完整抗体和其结合片段。通常，片段与其来源的完整抗体竞争性地与抗原特异性结合。任选，抗体或其结合片段可以与其它蛋白质化学结合，或者与其它蛋白质以融合蛋白质的形式表达。例如，所述抗体可以是单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。例如，所述抗体或其结合片段的结合蛋白质可以包括TIGIT。例如，所述抗体或其结合片段对TIGIT可以有特异性。
术语“抗原结合片段”是指完整抗体的一部分并且是指完整抗体的抗原决定可变区。例如，所述抗原结合片段的可以包括Fab、Fab'、F(ab')2、Fv片段和单链Fv片段、串联Fv片段、VHH、双特异性抗体。例如，所述抗原结合片段可以是VHH。例如，所述抗原结合片段可以结合TIGIT。例如，所述抗原结合片段对TIGIT可以有特异性。
在本申请中，术语“VHH”通常是指包含重链抗体的可变抗原结合结构域的抗体。VHH也可称为纳米抗体(Nanobody)(Nb)和/或单域抗体。例如，所述VHH可以结合TIGIT。例如，所述VHH对TIGIT可以有特异性。
在本申请中，所述抗体可包含通过二硫键相互连接的至少两条重(H)链和两条轻(L)链。每条重链由重链可变区(VH)和重链恒定区组成。术语“重链恒定区”由三个结构域CH1，CH2和CH3组成。每条轻链由轻链可变区(VL)和轻链恒定区组成。术语“轻链恒定区”由一个结构域CL组成。VH和VL区可以进一步细分为高变区，称为互补决定区(CDR)，散布有更保守的区域，称为构架区(FR)。每个VH和VL由以下列顺序从氨基末端到羧基末端排列的三个CDR和四个FR组成:FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合。
在本申请中，术语“TIGIT蛋白”或“TIGIT抗原”可以互换使用，并且包括TIGIT的任何功能活性片段、变体和同源物，其由细胞天然表达或在用TIGIT基因转染的细胞上表达。 在本申请中，TIGIT可以为人TIGIT，其在UniProt/Swiss-Prot中的登录号为Q495A1。例如，TIGIT可以为人TIGIT的功能活性片段。例如，所述“功能活性片段”可以包括保留至少一种天然存在的蛋白质的内源功能(例如，与本申请所述的抗原结合蛋白结合)的片段。例如，所述“功能活性片段”可以包括与本申请的抗原结合蛋白结合的结构域。在本申请中，TIGIT可在免疫细胞表面表达。例如，可在调节性T细胞(Treg)表面表达。
术语“功能活性片段”指与天然存在序列具有基本上同一的氨基酸序列或由基本上同一的核苷酸序列编码并能够具有天然存在序列的一种或多种活性的多肽。在本申请的上下文中，任何给定序列的功能活性片段是指其中残基的特定序列(无论是氨基酸或核苷酸残基)已经经过修饰而使得所述多肽或多核苷酸基本上保留至少一种内源功能的序列。可以通过天然存在的蛋白质和/或多核苷酸中存在的至少一个氨基酸残基和/或核苷酸残基的添加、缺失、取代、修饰、替换和/或变异来获得编码功能活性片段的序列，只要保持原来的功能活性即可。
在本申请中，术语“衍生物”通常是指本申请的多肽或多核苷酸而言包括自/对序列的一个(或多个)氨基酸残基的任何取代、变异、修饰、替换、缺失和/或添加，只要所得的多肽或多核苷酸基本上保留其至少一种内源功能。
在本申请中，术语“类似物”通常对多肽或多核苷酸而言，包括多肽或多核苷酸的任何模拟物，即拥有该模拟物模拟的多肽或多核苷酸的至少一种内源功能的化学化合物。
通常，可以进行氨基酸取代，例如至少1个(例如，1、2、3、4、5、6、7、8、9、10或20个以上)氨基酸取代，只要经修饰的序列基本上保持需要的活性或能力。氨基酸取代可包括使用非天然存在的类似物。
在本申请中，术语“同源物”通常是指与天然存在序列具有一定同源性的氨基酸序列或核苷酸序列。术语“同源性”可以等同于序列“同一性”。同源序列可以包括可以与主题序列是至少80％、85％、90％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％相同的氨基酸序列。通常，同源物将包含与主题氨基酸序列相同的活性位点等。同源性可以根据相似性(即具有相似化学性质/功能的氨基酸残基)来考虑，也可以在序列同一性方面表达同源性。在本申请中，提及的氨基酸序列或核苷酸序列的SEQ ID NO中的任一项具有百分比同一性的序列是指在所提及的SEQ ID NO的整个长度上具有所述百分比同一性的序列。为了确定序列同一性，可进行序列比对，其可通过本领域技术人员了解的各种方式进行，例如，使用BLAST、BLAST-2、ALIGN、NEEDLE或Megalign(DNASTAR)软件等。本领域技术人员能够确定用于比对的适当参数，包括在所比较的全长序列中实现最优比对所需要的任何算法。
用于本申请的蛋白质或多肽也可以具有氨基酸残基的缺失、插入或取代，所述氨基酸残基产生沉默的变化并导致功能上等同的蛋白质。可以根据残基的极性、电荷、溶解性、疏水性、亲水性和/或两性性质的相似性进行有意的氨基酸取代，只要保留内源性功能即可。例如，带负电荷的氨基酸包括天冬氨酸和谷氨酸；带正电荷的氨基酸包括赖氨酸和精氨酸；并且含具有相似亲水性值的不带电极性头基的氨基酸包括天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸和酪氨酸。
在本申请中，术语“IgG”是指属于基本上由公认的免疫球蛋白γ基因编码的抗体类别的多肽。在人类中，此类别包括IgG1、IgG2、IgG3和IgG4。在小鼠中，此类别包括IgG1、IgG2a、IgG2b和IgG3。
在本申请中，术语“多肽”、“肽”和“蛋白”在本文中可以互换，是指氨基酸残基的聚合物。这个术语可用于指一个或多个氨基酸残基是其相应的天然氨基酸的人工合成化学模拟物的氨基酸聚合物，也可用于指天然的氨基酸聚合物，那些含修饰残基的氨基酸聚合物以及非天然的氨基酸聚合物。例如，所述多肽可以包括所述的抗原结合蛋白。
在本申请中，术语“核酸分子”通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。
在本申请中，术语“细胞”通常是指可以或已经含有包括本申请所述的核酸分子的质粒或载体，或者能够表达本申请所述的多肽或本申请所述的抗原结合蛋白的个体细胞，细胞系或细胞培养物。所述细胞可以包括单个细胞的子代。由于天然的，意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述的多肽或抗原结合蛋白即可。所述细胞可以通过使用本申请所述的载体体外转染细胞而得到。所述细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。
在本申请中，术语“药物组合物”通常指适合施用于哺乳动物个体的化学或生物组合物。例如，所述药物组合物可以包括所述抗原结合蛋白，所述多肽，所述核酸分子，所述载体和/或所述细胞，以及可选的药学上可接受的载体。
在本申请中，术语“药物组合”通常指具有至少两种活性成分的组合。通常，药物组合中的不同活性成分可用于联合施用。例如，在所述药物组合中，两种活性成分可以混合放置，也可以分开放置。例如，在施用时，所述药物组合可以共同施用，也可以先后施用。
在本申请中，术语“和/或”应理解为意指可选项中的任一项或可选项的两项。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。在某些情形中，“包含”也涵盖仅包括指定的组分的情况。例如，包含也表示为也表示“由……组成”的含义。
发明详述
分离的抗原结合蛋白
一方面，本申请提供一种分离的抗原结合蛋白，其具有下述性质中的一种或多种：(1)能够特异性结合TIGIT蛋白；(2)能够与人TIGIT和食蟹猴TIGIT蛋白均有结合活性；(3)能够对细胞表面TIGIT蛋白与CD155的相互作用具有阻断效果；以及(4)能够抑制肿瘤的生长和/或增殖。例如，所述分离的抗原结合蛋白与TIGIT蛋白的结合活性可以通过流式细胞荧光分选技术(FACS)检测。
在某些实施方式中，所述抗原结合蛋白包括抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
在某些实施方式中，所述抗原结合片段为VHH。
在某些实施方式中，抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
CDR
抗体的CDR又称互补决定区，是可变区的一部分。该区域的氨基酸残基可以与抗原或抗原表位接触。抗体CDR可以通过多种编码系统来确定，如CCG、Kabat、Chothia、IMGT、AbM、综合考虑Kabat/Chothia等。这些编码系统为本领域内已知，具体可参见，例如，http://www.bioinf.org.uk/abs/index.html#kabatnum。本领域技术人员可以根据抗体的序列和结构，用不同的编码系统确定出CDR区。使用不同的编码系统，CDR区可能存在差别。在本申请中，所述CDR涵盖根据任何CDR划分方式划分得到的CDR序列；也涵盖其变体，所述变体包括所述CDR的氨基酸序列经过取代、缺失和/或添加一个或多个氨基酸。例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个、5个、6个、7个、8个或9个氨基酸取代、缺失和/或插入；也涵盖其同源物，所述同源物可以为与所述CDR的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的氨基酸序列。在某些实施方式中，本申请所述分离的抗原结合蛋白通过Kabat编码系统定义。
在本申请中，所述抗原结合蛋白可包含源自抗体重链可变区VH中的至少一个CDR。例如，所述VH可包含SEQ ID NO:132所示的氨基酸序列。例如，所述VH可包含SEQ ID NO:40-67中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包括HCDR3。例如，所述HCDR3可包含SEQ ID NO:127所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包括HCDR3。例如，所述HCDR3包含SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR2。例如，所述HCDR2可包含SEQ ID NO:128所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR2。例如，所述HCDR2包含SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:6和SEQ ID NO:7中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1。例如，所述HCDR1可包含SEQ ID NO:1所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1，HCDR2，和HCDR3。例如，所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:128所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:127所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1，HCDR2，和HCDR3。例如，所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:6和SEQ ID NO:7中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1，HCDR2，和HCDR3，且所述HCDR1、HCDR2、HCDR3可包含选自下组的任意一组氨基酸序列：
(1)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:2；和HCDR3：SEQ ID NO:4；
(2)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:4；
(3)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:8；
(4)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:9；
(5)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:10；
(6)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:11；
(7)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:12；
(8)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:13；
(9)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:14；
(10)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:15；
(11)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:16；
(12)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:17；
(13)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:18；
(14)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:19；
(15)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:20；
(16)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:5；HCDR3：SEQ ID NO:18；
(17)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:6；HCDR3：SEQ ID NO:18；以及
(18)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:7；HCDR3：SEQ ID NO:18。
在本申请中，所述抗原结合蛋白可包括HCDR3，且所述HCDR3可包含SEQ ID NO:68、SEQ ID NO:69和SEQ ID NO:70中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR2，且所述HCDR2可包含SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76和SEQ ID NO:77中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1，且所述HCDR1可包含SEQ ID NO:78、SEQ ID NO:79、SEQ ID NO:80、SEQ ID NO:81和SEQ ID NO:82中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1，HCDR2，和HCDR3。例如，所述抗原结合蛋白的所述HCDR1可包含SEQ ID NO:78、SEQ ID NO:79、SEQ ID NO:80、SEQ ID NO:81和SEQ ID NO:82中任一项所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76和SEQ ID NO:77中任一项所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:68、SEQ ID NO:69和SEQ ID NO:70中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1，HCDR2，和HCDR3，且所述HCDR1、 HCDR2、HCDR3可包含选自下组的任意一组氨基酸序列：
(1)HCDR1：SEQ ID NO:78；HCDR2：SEQ ID NO:71；和HCDR3：SEQ ID NO:68；
(2)HCDR1：SEQ ID NO:78；HCDR2：SEQ ID NO:72；和HCDR3：SEQ ID NO:68；
(3)HCDR1：SEQ ID NO:79；HCDR2：SEQ ID NO:72；和HCDR3：SEQ ID NO:68；
(4)HCDR1：SEQ ID NO:78；HCDR2：SEQ ID NO:73；和HCDR3：SEQ ID NO:68；
(5)HCDR1：SEQ ID NO:80；HCDR2：SEQ ID NO:73；和HCDR3：SEQ ID NO:68；
(6)HCDR1：SEQ ID NO:78；HCDR2：SEQ ID NO:71；和HCDR3：SEQ ID NO:68；
(7)HCDR1：SEQ ID NO:78；HCDR2：SEQ ID NO:74；和HCDR3：SEQ ID NO:68；
(8)HCDR1：SEQ ID NO:80；HCDR2：SEQ ID NO:75；和HCDR3：SEQ ID NO:68；
(9)HCDR1：SEQ ID NO:81；HCDR2：SEQ ID NO:76；和HCDR3：SEQ ID NO:69；以及
(10)HCDR1：SEQ ID NO:82；HCDR2：SEQ ID NO:77；和HCDR3：SEQ ID NO:70。
FR
本申请中，抗体框架区FR指抗体可变区中存在于分歧性更高的(即高变)CDR之间的部分。此类框架区典型地称为框架1至4(FR1、FR2、FR3和FR4)且提供用于在三维空间中呈现CDR的骨架，以形成抗原结合表面。
在本申请中，所述抗原结合蛋白可包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连。例如，所述H-FR1可包含SEQ ID NO:129所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的所述H-FR1可包含SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31和SEQ ID NO:32中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间。例如，所述H-FR2可包含SEQ ID NO:33所示的氨基酸序列。
在某些实施方式中，所述抗原结合蛋白包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3SEQ ID NO:130所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的所述H-FR3可包含SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36和SEQ ID NO:37中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连。例如，所述H-FR4可包含SEQ ID NO:131所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的所述H-FR4可包含SEQ ID NO:38或SEQ ID NO:39中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含H-FR1，且所述H-FR1可包含SEQ ID NO:133所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的所述H-FR1可包含SEQ ID NO:83、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91、SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、SEQ ID NO:97、SEQ ID NO:98和SEQ ID NO:99中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含H-FR2，且所述H-FR2可包含SEQ ID NO:134所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的所述H-FR2可包含SEQ ID NO:100、SEQ ID NO:101和SEQ ID NO:102中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含H-FR3，且所述H-FR3可包含SEQ ID NO:103SEQ ID NO:104、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:108和SEQ ID NO:109中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含H-FR4，且所述H-FR4可包含SEQ ID NO:38所示的氨基酸序列。
重链可变区
在本申请中，所述抗原结合蛋白可包括抗体重链可变区VH，且所述VH可包含SEQ ID NO:132所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的所述VH可包含SEQ ID NO:40-67中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可为VHH，且所述VHH可包含SEQ ID NO:40-67中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含VH，且所述VH可包含SEQ ID NO:110-126中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可为VHH，且所述VHH可包含SEQ ID NO:1110-126中任一项所示的氨基酸序列。
重链恒定区
在本申请中，所述的分离的抗原结合蛋白可以包括重链恒定区。所述重链恒定区是指包 含至少三个重链恒定结构域CH1、CH2、和CH3的区域。非限制性示例性重链恒定区包括γ、δ、和α。非限制性示例性重链恒定区还包括ε和μ。每个重链恒定区对应于一种抗体同种型。例如，包含γ恒定区的抗体为IgG抗体，包含δ恒定区的抗体为IgD抗体，包含α恒定区的抗体为IgA抗体。此外，包含μ恒定区的抗体为IgM抗体，包含ε恒定区的抗体为IgE抗体。某些同种型可以进一步细分为亚类。例如，IgG抗体包括但不限于，IgG1(包含γ1恒定区)、IgG2(包含γ2恒定区)、IgG3(包含γ3恒定区)、和IgG4(包含γ4恒定区)抗体；IgA抗体包括但不限于，IgA1(包含α1恒定区)和IgA2(包含α2恒定区)抗体；IgM包括但不限于，IgM1和IgM2。
在本申请中，所述的抗原结合蛋白可以包括抗体重链恒定区，所述抗体重链恒定区可以源自IgG。在本申请中，所述的抗原结合蛋白可以包括抗体重链恒定区，所述抗体重链恒定区可以源自人IgG。在本申请中，所述的抗原结合蛋白可以包含抗体重链恒定区，所述抗体重链恒定区可以源自人IgG1。
核酸分子
另一方面，本申请还提供了一种或多种核酸分子，所述核酸分子可以是任意长度的分离形式的核苷酸、脱氧核苷酸和/核糖核苷酸，可以编码所述分离的抗原结合蛋白。
载体
另一方面，本申请还提供了一种载体，所述载体可以包括所述的核酸分子。所述载体可以转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达。例如，载体可以包括启动子、转录子、增强子、复制子、选择元件和报告基因。例如，载体可以包括协助进入细胞的成分。为了使所述核酸分子在载体中复制，所述核酸分子的5’端和3’端还可以包含长末端重复序列。
细胞
另一方面，本申请还提供了细胞，所述细胞可以包括所述的分离的抗原结合蛋白、所述的嵌合抗原受体、所述的核酸分子和/或所述的载体。所述细胞可以包括单个细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始母细胞完全相同(在总DNA互补体的形态上或在基因组上)。
药物组合物
另一方面，本申请还提供了一种药物组合物，可以包括所述分离的抗原结合蛋白、所述多肽、所述核酸分子、所述载体和/或所述细胞，以及任选地药学上可接受的佐剂。
在某些实施方案中，所述药物组合物还可以包含一种或多种(药学上有效的)载剂、稳定 剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下优选地对接受者无毒。本发明的药物组合物可以包括液体、冷冻和冻干组合物。
在某些实施方案中，所述药学上可接受的佐剂可以包括与药物给药相容的任何和所有的溶剂、分散介质、包衣、等渗剂和吸收延迟剂，通常安全、无毒，且既不是生物学上也非其它方面不合需要的。
在某些实施方案中，所述药物组合物可以包含肠胃外、经皮、腔内、动脉内、鞘内和/或鼻内施用或直接注射到组织中。例如，所述药物组合物可以通过输注或注射施用于患者或者受试者。在某些实施方案中，所述药物组合物的施用可以通过不同的方式进行，例如静脉内、腹膜内、皮下、肌肉内、局部或真皮内施用。
药物组合
本申请所述的药物组合是指由多于一种活性成分的混合或组合产生的产品，并且包括活性成分的固定和非固定组合。术语“固定组合”是指活性成分例如本申请所述的抗原结合蛋白和一种或多种组合搭档均以单一实体或剂量的形式同时施用于患者。术语“非固定组合”是指活性成分例如本发明化合物和一种或多种组合搭档作为分开的实体同时、共同或依次地(没有特定时间限制)施用于患者，其中这种施用在患者体内提供治疗有效水平的两种或多种活性成分。
在本申请中，所述药物组合可包含本申请所述的抗原结合蛋白，及免疫检查点抑制剂。
在某些实施方式中，所述免疫检查点抑制剂包含靶向PD-L1的抗原结合蛋白。
例如，所述靶向PD-L1的抗原结合蛋白可包含HCDR1，HCDR2和HCDR3。例如，所述HCDR1可包含SEQ ID NO:135所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:136所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:137所示的氨基酸序列。
例如，所述靶向PD-L1的抗原结合蛋白可包含LCDR1，LCDR2和LCDR3。例如，所述LCDR1可包含SEQ ID NO:139所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:140所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:141所示的氨基酸序列。
在本申请中，所述药物组合中所述靶向PD-L1的抗原结合蛋白可包含VH。例如，所述VH可包含SEQ ID NO:138所示的氨基酸序列。
在本申请中，所述药物组合中所述靶向PD-L1的抗原结合蛋白可包含VL。例如，所述VL可包含SEQ ID NO:142所示的氨基酸序列。
在本申请中，所述药物组合中所述靶向PD-L1的抗原结合蛋白可包含VH和VL。例如， 所述VH可包含SEQ ID NO:138所示的氨基酸序列，且所述VL可包含SEQ ID NO:142所示的氨基酸序列。
制备方法
另一方面，本申请还提供了制备所述分离的抗原结合蛋白的方法。所述方法可以包括在使得所述抗原结合蛋白表达的条件下，培养所述细胞。
用途
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述的多肽、所述的核酸分子、所述的载体、所述的细胞、所述的药物组合物和/或所述的药物组合在制备药物中的用途，所述药物可以用于预防、缓解和/或治疗疾病和/或病症。
另一方面，本申请还提供了预防、缓解和/或治疗疾病和/或病症的方法，所述方法可以包括向受试者施用所述分离的抗原结合蛋白、所述的多肽、所述的核酸分子、所述的载体、所述的细胞、所述的药物组合物和/或所述的药物组合。
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述的多肽、所述的核酸分子、所述的载体、所述的细胞、所述的药物组合物和/或所述的药物组合，其用于预防、缓解和/或治疗疾病和/或病症。
在本申请中，所述疾病和/或病症包括与TIGIT异常表达相关的疾病和/或病症。
在本申请中，所述疾病和/或病症包括肿瘤。
在本申请中，所述肿瘤包括实体瘤。
在本申请中，肿瘤包括非实体瘤。
在某些实施方式中，所述肿瘤包括对免疫检查点敏感的肿瘤。
在某些实施方式中，所述肿瘤包括结肠癌、黑色素瘤，非小细胞肺癌，肾细胞癌和/或肝细胞癌。
另一方面，本申请还提供了一种阻断TIGIT蛋白和CD155相互作用的方法，所述方法包括向有需要的受试者施用所述的抗原结合蛋白，所述的多肽，所述的核酸分子，所述的载体，所述的细胞，所述的药物组合物，和/或所述的药物组合。
在某些实施方式中，所述方法为非诊断和非治疗目的的方法。
在某些实施方式中，所述方法为体外方法和/或离体方法。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的融合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1靶向人TIGIT纳米抗体的筛选
1.1免疫羊驼并构建噬菌体展示免疫纳米抗体库
首先表达重组人TIGIT的细胞外结构域(ECD)，该结构域连着Fc片段，方便后续的蛋白纯化。根据本领域已知的方案，使用TIGIT-Fc抗原对羊驼进行免疫，动物免疫外包给爱康得生物医学技术(苏州)有限公司，单次免疫剂量为2mg蛋白。第三次和第四次免疫结束后，分别采集5mL外周血，分离血清，采用ELISA检测免疫效果，ELISA免疫效价达到1:16000以上(包被抗原5ug/mL，OD值大于2.0)，冲击免疫后，采集150mL外周血。然后分离PBMC并使用QIAGEN公司提供的RNA提取试剂盒提取总RNA。最后使用Super-Script III FIRST STRANDSUPERMIX试剂盒将提取的RNA反转录成cDNA。在本领域已知的方案下，通过巢式PCR扩增重链抗体的可变区(VHH)，所用引物如下：第一轮PCR的引物：
CALL001：5′-GTCCTGGCTGCTCTTCTACAAGG-3′；(SEQ ID NO:143)
CALL002：5′-GGTACGTGCTGTTGAACTGTTCC-3′；(SEQ ID NO:144)
第二轮PCR的引物：
VHH-Back：5′-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3′；(SEQ ID NO:145)
VHH-For：5′-CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3′。(SEQ ID NO:146)
胶回收目标片段，并使用限制性内切酶(来自Thermo)PstⅠ和Eco91Ⅰ将其克隆至噬菌体展示的载体pMES4(GenBank GQ907248)中。将质粒脱盐后，电转至电转感受态大肠杆菌TG1中，构建噬菌体展示纳米抗体库NanoTIGI，并对文库进行多样性评价。通过梯度稀释点板，计算库容大小为2×109，之后随即挑取22个单克隆进行测序，确定突变率为81.8％(18/22)，多样性为1.6E9。
1.2靶向人TIGIT进行淘选
用人TIGIT-His蛋白(TIT-H52H5-100ug，ACRO)10μg/ml包被平板，4℃放置过夜。隔天用1×PBST(PBS中含有0.05％Tween20)清洗3次后，0.5％BSA室温封闭2小时，1×PBST清洗3次，加入100μl噬菌体(来自实施例1所构建的噬菌体展示免疫纳米抗体库NanoTIGI)进行筛选，1个小时后用1×PBST清洗15次，以洗掉不结合抗原的噬菌体，最后用pH＝2.2的Glycine-HCl进行洗脱，100μl/well，然后用pH＝8.0的Tris-HCl中和，取一半洗脱出来的噬菌体感染处于对数生长期的TG1，半小时后超感染M13KO7，过夜培养。隔天沉淀噬菌体用于下一轮的筛选。相似的筛选过程重复3轮，第二轮的时候还是用10μg/ml 的人TIGIT-His正筛，1×PBST清洗20次，第三轮筛选条件与第二轮相同，抗原量为10μg/ml，1×PBST清洗次数为40次。之后通过pool ELISA检测抗体的特异性富集。
1.3用噬菌体的酶联免疫方法(ELISA)筛选特异性单克隆抗体
挑取单菌落至12块96孔深孔板分别过夜培养，生产噬菌体，隔天使用人TIGIT-Fc，IgG1-Fc和0.2％BSA包被的384板，1×PBST(PBS中含有0.05％Tween20)清洗3次后，0.5％BSA室温封闭2小时，1×PBST清洗3次，加入100μl噬菌体上清后孵育1小时，洗涤后加入anti-M13-HRP(SinoBiological，货号：11973-MM05T-H，)孵育1小时，洗涤后加入TMB显色液(Invitrogen，货号：002023)，3-5分钟后加入2M磷酸终止显色反应，最后读取OD450。当样品孔OD值大于对照孔OD值5倍以上时，视为阳性孔。根据序列比对软件BioEdit分析各个克隆的氨基酸序列，共获得65条阳性序列。其中，TIGI7的VHH序列如SEQ ID NO:40所示，TIGI36的VHH序列如SEQ ID NO:41所示，TIGI40的VHH序列如SEQ ID NO:42所示，TIGI41的VHH序列如SEQ ID NO:43所示，TIGI42的VHH序列如SEQ ID NO:44所示，TIGI53的VHH序列如SEQ ID NO:45所示，TIGI44的VHH序列如SEQ ID NO:46，TIGI56的VHH序列如SEQ ID NO:47所示，TIGI60的VHH序列如SEQ ID NO:48所示，TIGI63的VHH序列如SEQ ID NO:49所示，TIGI64的VHH序列如SEQ ID NO:50所示，TIGI35的VHH序列如SEQ ID NO:110所示，TIGI37的VHH序列如SEQ ID NO:111所示，TIGI38的VHH序列如SEQ ID NO:112所示，TIGI39的VHH序列如SEQ ID NO:113所示，TIGI45的VHH序列如SEQ ID NO:114所示，TIGI46的VHH序列如SEQ ID NO:115所示，TIGI48的VHH序列如SEQ ID NO:116所示，TIGI51的VHH序列如SEQ ID NO:117所示，TIGI52的VHH序列如SEQ ID NO:118所示，TIGI54的VHH序列如SEQ ID NO:119所示，TIGI57的VHH序列如SEQ ID NO:120所示，TIGI58的VHH序列如SEQ ID NO:121所示，TIGI59的VHH序列如SEQ ID NO:122所示，TIGI61的VHH序列如SEQ ID NO:123所示，TIGI65的VHH序列如SEQ ID NO:124所示，TIGI33的VHH序列如SEQ ID NO:125所示，TIGI43的VHH序列如SEQ ID NO:126所示。
实施例2 TIGIT纳米抗体的体外特异性和亲和力评价
2.1细胞酶联免疫方法(ELISA)检测阳性抗体噬菌体上清的特异性结合
分别接种293T，293T-TIGIT于1块高吸附的384孔ELISA板，1E4/well，37℃培养过夜；第二天弃去上清，用4％多聚甲醛固定384孔板，常温静置20min；PBS清洗三次；2倍梯度稀释噬菌体，然后加入384孔；4℃静置2小时后，PBS清洗3次，加入稀释于5％Milk的二抗，anti-6×His-HRP；4℃静置1小时后，PBS清洗7次，TMB显色，3-5分钟后加入 2M磷酸终止显色反应，读取OD450。
2.2二价纳米抗体在EXPI293中的表达和纯化
将序列分析所得的65条纳米抗体的核苷酸序列亚克隆至真核表达载体pcDNA3.4中，然后转染至EXPI293，培养表达5天以制备二价抗体。收集上清液并通过protein A(金斯瑞，货号：L00695-80)纯化。
2.3酶联免疫方法(ELISA)检测候选TIGIT纳米抗体与人TIGIT的结合情况
用人TIGIT-Fc融合蛋白和5％Milk包被384孔板，4℃静置过夜。隔天加入5％Milk封闭2小时。将纯化的抗体配制成100nM，3倍稀释，共11个梯度。封闭完成后洗涤3次，加入稀释的二价抗体，室温震荡孵育1小时，洗涤后加入anti-human IgG Fc-HRP，室温震荡孵育1小时，洗涤后加入TMB显色液，3-5分钟后加入2M磷酸终止显色反应，读取OD450。
2.4流式细胞术检测TIGIT纳米抗体与细胞表面TIGIT的结合
复苏293T和293T-hTIGIT细胞传代一代；收获细胞，计数后调整细胞密度为1×106/ml，30μl/well(3×104/well)；32个VHH-TIGIT的抗体和阳性抗体Tiragolumab(VH的氨基酸序列如SEQ ID NO:147所示，VL的氨基酸序列如SEQ ID NO:148所示)，TIGI7，100nM，设PBS对照，30μl/well，设置两个复孔，混匀后4℃孵育1小时；含0.1％BSA的PBS洗涤二次，500g，5min，4℃，甩干；1：200稀释荧光二抗Goat pAb to Hu IgG[DyLight 650](abcam，货号：ab98593)，30μl/well，混匀后4℃孵育30分钟；含0.1％BSA的PBS洗涤二次，500g，5min，4℃，甩干；30μl/well重悬，上高通量流式检测仪(IQue)读值。结果如图1显示，28条纳米抗体可以特异性地结合细胞表面的TIGIT。
2.5通过流式检测TIGIT纳米抗体对细胞表面TIGIT与CD155相互作用的阻断效果
复苏293T-hTIGIT细胞并传代；收获细胞，计数后调整细胞密度为1×106/ml，30μl/well(3×104/well)；28个纳米抗体和阳性抗体Tiragolumab的浓度为800nM，15μl/well，与15μl12μg/ml的Biotin-CD155预混，之后加入293T细胞，30μl/well，混匀后4℃孵育1小时；含0.1％BSA的PBS洗涤二次，500g，5min，4℃，甩干；1：500稀释Streptavidin-APC conjugate(BD，货号：554067)，30ul/well，混匀后4℃孵育30分钟；含0.1％BSA的PBS洗涤二次，500g，5min，4℃，甩干；30μl/well重悬，上高通量流式检测仪(IQue)读值，结果如图2显示，共有26条纳米抗体可以阻断TIGIT和CD155的相互作用。
2.6 Fortebio检测具有阻断活性抗体的亲和力
稀释具有阻断活性的TIGIT纳米抗体至100nM，将抗体加入384孔板中。稀释人TIGIT-His抗原(北京百普赛斯生物科技股份有限公司，TIT-H52H5-100ug)至100nM，2倍 稀释，共7个梯度，加入384孔板。使用AHC探针(Sartorius，货号：18-5060)，设置结合时间为180sec，解离时间300sec，基线为60sec，再生循环三次，每次5sec。之后用软件拟合结合-解离曲线，计算抗体的亲和力，结果如图3A-图3AA，大部分纳米抗体的亲和力都在nM级别。
实施例3 TIGIT人源化小鼠Balb/c体内药效研究
使用TIGIT人源化小鼠Balb/c和CT26结肠癌模型，研究比较TIGI7，TIGI37，TIGI39，TIGI46和TIGI48的体内抑瘤作用。从集萃药康采购6-8周龄的人源化小鼠，饲养一周后接种CT26细胞，细胞数为1E6。一周后，待肿瘤体积长到80-100mm3，随机分组给药，所有组给药途径均为腹腔注射，每三天给药1次，连续给药6次，末次给药大约11天后结束实验。给药和观察期间每周测量3次小鼠体重和肿瘤体积，并记录测量值，结果如图4A，计算肿瘤体积抑制率(TGITV％)，实验结束时，动物安乐死，剥取肿瘤称重、拍照，计算瘤重抑制率(TGITW％)，实验结果如图4B。实验结果显示，TIGI7，TIGI37，TIGI39，TIGI46，TIGI48和Tiragolumab均有抑瘤作用，其瘤重抑瘤率分别是61.37％，47.06％，55.69％，50.98％，54.51％和64.51％；其肿瘤体积抑瘤率分别是62.98％，53.63％、59.80％、52.94％、60.86％，67.23％。
实施例4 TIGI7与YN035联合用药的体内药效实验
使用TIGIT人源化小鼠Balb/c和CT26结肠癌模型，研究TIGI7和YN035(VH的序列如SEQ ID NO:138所示，VL的序列如SEQ ID NO:142所示)联合用药的体内抑瘤作用。从集萃药康采购6-8周龄的人源化小鼠，饲养一周后接种CT26细胞，细胞数为1E6。一周后，待肿瘤体积长到80-100mm3，随机分组给药，所有组给药途径均为腹腔注射，每三天给药1次，连续给药6次，末次给药大约11天后结束实验。给药和观察期间每周测量3次小鼠体重和肿瘤体积，并记录测量值，计算肿瘤体积抑制率(TGITV％)。另外观测当天肿瘤体积超过3000mm3时，对单只小鼠进行安乐死，并计算各组的中位生存期。实验结果如图5A-5C，YN035(PD-L1单抗)的抑瘤率为55.97％，TIGI7单药的抑瘤率为59.59％，联合用药的抑瘤率为62.39％，其中位生存期分别是29.5天，32天和32.5天。因此判定YN035和TIGI7具有协同抑瘤作用。
实施例5 TIGI7的人源化，亲和力成熟和交叉活性改造
5.1 TIGI7的人源化
选择TIGI7对其框架区进行人源化。简单来说，从文献中获取人抗体DP-47序列，将DP-47与TIGI7分别在IMGT网站上(http://www.imgt.org/)进行编号，确定框架区和CDR，然后将TIGI7框架上的氨基酸换成DP-47框架区上对应的氨基酸。由于FR2对纳米抗体稳定性具有重要作用，因此保留不变。其中，hTIGI7的VHH序列如SEQ ID NO:51所示，hTIGI7.1的VHH序列如SEQ ID NO:52所示，hTIGI7.2的VHH序列如SEQ ID NO:53所示，hTIGI7.3的VHH序列如SEQ ID NO:54所示，hTIGI7.4的VHH序列如SEQ ID NO:55所示，hTIGI7.5的VHH序列如SEQ ID NO:56所示，hTIGI7.6的VHH序列如SEQ ID NO:57所示，hTIGI7.7的VHH序列如SEQ ID NO:58所示，hTIGI7.8的VHH序列如SEQ ID NO:59所示，hTIGI7.9的VHH序列如SEQ ID NO:60所示，hTIGI7.10的VHH序列如SEQ ID NO:61所示，hTIGI7.11的VHH序列如SEQ ID NO:62所示，hTIGI7.12的VHH序列如SEQ ID NO:63所示，hTIGI7.13的VHH序列如SEQ ID NO:64所示，将人源化后的序列克隆到表达载体pcDNA3.4中，通过瞬时转染EXPI293产生抗体蛋白，并通过protein A纯化。
5.2 hTIGI7的亲和力和稳定性鉴定
将正常培养的293T-hTIGIT细胞消化离心后，用含0.1％BSA的PBS重悬，取一96孔尖底板，向每个孔中加入4E4个细胞，并定容至30μl。将相应抗体稀释至100nM，之后再以3倍稀释7个梯度，最后一个梯度不加抗体。取30μl抗体溶液，与细胞悬液混匀，4℃下孵育1h。用含0.1％BSA的PBS洗涤细胞2遍，每次500g离心5min，将孔内液体尽量甩干。将anti-human IgG Fc-650二抗(abcam，货号：ab98593)用含0.1％BSA的PBS以1：200倍稀释，向每孔加入30μl并与细胞混匀，4℃下孵育30min。用含0.1％BSA的PBS洗涤细胞3遍，每次500g离心5min，最后用30μl含0.1％BSA的PBS重悬细胞，上机，读取数据后用Graphpad做四参数拟合曲线，计算EC50，结果如图6A。稳定性鉴定则通过跑HPLC-SEC来分析抗体的聚集程度。结果如图6B，人源化后的TIGI7结合活性有所下降，但SEC显示纳米抗体无聚集，具备良好的成药性。
5.3 hTIGI7的亲和力成熟和交叉活性改造
根据文献nature protocol的文献(Nature protocols,2007,2(6):1368-1386.)构建hTIGI7亲和力成熟的噬菌体展示抗体库。简单来说就是将hTIGI7的核苷酸序列克隆至HP153载体，之后转化到大肠杆菌CJ236中，培养噬菌体，提取包含尿嘧啶的单链DNA。在体外将人工合成的突变引物与单链DNA混合互补，制备CCC-dsDNA。将CCC-dsDNA脱盐后电转化至M13KO7预感染的TG1中，之后过夜放大培养，隔天收集噬菌体，用来筛选具有人猴交叉活性的TIGIT纳米抗体。筛选过程与实施例1.2类似，第一轮所用抗原为100nM的 cynomolgus TIGIT，第二轮所用抗原为100nM的cynomolgus TIGIT，第三轮为10nM的human TIGIT，第四轮为1nM Cynomolgus TIGIT，第五轮为0.01nM human TIGIT，清洗次数均为50次。筛选结束后，通过噬菌体单克隆ELISA挑取可同时结合人和猴TIGIT的阳性序列。测序后共获得13条纳米抗体。
5.4鉴定亲和力成熟和交叉活性改造后的纳米抗体。
将正常培养的293T-human TIGIT和293T-cynomolgus TIGIT细胞消化离心后，用含0.1％BSA的PBS重悬，取一96孔尖底板，向每个孔中加入4E4个细胞，每孔30μl。将相应抗体hTIGI7.1-hTIGI7.13稀释至200nM，之后再以3倍稀释11个梯度，最后一个梯度不加抗体。取30μl抗体溶液，与细胞悬液混匀，4℃下孵育1h。用含0.1％BSA的PBS洗涤细胞2遍，每次500g离心5min，将孔内液体甩干。将anti-human IgG Fc-650二抗(abcam，货号：ab98593)用含0.1％BSA的PBS以1：200倍稀释，向每孔加入30μl并与细胞混匀，4℃下孵育30min。用含0.1％BSA的PBS洗涤细胞3遍，每次500g，离心5min，最后用30μl含0.1％BSA的PBS重悬细胞，上机，读取数据后用Graphpad做四参数拟合曲线，计算EC50，结果如图7A和7B。另外通过SEC检测抗体的聚集情况，从而判定其成药性，结果图7C-7O所示。结果显示，相较于TIGI7，hTIGI7.11亲和力更高，而且跟human TIGIT和cynomolgus TIGIT的EC50一致，曲线的峰高也比较接近。而且所有抗体均为显示任何聚集，成药性良好。
5.5对hTIGI7进行高风险位点移除
利用abYsis网站上Annotate的功能对hTIGI7.11的CDR区进行分析，发现在CDR2上有一个高风险位点DS，会引起抗体异构化，因此将其中的天冬氨酸突变成谷氨酸，苏氨酸或丙氨酸，分别命名为hTIGI7.11E(VHH序列为SEQ ID NO:65)，hTIGI7.11T(VHH序列为SEQ ID NO:66)，hTIGI7.11A(VHH序列为SEQ ID NO:67)。将突变后的序列亚克隆至真核表达载体pcDNA3.4，之后瞬时转染到EXPI293表达，用protein A纯化。将纯化后的抗体与293T-human TIGIT和293T-cynomolgus TIGIT进行流式验证，结果如图8A和8B，发现突变后的抗体并未改变其亲和力。另外，为了检测hTIGI7.11E，hTIGI7.11T，hTIGI7.11A的稳定性，将2nM抗体分别在20℃，30℃，40℃，50℃，60℃，70℃，80℃，90℃下孵育1小时，然后做ELISA验证其结合活性，结果如图8C，发现突变后的hTIGI7.11E的稳定性与hTIGI7.11最相似。反复冻融三次后，检测抗体的SEC和流式结合活性，结果如图8D和8E，突变后的三个抗体并未有任何差异。
5.6验证hTIGI7.11E与激活后cynomolgus PBMC上TIGIT的结合活性
先用500ng/ml的SEA与PBMC共孵育48小时，用PBS清洗3次，之后取1E5的细胞与200nM的hTIGI7.11E，Caplacizumab和IgG1-Fc在4℃条件下孵育1小时，用含0.1％BSA的PBS洗涤细胞3遍，将anti-human IgG Fc-650二抗(abcam，货号：ab98593)用含0.1％BSA的PBS以1：200倍稀释，向每孔加入30μl并与细胞混匀，4℃下孵育30min，用含0.1％BSA的PBS洗涤细胞3遍，每次500g，离心5min，最后用30μl含0.1％BSA的PBS重悬细胞，上流式细胞仪(IQue)检测。结果如图9A和9B，相较于阴性抗体Caplacizumab和IgG1-Fc，hTIGI7.11E和阳性对照抗体2A3可以特异性地结合cynomolgus PBMC上的TIGIT。
5.7用Fortebio验证hTIGI7.11E与Tiragolumab和2A3的抗原结合表位
先将三种抗体分别稀释到100nM，加入384孔板。一共使用8根AHC探针(Sartorius，货号：18-5060)，每根探针为一组，共8组。8根探针首先用100nM人TIGIT进行loading，然后依次跑抗体。第一组的第一个抗体为hTIGI7.11E，第二个抗体为hTIGI7.11E；第二组的第一个抗体为hTIGI7.11E，第二个抗体Tiragoluamab；第三组的第一个抗体为Tiragoluamab，第二个抗体Tiragoluamab；第四组的第一个抗体为Tiragoluamab，第二个抗体hTIGI7.11E；第五组的第一个抗体为hTIGI7.11E，第二个抗体hTIGI7.11E；第六组的第一个抗体为hTIGI7.11E，第二个抗体2A3；第七组的第一个抗体为2A3，第二个抗体2A3；第八组的第一个抗体为2A3，第二个抗体hTIGI7.11E。Baseline的时间为60sec，结合的时间为300sec。结果如图10A-10D，hTIGI7.11E，Tiragolumab和2A3均可以阻断TIGIT与CD155的相互作用，其中hTIGI7.11E与Tiragolumab具有相同的抗原结合表位，hTIGI7.11E与2A3具有不同的抗原结合表位。
5.8比较hTIGI7.11E与Tiragolumab和2A3的阻断活性
复苏293T-hTIGIT细胞传代；收获细胞，计数后调整细胞密度为1×106/ml，30μl/well(3×104/well)；hTIGI7.11E和2A3的抗体和阳性抗体Tiragolumab以800nM为最高浓度，3倍比，11个梯度，设PBS对照，15μl/well，之后加入12ug/ml的Biotin-CD155，15μl/well，混匀后4℃孵育1小时；含0.1％BSA的PBS洗涤二次，500g，5min，4℃，甩干；1：500稀释Streptavidin,Alexa FluorTMAPC conjugate，30ul/well，混匀后4℃孵育30分钟；含0.1％BSA的PBS洗涤二次，500g，5min，4℃，甩干；30μl/well重悬，上高通量流式检测仪读值，采用Graphpad做四参数拟合曲线。结果如图11，hTIGI7.11E的IC50优于Tiragolumab和2A3大概2-3倍左右。
5.9检测hTIGI7.11E的ADCC活性
靶细胞为：293T-human TIGIT细胞，为实验室前期构建好的可以稳定表达人TIGIT和luciferase荧光素酶的293T细胞株；靶细胞计数离心后用“DMEM+10％FBS”的培养基重悬，2E5/ml的细胞密度，50μl/孔，即5000/孔靶细胞；从公司库里出库一支冻存的PBMC，8E7cells；用PBS润洗3次，500g/5min，400g/5min，300g/5min，室温离心。后计数；效应细胞同样用“DMEM+10％FBS”的培养基重悬，1.1E7/ml的细胞密度，50μl/孔，即1.75E5/孔靶细胞，即效靶比E：T＝35：1；抗体为：hTIGIT7.11E、IgG1-FC、Tiragolumab，抗体起始工作浓度为100nM，8倍比梯度稀释，共形成7个浓度梯度，50μl/孔；将靶细胞，效应细胞和抗体一起加入白底不透光的96孔细胞培养板中，同时设置靶细胞单独孔作为对照，37℃培养箱培养48h后，用Tecan酶标仪检测板中荧光素酶的含量；裂解百分比(％)＝(靶单独孔-实验孔)/靶单孔×100。结果如图12，hTIGI7.11E的ADCC活性与Tiragoluamab和2A3相当。
实施例6验证hTIGI7.11E的体内药效
将CT26细胞以1×106个/0.1mL浓度接种于Balb/c-hTIGIT人源化小鼠的右侧皮下，待肿瘤生长到大约80-100mm3时按肿瘤体积挑选并随机分组，所有组给药途径均为腹腔注射，每三天给药1次，连续给药6次，末次给药11天后结束实验。给药和观察期间每周测量3次小鼠体重和肿瘤体积，并记录测量值，计算肿瘤体积抑制率(TGITV％)。实验结束时，动物安乐死。结果如图13，在第17天的时候，相较于对照组IgG1-Fc，中高浓度的hTIGI7.11E均能显著地抑制肿瘤生长。

Claims (67)

  1. 分离的抗原结合蛋白，其具有下述性质中的一种或多种：
    (1)能够特异性结合TIGIT蛋白；
    (2)能够与人TIGIT和食蟹猴TIGIT蛋白均有结合活性；
    (3)能够对细胞表面TIGIT蛋白与CD155的相互作用具有阻断效果；以及
    (4)能够抑制肿瘤的生长和/或增殖。
  2. 根据权利要求1所述的抗原结合蛋白，其包括抗体或其抗原结合片段。
  3. 根据权利要求2所述的抗原结合蛋白，其中所述抗原结合片段包括Fab、Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
  4. 根据权利要求2-3中任一项所述的抗原结合蛋白，其中所述抗原结合片段为VHH。
  5. 根据权利要求2-4中任一项所述的抗原结合蛋白，其中所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
  6. 根据权利要求1-5中任一项所述的抗原结合蛋白，其包含源自抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:132所示的氨基酸序列。
  7. 根据权利要求1-6中任一项所述的抗原结合蛋白，其包括HCDR3，且所述HCDR3包含SEQ ID NO:127所示的氨基酸序列。
  8. 根据权利要求1-7中任一项所述的抗原结合蛋白，其包括HCDR3，且所述HCDR3包含SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20中任一项所示的氨基酸序列。
  9. 根据权利要求1-8中任一项所述的抗原结合蛋白，其包含HCDR2，且所述HCDR2包含SEQ ID NO:128所示的氨基酸序列。
  10. 根据权利要求1-9中任一项所述的抗原结合蛋白，其包含HCDR2，且所述HCDR2包含SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:6和SEQ ID NO:7中任一项所示的氨基酸序列。
  11. 根据权利要求1-10中任一项所述的抗原结合蛋白，其包含HCDR1，且所述HCDR1包含SEQ ID NO:1所示的氨基酸序列。
  12. 根据权利要求1-11中任一项所述的抗原结合蛋白，其包含HCDR1，HCDR2，和HCDR3，所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:128所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:127所示的氨基酸序列。
  13. 根据权利要求1-12中任一项所述的抗原结合蛋白，其包含HCDR1，HCDR2，和HCDR3，所述HCDR1包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ  ID NO:2、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:6和SEQ ID NO:7中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20中任一项所示的氨基酸序列。
  14. 根据权利要求1-13中任一项所述的抗原结合蛋白，其包含HCDR1，HCDR2，和HCDR3，且所述HCDR1、HCDR2、HCDR3包含选自下组的任意一组氨基酸序列：
    (1)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:2；和HCDR3：SEQ ID NO:4；
    (2)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:4；
    (3)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:8；
    (4)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:9；
    (5)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:10；
    (6)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:11；
    (7)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:12；
    (8)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:13；
    (9)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:14；
    (10)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:15；
    (11)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:16；
    (12)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:17；
    (13)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:18；
    (14)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:19；
    (15)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:3；和HCDR3：SEQ ID NO:20；
    (16)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:5；HCDR3：SEQ ID NO:18；
    (17)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:6；HCDR3：SEQ ID NO:18；以及
    (18)HCDR1：SEQ ID NO:1；HCDR2：SEQ ID NO:7；HCDR3：SEQ ID NO:18。
  15. 根据权利要求11-14中任一项所述的抗原结合蛋白，其包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:129所示的氨基酸序列。
  16. 根据权利要求15所述的抗原结合蛋白，其中所述H-FR1包含SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID  NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31和SEQ ID NO:32中任一项所示的氨基酸序列。
  17. 根据权利要求11-16中任一项所述的抗原结合蛋白，其包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:33所示的氨基酸序列。
  18. 根据权利要求9-17中任一项所述的抗原结合蛋白，其包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3SEQ ID NO:130所示的氨基酸序列。
  19. 根据权利要求18所述的抗原结合蛋白，其中所述H-FR3包含SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36和SEQ ID NO:37中任一项所示的氨基酸序列。
  20. 根据权利要求7-20中任一项所述的抗原结合蛋白，其包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:131所示的氨基酸序列。
  21. 根据权利要求20所述的抗原结合蛋白，其中所述H-FR4包含SEQ ID NO:38或SEQ ID NO:39中任一项所示的氨基酸序列。
  22. 根据权利要求1-21中任一项所述的抗原结合蛋白，其包括抗体重链可变区VH，且所述VH包含SEQ ID NO:132所示的氨基酸序列。
  23. 根据权利要求22所述的抗原结合蛋白，其中所述VH包含SEQ ID NO:40-67中任一项所示的氨基酸序列。
  24. 根据权利要求1-23中任一项所述的抗原结合蛋白，其为VHH，且所述VHH包含SEQ ID NO:40-67中任一项所示的氨基酸序列。
  25. 根据权利要求1-5中任一项所述的抗原结合蛋白，其包括HCDR3，且所述HCDR3包含SEQ ID NO:68、SEQ ID NO:69和SEQ ID NO:70中任一项所示的氨基酸序列。
  26. 根据权利要求1-5，和25中任一项所述的抗原结合蛋白，其包含HCDR2，且所述HCDR2包含SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76和SEQ ID NO:77中任一项所示的氨基酸序列。
  27. 根据权利要求1-5，和25-26中任一项所述的抗原结合蛋白，其包含HCDR1，且所述HCDR1包含SEQ ID NO:78、SEQ ID NO:79、SEQ ID NO:80、SEQ ID NO:81和SEQ ID NO:82中任一项所示的氨基酸序列。
  28. 根据权利要求1-5和25-27中任一项所述的抗原结合蛋白，其包含HCDR1，HCDR2，和HCDR3，所述HCDR1包含SEQ ID NO:78、SEQ ID NO:79、SEQ ID NO:80、SEQ ID NO:81和SEQ ID NO:82中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76 和SEQ ID NO:77中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:68、SEQ ID NO:69和SEQ ID NO:70中任一项所示的氨基酸序列。
  29. 根据权利要求1-5和25-28中任一项所述的抗原结合蛋白，其包含HCDR1，HCDR2，和HCDR3，且所述HCDR1、HCDR2、HCDR3包含选自下组的任意一组氨基酸序列：
    (1)HCDR1：SEQ ID NO:78；HCDR2：SEQ ID NO:71；和HCDR3：SEQ ID NO:68；
    (2)HCDR1：SEQ ID NO:78；HCDR2：SEQ ID NO:72；和HCDR3：SEQ ID NO:68；
    (3)HCDR1：SEQ ID NO:79；HCDR2：SEQ ID NO:72；和HCDR3：SEQ ID NO:68；
    (4)HCDR1：SEQ ID NO:78；HCDR2：SEQ ID NO:73；和HCDR3：SEQ ID NO:68；
    (5)HCDR1：SEQ ID NO:80；HCDR2：SEQ ID NO:73；和HCDR3：SEQ ID NO:68；
    (6)HCDR1：SEQ ID NO:78；HCDR2：SEQ ID NO:71；和HCDR3：SEQ ID NO:68；
    (7)HCDR1：SEQ ID NO:78；HCDR2：SEQ ID NO:74；和HCDR3：SEQ ID NO:68；
    (8)HCDR1：SEQ ID NO:80；HCDR2：SEQ ID NO:75；和HCDR3：SEQ ID NO:68；
    (9)HCDR1：SEQ ID NO:81；HCDR2：SEQ ID NO:76；和HCDR3：SEQ ID NO:69；以及
    (10)HCDR1：SEQ ID NO:82；HCDR2：SEQ ID NO:77；和HCDR3：SEQ ID NO:70。
  30. 根据权利要求1-5和27-29中任一项所述的抗原结合蛋白，其包含H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:133所示的氨基酸序列。
  31. 根据权利要求30所述的抗原结合蛋白，其中所述H-FR1包含SEQ ID NO:83、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91、SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、SEQ ID NO:97、SEQ ID NO:98和SEQ ID NO:99中任一项所示的氨基酸序列。
  32. 根据权利要求1-5和26-31中任一项所述的抗原结合蛋白，其包含H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:134所示的氨基酸序列。
  33. 根据权利要求32所述的抗原结合蛋白，其中所述H-FR2包含SEQ ID NO:100、SEQ ID NO:101和SEQ ID NO:102中任一项所示的氨基酸序列。
  34. 根据权利要求1-5和26-33中任一项所述的抗原结合蛋白，其包含H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3SEQ ID NO:103SEQ ID NO:104、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:108和SEQ ID NO: 109中任一项所示的氨基酸序列。
  35. 根据权利要求1-5和25-34中任一项所述的抗原结合蛋白，其包含H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:38所示的氨基酸序列。
  36. 根据权利要求1-5和25-35中任一项所述的抗原结合蛋白，其包含VH，且所述VH包含SEQ ID NO:110-126中任一项所示的氨基酸序列。
  37. 根据权利要求1-5和25-36中任一项所述的抗原结合蛋白，其为VHH，且所述VHH包含SEQ ID NO:110-126中任一项所示的氨基酸序列。
  38. 根据权利要求1-37中任一项所述的抗原结合蛋白，其包含抗体重链恒定区，所述抗体重链恒定区源自IgG。
  39. 根据权利要求1-38中任一项所述的抗原结合蛋白，其包含抗体重链恒定区，所述抗体重链恒定区源自人IgG。
  40. 根据权利要求1-39中任一项所述的抗原结合蛋白，其包含抗体重链恒定区，所述抗体重链恒定区源自人IgG1。
  41. 多肽，其包含权利要求1-40中任一项所述的抗原结合蛋白。
  42. 一种或多种分离的核酸分子，其编码权利要求1-40中任一项所述的抗原结合蛋白。
  43. 载体，其包含权利要求41所述的核酸分子。
  44. 细胞，其包含权利要求42所述的载体。
  45. 制备权利要求1-40中任一项所述的抗原结合蛋白的方法，所述方法包括在使得所述抗原结合蛋白表达的条件下，培养权利要求44所述的细胞。
  46. 药物组合物，其包含权利要求1-40中任一项所述的抗原结合蛋白，以及任选地药学上可接受的载体。
  47. 药物组合，其包含权利要求1-40中任一项所述的抗原结合蛋白，以及免疫检查点抑制剂。
  48. 根据权利要求47所述的药物组合，其中所述免疫检查点抑制剂包括靶向PD-L1的抗原结合蛋白。
  49. 根据权利要求48所述的药物组合，其中所述靶向PD-L1的抗原结合蛋白包含HCDR1，HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:135所示的氨基酸序列，所述HCDR2包含SEQ ID NO:136所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:137所示的氨基酸序列。
  50. 根据权利要求48-49中任一项所述的药物组合，其中所述靶向PD-L1的抗原结合蛋白包含LCDR1，LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:139所示的氨基酸序列， 所述LCDR2包含SEQ ID NO:140所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:141所示的氨基酸序列。
  51. 根据权利要求48-50中任一项所述的药物组合，其中所述靶向PD-L1的抗原结合蛋白包含VH，且所述VH包含SEQ ID NO:138所示的氨基酸序列。
  52. 根据权利要求48-51中任一项所述的药物组合，其中所述靶向PD-L1的抗原结合蛋白包含VL，且所述VL包含SEQ ID NO:142所示的氨基酸序列。
  53. 权利要求1-40中任一项所述的抗原结合蛋白，权利要求41所述的多肽，权利要求42所述的核酸分子，权利要求43所述的载体，权利要求44所述的细胞，权利要求46所述的药物组合物，和/或权利要求47-52中任一项所述的药物组合在制备药物中的用途，所述药物用于预防、治疗和/或缓解疾病和/或病症。
  54. 根据权利要求53所述的用途，其中所述疾病和/或病症包括与TIGIT异常表达相关的疾病和/或病症。
  55. 根据权利要求53-54中任一项所述的用途，其中所述疾病和/或病症包括肿瘤。
  56. 根据权利要求55所述的用途，其中所述肿瘤包括实体瘤。
  57. 根据权利要求55所述的用途，其中所述肿瘤包括非实体瘤。
  58. 根据权利要求55-57中任一项所述的方法，其中所述肿瘤包括对免疫检查点敏感的肿瘤。
  59. 根据权利要求55-58中任一项所述的方法，其中所述肿瘤包括结肠癌、黑色素瘤，非小细胞肺癌，肾细胞癌和/或肝细胞癌。
  60. 一种预防、治疗和/或缓解疾病和/或病症的方法，所述方法包括向有需要的受试者施用权利要求1-40中任一项所述的抗原结合蛋白，权利要求41所述的多肽，权利要求42所述的核酸分子，权利要求43所述的载体，权利要求44所述的细胞，权利要求46所述的药物组合物，和/或权利要求47-52中任一项所述的药物组合。
  61. 根据权利要求60所述的方法，其中所述疾病和/或病症包括与TIGIT异常表达相关的疾病和/或病症。
  62. 根据权利要求60-61中任一项所述的方法，其中所述疾病和/或病症包括肿瘤。
  63. 根据权利要求62所述的方法，其中所述肿瘤包括实体瘤。
  64. 根据权利要求62所述的方法，其中所述肿瘤包括非实体瘤。
  65. 根据权利要求62-64中任一项所述的方法，其中所述肿瘤包括对免疫检查点敏感的肿瘤。
  66. 根据权利要求62065中任一项所述的方法，其中所述肿瘤包括结肠癌、黑色素瘤，非小细胞肺癌，肾细胞癌和/或肝细胞癌。
  67. 一种阻断TIGIT蛋白和CD155相互作用的方法，所述方法包括向有需要的受试者施用 权利要求1-40中任一项所述的抗原结合蛋白，权利要求41所述的多肽，权利要求42所述的核酸分子，权利要求43所述的载体，权利要求44所述的细胞，权利要求46所述的药物组合物，和/或权利要求47-52中任一项所述的药物组合。
PCT/CN2023/073041 2022-01-24 2023-01-19 靶向tigit的抗原结合蛋白及其用途 WO2023138639A1 (zh)

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WO2021008523A1 (zh) * 2019-07-15 2021-01-21 上海君实生物医药科技股份有限公司 抗tigit抗体及其应用
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US20170165366A1 (en) * 2015-10-01 2017-06-15 Potenza Therapeutics, Inc. Anti-TIGIT Antigen-Binding Proteins and Methods of Use Thereof
US20210095028A1 (en) * 2018-01-15 2021-04-01 Nanjing Legend Biotech Co., Ltd. Antibodies and variants thereof against tigit
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