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WO2022111559A1 - 双特异性抗体及其用途 - Google Patents

双特异性抗体及其用途 Download PDF

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WO2022111559A1
WO2022111559A1 PCT/CN2021/133003 CN2021133003W WO2022111559A1 WO 2022111559 A1 WO2022111559 A1 WO 2022111559A1 CN 2021133003 W CN2021133003 W CN 2021133003W WO 2022111559 A1 WO2022111559 A1 WO 2022111559A1
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antibody
seq
binding
amino acid
acid sequence
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PCT/CN2021/133003
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English (en)
French (fr)
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朱向阳
崔小培
徐锦根
于海佳
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上海华奥泰生物药业股份有限公司
华博生物医药技术（上海）有限公司
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Priority to JP2023531505A priority Critical patent/JP2023550780A/ja
Priority to EP21897059.8A priority patent/EP4253424A4/en
Priority to US18/038,627 priority patent/US20240010730A1/en
Publication of WO2022111559A1 publication Critical patent/WO2022111559A1/zh

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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • 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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present application relates to the field of biomedicine, in particular to an anti-PD-L1/TIGIT bispecific antibody and its use.
  • Tumors can be divided into two categories: benign tumors and malignant tumors according to the cell characteristics of new organisms and the degree of harm to the body. Among them, malignant tumors are the major diseases that endanger human health in today's society, and the mortality rate ranks second.
  • Conventional treatments for solid tumors include surgery, chemotherapy, radiation therapy, molecularly targeted drug therapy, and immunotherapy. Many patients with solid tumors are found at an advanced stage and have lost the opportunity for surgical treatment, and most patients are weak and often cannot bear the strong side effects caused by radiotherapy and chemotherapy.
  • Biological therapeutic drugs, especially cancer immunotherapy represented by immune checkpoint antibodies such as PD-1 are more and more widely used due to their low toxicity and high effectiveness, especially for metastatic, relapsed, and refractory disease. It may be the only option for patients with advanced solid tumors who are refractory to sexual or existing standard chemotherapy.
  • bispecific antibodies are a hot topic in antibody drug development, they face many challenges, such as preclinical evaluation models, low expression levels, poor stability, complex processes, and large differences in quality control. Development is difficult. Therefore, there is an urgent need in the art to develop an anti-tumor drug with good specificity, good curative effect and easy preparation.
  • the present application provides a multispecific antibody, which has one or more of the following properties: (1) can specifically bind to human PD-L1 and human TIGIT with high affinity; (2) can simultaneously bind to PD -L1 protein and TIGIT protein; (3) can keep the binding configuration of the multispecific antibody and the binding target unchanged, and maintain molecular stability; (4) in the preliminary culture conditions, the purity of the multispecific antibody is above 90% (5) can maintain the binding affinity of the binding target, and can exert the synergistic effect of dual targets compared with single anti-PD-L1 antibody (such as HB0023) and anti-TIGIT antibody (such as HB0030); and (6) effectively kill Tumor cells (especially tumors with high PD-L1 expression), eg, solid tumors.
  • the preparation method of the multispecific antibody of the present application is simple and feasible, and has a good application prospect.
  • the application provides a multispecific antibody comprising a first targeting moiety capable of specifically binding a TIGIT protein, wherein the first targeting moiety comprises an isolated antigen-binding protein that binds
  • the protein comprises a heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3, and the HCDR2 comprises the amino acid shown in SEQ ID NO:4 or 42 sequence, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 5 or 43.
  • the multispecific antibody comprises a light chain variable region VL
  • the VL comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 6 or 52 , the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 8 or 53.
  • the multispecific antibody comprises a heavy chain variable region VH, wherein the VH comprises the amino acids set forth in any one of SEQ ID NOs: 110, 1, 9, 12, 14 and 32 sequence.
  • the multispecific antibody comprises a light chain variable region VL, wherein the VL comprises any one of SEQ ID NOs: 111, 2, 10, 11, 13, 33 and 34 amino acid sequence.
  • the multispecific antibody further includes a second targeting moiety.
  • the second targeting moiety is capable of specifically binding a tumor-associated antigen.
  • the second targeting moiety is capable of specifically binding PD-L1 protein.
  • the second targeting moiety comprises an antibody or antigen-binding fragment
  • the antibody capable of binding the PD-L1 protein comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NOs: The amino acid sequences shown in ID NOs: 100, 101 and 102.
  • the antibody capable of binding PD-L1 protein comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 103, 104 and 105, respectively.
  • the multispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the heavy chain of the antibody capable of binding PD-L1 protein Variable region VH, the heavy chain variable region VH capable of binding TIGIT protein and the light chain variable region VL capable of binding TIGIT protein; and the second polypeptide chain comprises the PD-L1 protein capable of binding
  • the light chain variable region VL of the antibody, or the first polypeptide chain comprises the heavy chain variable region VH of the antibody capable of binding the TIGIT protein, and the heavy chain variable region capable of binding the PD-L1 protein VH and the light chain variable region VL capable of binding the PD-L1 protein; and the second polypeptide chain comprises the light chain variable region VL of an antibody capable of binding the TIGIT protein.
  • the VH of the antibody capable of binding TIGIT protein and the VL of the antibody capable of binding TIGIT protein in the first polypeptide chain constitute a scFv, or the first polypeptide chain contains The VH of the antibody capable of binding to the PD-L1 protein and the VL of the antibody capable of binding the PD-L1 protein constitute a scFv.
  • the first polypeptide chain comprises an amino acid sequence selected from any one of the group consisting of SEQ ID NOs: 78-83 and 85-87.
  • the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 84 or 88.
  • the application provides isolated one or more nucleic acid molecules encoding the multispecific antibodies.
  • the application provides a vector comprising the nucleic acid molecule.
  • the application provides cells comprising said nucleic acid molecule or a vector according to said.
  • the present application provides a method for preparing the multispecific antibody, the method comprising culturing the cell under conditions such that the multispecific antibody is expressed.
  • the application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the multispecific antibody, the nucleic acid molecule, the vector and/or the cell, and optionally a pharmaceutically acceptable adjuvant agent.
  • the present application provides the use of the multispecific antibody, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition in preparing a medicine, the medicine For the prevention, alleviation and/or treatment of a disease or disorder.
  • Figure 1 shows the binding activity of TIGIT chimeric antibodies to cells expressing human TIGIT.
  • Figure 2 shows the binding activity of TIGIT humanized antibodies to cells expressing human TIGIT.
  • Figure 3 shows the blocking activity of TIGIT chimeric antibodies against binding of human TIGIT to its ligand CD155.
  • Figure 4 shows the blocking activity of TIGIT humanized antibodies on binding of human TIGIT to its ligand CD155.
  • FIG. 5 shows the efficacy detection of TIGIT antibody in animals.
  • FIG. 6 shows the comparison of the efficacy of TIGIT antibody with the control antibody Tiragolumab in animals.
  • Figures 7A-7B show exemplary structures of the multispecific antibodies described herein.
  • FIG 8 shows the results of SDS-PAGE electrophoresis of the multispecific antibody TIGIT-IgG-PDL1-scFv described in the present application.
  • Figure 9 shows the results of SDS-PAGE electrophoresis of the multispecific antibody PDL1-IgG-TIGIT-scFv described in the present application.
  • Figure 10 shows the inhibitory effect of the multispecific antibody PDL1-IgG-TIGIT-scFv described in the present application on PDL1 function.
  • FIG 11 shows the inhibitory effect of the multispecific antibody TIGIT-IgG-PDL1-scFv described in the present application on the function of TIGIT.
  • Figure 12 shows the therapeutic effect of the multispecific antibody described in this application on a mouse tumor model.
  • TIGIT is referred to as "Ig and ITIM domain-containing T cell immune receptor", generally a member of the PVR (poliovirus receptor) family of immunoglobulins that bind PVR/ CD155 and Nectin-2/CD112.
  • PVR poliovirus receptor
  • TIGIT herein can refer to TIGIT proteins from any vertebrate source, including mammals, such as primates (eg, humans, rhesus monkeys, and cynomolgus monkeys) and rodents (eg, mice and rats).
  • the term encompasses full-length TIGIT or fragments thereof (such as mature fragments thereof lacking a signal peptide), unprocessed TIGIT, any form of TIGIT that results from processing in a cell, and artificially synthesized TIGIT.
  • the term also encompasses variants of TIGIT, such as splice variants or allelic variants.
  • the TIGIT is human TIGIT, and the amino acid sequence of human TIGIT comprises the amino acid sequence shown in UniProt Accession No. Q495A1.
  • the TIGIT is a cynomolgus monkey TIGIT and the amino acid sequence of the cynomolgus monkey TIGIT comprises the amino acid sequence shown in UniProt accession number G7NXM4.
  • the TIGIT is mouse TIGIT, and the amino acid sequence of mouse TIGIT comprises the amino acid sequence set forth in UniProt Accession No. P86176.
  • the term "PD-L1" generally refers to programmed cell death 1 ligand 1, also known as B7 homolog 1, B7-H1, cluster of differentiation 274, (3)274 or CD274, which is associated with PD-1 binding downregulates T cell activation and cytokine secretion.
  • P-L1 includes any native PD-L1 from any vertebrate source, including mammals, such as primates (eg, humans and cynomolgus monkeys) and rodents (eg, mice and rats) ).
  • the term encompasses "full length", unprocessed PD-L1 as well as any form of PD-L1 produced by cellular processing.
  • PD-L1 can exist as a transmembrane protein or as a soluble protein.
  • "PD-L1" includes intact PD-L1 and fragments thereof, and also includes functional variants, isoforms, species homologues, derivatives, analogs of PD-L1, and functional variants, isoforms, derivatives, and analogs of PD-L1, as well as those having at least one in common with PD-L1.
  • Epitope analogs The basic structure of PD-L1 includes four domains: extracellular Ig-like V-type domain and Ig-like C2-type domain, transmembrane domain and cytoplasmic domain.
  • Exemplary human PD-L1 amino acid sequences can be found under NCBI Accession No. NP_001254653 or UniProt Accession No. Q9NZQ7.
  • antigen-binding protein generally refers to a protein comprising an antigen-binding moiety, and optionally a scaffold or backbone moiety that allows the antigen-binding moiety to adopt a conformation that facilitates the binding of the antigen-binding protein to the antigen.
  • antigen binding proteins include, but are not limited to, antibodies, antigen binding fragments (Fab, Fab', F(ab) 2 , Fv fragments, F(ab') 2 , scFv, di-scFv and/or dAb), immunoconjugation antibodies, multispecific antibodies (eg, bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • Fab antigen binding fragments
  • Fv fragments F(ab') 2
  • scFv di-scFv and/or dAb
  • immunoconjugation antibodies eg, multispecific antibodies (eg, bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • Fab generally refers to a fragment containing the variable domain of the heavy chain and the variable domain of the light chain, and also containing the constant domain of the light chain and the first constant domain (CH1) of the heavy chain
  • Fab' generally refers to a fragment that differs from Fab by adding a small number of residues (including one or more cysteines from the antibody hinge region) to the carboxy terminus of the heavy chain CH1 domain
  • F(ab"') 2 generally refers to a dimer of Fab', an antibody fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region.
  • Fv generally refers to the smallest antibody fragment containing the entire antigen recognition and binding site.
  • the fragment may consist of a heavy chain variable region and a light chain variable region in a tightly non-covalently bound dimer;
  • dsFv generally refers to disulfide-stabilized Fv fragments, The bond between its single light chain variable region and single heavy chain variable region is a disulfide bond.
  • dAb fragment generally refers to antibody fragments consisting of VH domains.
  • scFv generally refers to a monovalent molecule formed by covalently linking and pairing one heavy chain variable domain and one light chain variable domain of an antibody through a flexible peptide linker; such scFv molecules may have a general Structure: NH2 -VL-Linker-VH-COOH or NH2 -VH-Linker-VL-COOH.
  • an “antibody” is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies comprising two light chains and two heavy chains), polyclonal antibodies , multispecific antibodies (eg bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies and camelized single domain antibodies.
  • An “antibody” may generally comprise a protein comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region. In certain naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region comprises three domains, CH1, CH2 and CH3.
  • each light chain comprises a light chain variable region (VL) and a light chain constant region.
  • the light chain constant region contains one domain, CL.
  • the VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which alternate with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL contains three CDRs and four framework regions (FRs), arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • variable domains of native heavy and light chains each comprise four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4) , mostly adopt a ⁇ -sheet configuration, connected by three CDRs, forming loop connections, and in some cases forming part of the ⁇ -sheet structure.
  • the CDRs in each chain are brought together in close proximity by the FR regions and together with the CDRs from the other chain form the antigen-binding site of the antibody.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • variable generally refers to the fact that some portion of the sequence of the variable domains of an antibody varies strongly which contributes to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments in the light and heavy chain variable regions, called complementarity determining regions (CDRs) or hypervariable regions (HVRs). The more highly conserved portion of the variable domain is called the framework (FR).
  • CDRs complementarity determining regions
  • HVRs hypervariable regions
  • the CDRs of antibodies can be defined by a variety of methods, such as the Kabat definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health, Besse Star, Maryland (1991)), Chothia definition rules based on the location of structural loop regions (see, A1-Lazikani et al., JMol Biol 273:927-48, 1997) and concepts based on IMGT Ontology (IMGT-ONTOLOGY) and IMGT Scientific Chart Rules for IMGT Definition Rules.
  • IMGT refers to the International ImMunoGeneTics Information System, a global reference database for immunogenetics and immunoinformatics (http://www.imgt.org). IMGT specializes in immunoglobulins (IG) or antibodies from humans and other vertebrates, T cell receptors (TR), major histocompatibility (MH), and the immunoglobulin superfamily from vertebrates and invertebrates (IgSF), MH superfamily (MhSF), and immune system-related proteins (RPI).
  • IG immunoglobulins
  • TR T cell receptors
  • MH major histocompatibility
  • IgSF immunoglobulin superfamily from vertebrates and invertebrates
  • MhSF MH superfamily
  • RPI immune system-related proteins
  • kabat's antibody coding rules generally refers to a numbering system for amino acid residues in the variable regions of the heavy and light chains of antibodies or antigen-binding portions thereof (Kabat et al. (1971) Ann. NY) Acad. Sci. 190:382-391 and Kabat, E.A. et al. (1991) Sequence of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Human Health Services, NIH Bulletin No. 91-3242).
  • CDR1 is amino acids 31-35 of the heavy chain variable region
  • CDR2 is amino acids 50-65 of the heavy chain variable region
  • CDR3 is the 95th amino acid of the heavy chain variable region - Amino acid at position 102.
  • CDR1 is amino acids 24-34 of the light chain variable region
  • CDR21 is amino acids 50-56 of the light chain variable region
  • isolated antigen binding protein generally refers to an antigen binding protein that has been identified, isolated and/or recovered from components of the environment in which it is produced (eg, native or recombinant). Contaminant components of its producing environment are often substances that interfere with its research, diagnostic or therapeutic use, and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • An isolated antigen binding protein or antibody will generally be prepared by at least one purification step.
  • multispecific antibody generally refers to an antibody having variable regions that recognize more than one epitope on one or more antigens.
  • Multispecific antibodies include, but are not limited to, full-length antibodies, antibodies with two or more VL and VH domains, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, and tribodies Antibodies, antibody fragments that have been covalently or non-covalently linked.
  • a multispecific antibody may be a "bispecific antibody” that recognizes two different epitopes on the same or different antigens.
  • the term "monoclonal antibody” generally refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies in the population are identical except for possible minor natural mutations.
  • Monoclonal antibodies are usually highly specific for a single antigenic site.
  • each monoclonal antibody is directed against a single determinant on the antigen.
  • the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without contamination by other immunoglobulins.
  • the modifier "monoclonal” denotes a characteristic of an antibody obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring the production of the antibody by any particular method.
  • the monoclonal antibodies used herein can be produced in hybridoma cells, or can be produced by recombinant DNA methods.
  • chimeric antibody generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species.
  • the variable regions are derived from antibodies from experimental animals such as rodents ("parental antibodies”), and the constant regions are derived from human antibodies, such that the resulting chimeric antibody is more robust in human subjects than the parental (eg, mouse-derived) antibody Reduced likelihood of triggering an adverse immune response.
  • humanized antibody generally refers to an antibody in which some or all of the amino acids other than the CDR regions of a non-human antibody (eg, a mouse antibody) have been replaced by corresponding amino acids derived from human immunoglobulins. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions are also permissible as long as they still retain the ability of the antibody to bind to a particular antigen.
  • a humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region.
  • a "humanized antibody” retains antigenic specificity similar to the original antibody.
  • “Humanized” forms of non-human (eg, murine) antibodies may minimally comprise chimeric antibodies that contain sequences derived from non-human immunoglobulins.
  • CDR region residues in a human immunoglobulin can be substituted with a non-human species (donor antibody) (such as mouse, rat) having the desired properties, affinity and/or ability , rabbit or non-human primate) CDR region residue replacement.
  • donor antibody such as mouse, rat
  • FR region residues of the human immunoglobulin can be replaced with corresponding non-human residues.
  • humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve antibody properties, such as binding affinity.
  • Fully human antibody generally refers to the antibody expressed by the human antibody gene-encoding gene transferred into a genetically engineered antibody gene-deficient animal. All parts of an antibody, including the variable and constant regions of the antibody, are encoded by genes of human origin. Fully human antibodies can greatly reduce the immune side effects caused by heterologous antibodies to the human body. Methods for obtaining fully human antibodies in the art include phage display technology, transgenic mouse technology, ribosome display technology and RNA-polypeptide technology.
  • binding generally refer to a measurable and reproducible interaction, such as binding between an antigen and an antibody, which can be determined in the presence of a molecule
  • a target in the context of a heterogeneous population (including biological molecules).
  • an antibody binds to an epitope through its antigen binding domain, and this binding requires some complementarity between the antigen binding domain and the epitope.
  • an antibody that specifically binds a target is an antibody that binds to that target with greater affinity, avidity, easier, and/or for a greater duration than it binds to other targets.
  • an antibody is said to "specifically bind” to an antigen when it binds to an epitope more readily through its antigen-binding domain than it would bind to a random, unrelated epitope.
  • Epitope refers to a specific group of atoms (eg, sugar side chains, phosphoryl groups, sulfonyl groups) or amino acids on an antigen to which an antigen binding protein (eg, an antibody) binds.
  • KD KD
  • KD KD
  • KD the equilibrium dissociation constant
  • KD being the dissociation rate constant ( kdis , also known as the “off-rate” ) (koff)” or “kd”
  • kd the dissociation rate constant
  • kon the on-rate constant
  • KD equilibrium dissociation constant
  • association and dissociation rate constants are well known in the art and include, but are not limited to, Biofilm Interferometry (BLI), Radioimmunoassay (RIA), Equilibrium Dialysis, Surface Plasmon Resonance (SPR), Fluorescence Resonance Energy Transfer (FRET) , co-immunoprecipitation (Co-IP) and protein chip technology.
  • BBI Biofilm Interferometry
  • RIA Radioimmunoassay
  • SPR Surface Plasmon Resonance
  • FRET Fluorescence Resonance Energy Transfer
  • Co-IP co-immunoprecipitation
  • the measured affinity for a particular protein-protein interaction may vary if measured under different conditions (eg, salt concentration, pH).
  • reference antibody generally refers to an antibody with which the antigen binding protein described in this application competes for binding to the antigen TIGIT.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • cytotoxic effector cells eg, NK cells , neutrophils, and macrophages
  • FcRs Fc receptors
  • Primary cells that mediate ADCC eg, NK cells
  • monocytes express Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII (see table in Ravetch and Kinet, Annu. Rev. Immunol.
  • in vitro and/or in vivo cytotoxicity assays can be performed to assess ADCC activity of a molecule of interest, eg, in vitro ADCC assays can be performed as described in US Pat. No. 5,500,362 or No. 5,821,337 or US Pat. No. 6,737,056 (Presta) recorded. Useful effector cells for such assays include PBMC and NK cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, eg, in animal models such as disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998). For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks Fc ⁇ R binding (and thus likely lacks ADCC activity), but retains FcRn binding ability.
  • Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks Fc ⁇ R binding (and thus likely lacks ADCC activity), but retains F
  • ADCC activity can be reduced by modifying the Fc region.
  • sites that affect binding to Fc receptors can be removed, eg, to remove sites that are not salvage receptor binding sites.
  • the Fc region can be modified to remove the ADCC site.
  • ADCC sites are known in the art, see eg, Sarmay et al. (1992) Molec. Immunol. 29(5):633-9 for ADCC sites of IgGl.
  • the term "between” generally means that the C-terminus of a certain amino acid fragment is directly or indirectly connected to the N-terminus of the first amino acid fragment, and its N-terminus is directly or indirectly connected to the C-terminus of the second amino acid fragment.
  • indirect connection In the light chain, for example, the N-terminus of the L-FR2 is directly or indirectly linked to the C-terminus of the LCDR1, and the C-terminus of the L-FR2 is directly or indirectly linked to the N-terminus of the LCDR2.
  • the N-terminus of the L-FR3 is directly or indirectly linked to the C-terminus of the LCDR2, and the C-terminus of the L-FR3 is directly or indirectly linked to the N-terminus of the LCDR3.
  • the N-terminus of the H-FR2 is directly or indirectly linked to the C-terminus of the HCDR1
  • the C-terminus of the H-FR2 is directly or indirectly linked to the N-terminus of the HCDR2.
  • the N-terminus of the H-FR3 is directly or indirectly linked to the C-terminus of the HCDR2
  • the C-terminus of the H-FR3 is directly or indirectly linked to the N-terminus of the HCDR3.
  • first amino acid fragment" and "second amino acid fragment” can be any amino acid fragment that is the same or different.
  • isolated antigen binding protein generally refers to an antigen binding protein that has been identified, isolated and/or recovered from components of the environment in which it is produced (eg, native or recombinant). Contaminant components of its producing environment are often substances that interfere with its research, diagnostic or therapeutic use, and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • An isolated antigen binding protein or antibody will generally be prepared by at least one purification step.
  • the isolated antigen binding proteins described herein generally do not bind antigens other than TIGIT antigens.
  • isolated nucleic acid molecule or isolated polynucleotide
  • isolated nucleic acid molecule generally refers to DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, which is not related to the polynucleus found in nature All or a portion of the nucleotides are associated, or linked, to polynucleotides to which they are not linked in nature.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells.
  • the vectors may include vectors primarily for the insertion of DNA or RNA into cells, vectors primarily for replication of DNA or RNA, and vectors primarily for expression of transcription and/or translation of DNA or RNA.
  • the carrier also includes a carrier having a variety of the above-mentioned functions.
  • the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell.
  • the vector can produce the desired expression product by culturing a suitable host cell containing the vector.
  • the term "cell” generally refers to an individual cell, cell line or cell that can or already contains a plasmid or vector comprising a nucleic acid molecule described herein, or that is capable of expressing an antibody or antigen-binding fragment thereof described herein. cell culture.
  • the cells may include progeny of a single host cell. Due to natural, accidental or intentional mutations, the progeny cells may not necessarily be morphologically or genomically identical to the original parental cells, but are capable of expressing the antibodies or antigen-binding fragments thereof described herein.
  • the cells can be obtained by transfecting cells in vitro using the vectors described herein.
  • the cells may be prokaryotic cells (eg E.
  • the cells can be mammalian cells.
  • the mammalian cells can be CHO-K1 cells.
  • the term "recombinant cell” generally refers to a cell into which a recombinant expression vector has been introduced.
  • the recombinant host cells include not only certain specific cells, but also progeny of these cells.
  • the term "pharmaceutically acceptable adjuvant” generally includes pharmaceutically acceptable carriers, excipients or stabilizers which are free of the cells or mammals to which they are exposed at the doses and concentrations employed. poisonous.
  • the physiologically acceptable carrier is a pH buffered aqueous solution.
  • physiologically acceptable carriers can include buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN TM , polyethylene glycol (PEG ) and PLURONICS TM .
  • buffers such as phosphates, citrates, and other organic acids
  • antioxidants including ascorbic acid
  • administer refers to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid.
  • administering can refer to therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of cells includes contact of reagents with cells, as well as contact of reagents with fluids, and contact of fluids with cells.
  • administering and “treating” also mean in vitro and ex vivo treatment by an agent, diagnostic, binding composition, or by another cell.
  • Treatment when applied to a human, animal or research subject, means therapeutic treatment, prophylactic or preventive measures, research and diagnosis; includes TIGIT conjugates with human or animal, subject, cell, tissue, physiological region chamber or contact with physiological fluids.
  • treating refers to the administration of an internal or external therapeutic agent, including any one of the TIGIT antigen binding proteins of the present application and compositions thereof, to a patient having one or more disease symptoms for which the Therapeutic agents have a therapeutic effect on these symptoms.
  • a patient is administered to a patient in an amount of the therapeutic agent effective to alleviate one or more symptoms of the disease (therapeutically effective amount). Desired effects of treatment include a reduction in the rate of disease progression, amelioration or amelioration of the disease state, and regression or improved prognosis.
  • one or more symptoms associated with cancer are alleviated or eliminated, including but not limited to, reducing (or destroying) cancer cell proliferation, reducing disease-derived symptoms, and improving the quality of life of those individuals with the disease , reducing the dosage of other drugs required to treat the disease, delaying the progression of the disease, and/or prolonging the survival of the individual, the individual is successfully "treated”.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the application provides an isolated antigen binding protein, which can comprise at least one CDR in the VH of the heavy chain variable region, and the VH can comprise the amino acid sequence shown in SEQ ID NO:55.
  • the application provides an isolated antigen binding protein, which can comprise at least one CDR in the VH of the heavy chain variable region, and the VH can comprise the amino acid sequence shown in SEQ ID NO:54.
  • the VH of the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3.
  • the antigen binding protein described in the present application may comprise HCDR1, wherein the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 3: GYSITSDYA.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding protein described in the present application may comprise HCDR2, wherein the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 56: IX 2 X 3 SGX 6 X 7 , wherein X 2 is S or T, and X 3 is S or Y, X6 is A or S, and X7 is P or T.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the HCDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 4 and 42.
  • the antigen binding protein described in the present application may comprise HCDR2, wherein the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 4: ITSSGST.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding protein described in the present application may comprise HCDR3, wherein the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 57: AX 2 LX 4 X 5 X 6 X 7 YX 9 X 10 AMDY, wherein X 2 is R or S, X4 is D or G, X5 is F or T, X6 is D or G, X7 is N or Y, X9 is G or absent , and X10 is G or absent.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the HCDR3 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 5 and 43.
  • the antigen binding protein described in the present application may comprise HCDR3, wherein the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 5: ARLDFGNYGGAMDY.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • antigen binding proteins described herein may also comprise the framework regions H-FR1, H-FR2, H-FR3 and H-FR4.
  • the antigen binding protein described in the present application may comprise a framework region H-FR1, wherein the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 58: X 1 VQLQESGPGLVKPSX 16 X 17 LSLTCTVX 25 , wherein X 1 is D or Q, X 16 is E or Q, X 17 is S or T, and X 25 is S or T.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the H-FR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 23 and 35.
  • the antigen binding protein described in the present application may comprise the framework region H-FR1, wherein the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 23: QVQLQESGPGLVKPSETLSLTCTVS.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding protein described in the present application may comprise a framework region H-FR2, wherein the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 60: WX 2 WIRQX 7 PGX 10 X 11 X 12 EWX 15 GY; wherein , X 2 is I or N, X 7 is F or P, X 10 is K or N, X 11 is G, R or K, X 12 is L or V, and X 15 is I or M.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the H-FR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 24-26 and 36-37.
  • the antigen binding protein described in the present application can comprise the framework region H-FR2, wherein the H-FR2 can comprise the amino acid sequence shown in SEQ ID NO: 59: WIWIRQPPGX 10 X 11 LEWIGY; wherein X 10 is K or N, X 11 is G or K.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the H-FR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 24-26.
  • the antigen binding protein described in the present application may comprise a framework region H-FR3, wherein the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 61: X 1 YNPSLKSRX 10 X 11 X 12 X 13 X 14 DTSKNQFX 22 LX 24 LX 26 X 27 VTX 30 X 31 DTATYYC, where X 1 is R, S or Y, X 10 is I or V, X 11 is S or T, X 12 is F or I, and X 13 is S or T, X 14 is R or V, X 22 is F or S, X 24 is K or Q, X 26 is S or T, X 27 is F or S, X 30 is A or T, and X 31 is A or E .
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the H-FR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 27, 38 and 39.
  • the antigen binding protein described herein may comprise a framework region H-FR3, wherein the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 27: YYNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYC.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding protein described in the present application may comprise a framework region H-FR4, wherein the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 62: WGQGTX 6 VX 8 VSS, wherein X 6 is L or S, and X 8 is I or T.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the H-FR4 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 28, 40 and 41.
  • the antigen binding protein described in the present application may comprise the framework region H-FR4, wherein the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 28: WGQGTLVTVSS.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding protein described herein may comprise a heavy chain variable region VH, wherein the VH may comprise the amino acid sequence shown in SEQ ID NO: 55: X 1 VQLQESGPGLVKPSX 16 X 17 LSLTCTVX 25 GYSITSDYAWX 36 WIRQX 41 PGX 44 X 45 X 46 EWX 49 GYIX 53 X 54 SGX 57 X 58 X 59 YNPSLKSRX 68 X 69 X 70 X 71 X 72 DTSKNQFX 80 LX 82 LX 84 X 85 VTX 88 X 89 DTATYYCAX 98 LX 100 X 101 X 102 X 103 YX 06 105 X AMDYWGQGTX 116 VX 118 VSS, where X 1 is D or Q, X 16 is E or Q, X 17 is S or T, X 25 is S or T, X 36 is I or N, X 41 is F or
  • the antigen binding protein can comprise a heavy chain variable region VH, wherein the VH can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 1, 9, 12, 14 and 32.
  • the antigen binding protein described herein may comprise a heavy chain variable region VH, wherein the VH may comprise the amino acid sequence set forth in SEQ ID NO: 54: QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWIWIRQPPGX 44 X 45 LEWIGYITSSGSTYYNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYCARLDFGNYGGAMDYWGQGTLVTVSS , wherein X44 is K or N, and, X 45 is G or K.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding protein can comprise a heavy chain variable region VH, wherein the VH can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 9, 12 and 14.
  • the antigen binding proteins described herein may comprise heavy chain constant regions, which may be derived from human IgG, eg, human IgGl.
  • the Fc region of human IgGl can be modified to achieve desired properties (eg, ADCC KO).
  • the modification may be an amino acid mutation.
  • the modification to IgG1 may be L234A/L235A, ie, amino acids at positions 234 and 235 are mutated from leucine (L) to alanine (A), respectively, according to EU numbering.
  • the heavy chain constant regions of the antigen binding proteins described herein can be derived from wild-type human IgG1.
  • the antigen binding proteins described herein may comprise a heavy chain constant region, which may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 35-36.
  • the isolated antigen-binding protein described in the present application may comprise at least one CDR in the VL of the light chain variable region, and the VL may comprise the amino acid sequence shown in SEQ ID NO:64.
  • the isolated antigen-binding protein described in this application may comprise at least one CDR in the VL of the light chain variable region, and the VL may comprise the amino acid sequence shown in SEQ ID NO:63.
  • the VL of the antigen binding protein may comprise LCDR1, LCDR2 and LCDR3.
  • the antigen binding protein described in the present application may comprise LCDR1, wherein the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 65: QHVSX 5 A, wherein X 5 is N or T.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the LCDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 6 and 52.
  • the antigen binding protein described in the present application may comprise LCDR1, wherein the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:6: QHVSTA.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding protein described in the present application may comprise LCDR2, wherein the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 7: SAS.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding protein described in the present application can comprise LCDR3, wherein the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 66: QQX 3 YX 5 X 6 PX 8 T, wherein X 3 is H or Y, X 5 is I or S, X6 is L or T, and X8 is W or Y.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the LCDR3 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 8 and 53.
  • the antigen binding protein described in the present application may comprise LCDR3, wherein the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 8: QQHYITPYT.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding proteins described herein may also comprise framework regions L-FR1, L-FR2, L-FR3 and L-FR4.
  • the antigen binding protein described in the present application may comprise a framework region L-FR1, wherein the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 68: DIX 3 MTQSX 8 X 9 X 10 X 11 X 12 X 13 SX 15 GDRVX 20 ITCX 24 AS; wherein X 3 is Q or V, X 8 is H or P, X 9 is K or S, X 10 is F or S, X 11 is L or M, and X 12 is F or S , X 13 is A or T, X 15 is I or V, X 20 is S or T, and X 24 is K or R.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the L-FR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 15, 16, 44 and 45.
  • the antigen binding protein described in the present application may comprise a framework region L-FR1, wherein the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 67: DIQMTQSPSSLSASVGDRVTITCX 24 AS; wherein X 24 is K or R.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the L-FR1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 15 and 16.
  • the antigen binding protein described in the present application may comprise a framework region L-FR2, wherein the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 70: X 1 X 2 WYQQKPGX 10 X 11 PKLLIX 17 ; wherein X 1 is L or V, X 2 is A or N, X 10 is K or Q, X 11 is A or S, and X 17 is H or Y.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the L-FR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 17, 18, 46 and 47.
  • the antigen binding protein described in the present application may comprise a framework region L-FR2, wherein the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 69: X 1 X 2 WYQQKPGKAPKLLIY; wherein X 1 is L or V, and X 2 is A or N.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the L-FR2 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 17 and 18.
  • the antigen binding protein described herein may comprise a framework region L-FR3, wherein the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 72: YX 2 X 3 X 4 GVPX8RFX 11 GX 13 X 14 SGTDFTX 21 TIX 24 SX 26 QX 28 EDX 31 AX 33 YYC; where X 2 is L or R, X 3 is Q or Y, X 4 is S or T, X 8 is D or S, and X 11 is I, S or T, X13 is R or S, X14 is G or R, X21 is F or L, X24 is N or S, X26 is L or V, X28 is A or P, X33 is F or L, and X 14 is T or V.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the L-FR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 19-21 and 48-50.
  • the antigen-binding protein described in the present application may comprise a framework region L-FR3, wherein the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 71: YX 2 X 3 SGDPSRFSGSX 14 SGTDFTLTISSLQPEDFATYYC; wherein X 2 is L or R, X3 is Q or Y, and X14 is G or R.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the L-FR3 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 19-21.
  • the antigen binding protein described in the present application may comprise a framework region L-FR4, wherein the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 73: FGX 3 GTKLEIK, wherein X 3 is G or Q.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the L-FR4 can include the amino acid sequence set forth in any one of SEQ ID NOs: 22 and 51.
  • the antigen binding protein described in the present application may comprise the framework region L-FR4, wherein the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 22: FGQGTKLEIK.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding protein described in the present application may comprise a light chain variable region VL, wherein the VL may comprise the amino acid sequence shown in SEQ ID NO: 64: DIX 3 MTQSX 8 X 9 X 10 X 11 X 12 X1 3 SX 15 GDRVX 20 ITCX 24 ASQHVSX 31 AX 33 X 34 WYQQKPGX 42 X 43 PKLLIX 49 SASYX 54 X 55 X 56 GVPX 60 RFX 63 GX 65 X 66 SGTDFTX 73 TIX 76 SX 78 QX 80 EDX 83 AX 85 YYCQQX 91 YX 93 TFGX 100 GTKLEIK, wherein X3 is Q or V, X8 is H or P, X9 is K or S, X10 is F or S, and X11 is L or M, X12 is F or S, X 13 is A or T, X 15 is I or V, X 20
  • the antigen binding protein can comprise a light chain variable region VL, wherein the VL can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 2, 10, 11, 13, 33 and 34.
  • the antigen binding protein described in the present application may comprise a light chain variable region VL, wherein the VL may comprise the amino acid sequence shown in SEQ ID NO: 63: DIQMTQSPSSLSASVGDRVTITCX 24 ASQHVSTAX 33 X 34 WYQQKPGKAPKLLIYSASYX 54 X 55 SGVPSRFSGSX 66 SGTDFTLTISSLQPEDFATYYCQQHYITPYTFGQGTKLEIK, wherein, X 24 is K or R, X 33 is L or V, X 34 is A or N, X 54 is L or R, X 55 is Q or Y, and X 66 is G or R.
  • the sequence may be a sequence determined according to IMGT definition rules.
  • the antigen binding protein can comprise a light chain variable region VL, wherein the VL can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 10, 11 and 13.
  • the antigen binding proteins described herein may comprise a light chain constant region, which may comprise human light chain constant region sequences, eg, may comprise a human kappa light chain constant region.
  • the light chain constant region of the isolated antigen binding proteins described herein may comprise the amino acid sequence set forth in SEQ ID NO:31.
  • the isolated antigen binding protein may comprise antibody heavy chain variable region CDRs - HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 3, and the HCDR2 may comprise The amino acid sequence shown in SEQ ID NO:56, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:57.
  • the isolated antigen binding protein may comprise the antibody heavy chain variable region CDRs - HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 3, the HCDR2 The amino acid sequence set forth in any one of SEQ ID NOs: 4 and 42 may be included, and the HCDR3 may include the amino acid sequence set forth in any one of SEQ ID NOs: 5 and 43.
  • the isolated antigen binding protein may comprise antibody heavy chain variable region CDRs - HCDR1, HCDR2 and HCDR3, and the HCDR1, the HCDR2 and the HCDR3 may respectively comprise SEQ ID NO: 3, The amino acid sequences shown in SEQ ID NO:4 and SEQ ID NO:5.
  • the isolated antigen binding protein may comprise antibody light chain variable region CDRs---LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 65, and the LCDR2 may comprise The amino acid sequence shown in SEQ ID NO:7, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:66.
  • the isolated antigen-binding protein may comprise antibody light chain variable region CDRs - LCDR1, LCDR2 and LCDR3, and the LCDR1 may comprise any one of SEQ ID NOs: 6 and 52.
  • the amino acid sequence, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 7, and the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 8 and 53.
  • the isolated antigen binding protein may comprise antibody light chain variable region CDRs---LCDR1, LCDR2 and LCDR3, and the LCDR1, the LCDR2 and the LCDR3 may respectively comprise SEQ ID NO: 6, The amino acid sequences shown in SEQ ID NO:7 and SEQ ID NO:8.
  • the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 3, and the HCDR2 may comprise SEQ ID NO The amino acid sequence shown in: 56, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 57, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 65, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 7, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 66.
  • the isolated antigen binding protein can comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 can comprise the amino acid sequence shown in SEQ ID NO: 3, and the HCDR2 can comprise SEQ ID NO: 3
  • the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 5 and 43
  • the LCDR1 may comprise SEQ ID NOs: 6 and 52
  • the amino acid sequence shown in any one, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 7, and the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 8 and 53.
  • the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3
  • SEQ ID NO: 3 The amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 can be respectively included in sequence.
  • the isolated antigen binding proteins described herein can comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and The LCDR3 can respectively comprise the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:52, SEQ ID NO:7 and SEQ ID NO:53 in sequence.
  • the isolated antigen binding protein may comprise a heavy chain variable region VH and a light chain variable region VL
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 55
  • the VL may comprise The amino acid sequence shown in SEQ ID NO:64.
  • the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL
  • the VH can comprise SEQ ID NO: 110, SEQ ID NO: 1, SEQ ID NO: 9.
  • the amino acid sequence shown in any one of SEQ ID NO: 12, SEQ ID NO: 32 and SEQ ID NO: 14, and the VL may comprise SEQ ID NO: 111, SEQ ID NO: 2, SEQ ID NO: : the amino acid sequence shown in any one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 13.
  • the isolated antigen binding protein may comprise a heavy chain variable region VH and a light chain variable region VL
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 54
  • the VL may comprise The amino acid sequence shown in SEQ ID NO:63.
  • the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL
  • the VH can comprise SEQ ID NO: 110, SEQ ID NO: 9, SEQ ID NO: 12 and the amino acid sequence shown in any one of SEQ ID NO: 14, and the VL may comprise any one of SEQ ID NO: 111, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 13 amino acid sequence shown.
  • the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL
  • the VH can comprise the amino acid sequence set forth in SEQ ID NO:9
  • the VL can comprise SEQ ID NO: : the amino acid sequence shown in 10.
  • the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL
  • the VH can comprise the amino acid sequence set forth in SEQ ID NO:9
  • the VL can comprise SEQ ID NO: : the amino acid sequence shown in 11.
  • the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL
  • the VH can comprise the amino acid sequence set forth in SEQ ID NO: 12
  • the VL can comprise SEQ ID NO: : the amino acid sequence shown in 13.
  • the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL
  • the VH can comprise the amino acid sequence set forth in SEQ ID NO: 14
  • the VL can comprise SEQ ID NO: : the amino acid sequence shown in 11.
  • the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL
  • the VH can comprise the amino acid sequence set forth in SEQ ID NO: 1
  • the VL can comprise SEQ ID NO: : the amino acid sequence shown in 2.
  • the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL
  • the VH can comprise the amino acid sequence set forth in SEQ ID NO: 32
  • the VL can comprise SEQ ID NO: : amino acid sequence shown in 34.
  • the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL
  • the VH can comprise the amino acid sequence set forth in SEQ ID NO: 32
  • the VL can comprise SEQ ID NO: : amino acid sequence shown in 33.
  • the isolated antigen binding protein described herein may further comprise a heavy chain constant region, which may be derived from a human IgG1 constant region, and a light chain constant region, which may be derived from a human kappa light chain constant region .
  • the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL, the VH can comprise the amino acid sequence set forth in SEQ ID NO:9, and the VL can comprise SEQ ID NO: The amino acid sequence shown in: 10; and, the isolated antigen-binding protein may comprise a heavy chain constant region and a light chain constant region, and the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 29, and the The light chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 31, and the isolated antigen binding protein may be referred to as HB0030.
  • the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL, the VH can comprise the amino acid sequence set forth in SEQ ID NO:9, and the VL can comprise SEQ ID NO: The amino acid sequence shown in: 11; and, the isolated antigen-binding protein may comprise a heavy chain constant region and a light chain constant region, the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 29, and the The light chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 31.
  • the isolated antigen binding protein may be referred to as HB0031.
  • the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL
  • the VH can comprise the amino acid sequence set forth in SEQ ID NO: 12
  • the VL can comprise SEQ ID NO: The amino acid sequence shown in: 13
  • the isolated antigen-binding protein may comprise a heavy chain constant region and a light chain constant region
  • the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 29
  • the light chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 31, and the isolated antigen binding protein may be referred to as HB0032.
  • the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL, the VH can comprise the amino acid sequence set forth in SEQ ID NO: 14, and the VL can comprise SEQ ID NO: The amino acid sequence shown in: 11; and, the isolated antigen-binding protein may comprise a heavy chain constant region and a light chain constant region, the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 29, and the The light chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 31, and the isolated antigen binding protein may be referred to as HB0033.
  • the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL, the VH can comprise the amino acid sequence set forth in SEQ ID NO: 1, and the VL can comprise SEQ ID NO: The amino acid sequence shown in: 2; and, the isolated antigen-binding protein may comprise a heavy chain constant region and a light chain constant region, and the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 30, and the The light chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 31.
  • the isolated antigen binding protein may be referred to as 900424.
  • the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL
  • the VH can comprise the amino acid sequence set forth in SEQ ID NO: 32
  • the VL can comprise SEQ ID NO:
  • the isolated antigen-binding protein may comprise a heavy chain constant region and a light chain constant region
  • the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 30
  • the The light chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 31, and the isolated antigen binding protein may be referred to as 900423.
  • the isolated antigen binding protein can comprise a heavy chain variable region VH and a light chain variable region VL
  • the VH can comprise the amino acid sequence set forth in SEQ ID NO: 32
  • the VL can comprise SEQ ID NO: The amino acid sequence shown in: 33
  • the isolated antigen-binding protein may comprise a heavy chain constant region and a light chain constant region
  • the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 30
  • the light chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 31, and the isolated antigen binding protein may be referred to as 900428.
  • the isolated antigen-binding protein described in this application can compete with a reference antibody for binding to the TIGIT protein, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, and the reference antibody has a heavy chain variable region and a light chain variable region.
  • the chain variable region may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 56; the HCDR3 may comprise SEQ ID NO: 56
  • the amino acid sequence shown in ID NO: 57 the light chain variable region of the reference antibody can comprise LCDR1, LCDR2 and LCDR3, the LCDR1 can comprise the amino acid sequence shown in SEQ ID NO: 65; the LCDR2 can comprise The amino acid sequence shown in SEQ ID NO:7; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:66.
  • the isolated antigen-binding protein described in this application can compete with a reference antibody for binding to the TIGIT protein, wherein the reference antibody may comprise a heavy chain variable region and a light chain variable region, and the reference antibody has a heavy chain variable region and a light chain variable region.
  • the chain variable region may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 4; the HCDR3 may comprise SEQ ID NO: 4
  • the amino acid sequence shown in ID NO: 5 the light chain variable region of the reference antibody can comprise LCDR1, LCDR2 and LCDR3, the LCDR1 can comprise the amino acid sequence shown in SEQ ID NO: 6; the LCDR2 can comprise The amino acid sequence shown in SEQ ID NO:7; the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:8.
  • the protein, polypeptide and/or amino acid sequence involved in this application should also be understood to include at least the following scope: variants or homologues with the same or similar functions as the protein or polypeptide.
  • the variant may be one in which one or more amino acids have been substituted, deleted, or added to the amino acid sequence of the protein and/or the polypeptide (eg, the antigen-binding protein described herein).
  • protein or peptide may comprise at least 1, such as 1-30, 1-20, or 1-10, and for example, 1, 2, 3, 4, or 5 amino acid substitutions that have been made , a protein or polypeptide with amino acid changes, deletions and/or insertions.
  • the functional variant may substantially retain the biological properties of the protein or the polypeptide prior to alteration (eg, substitution, deletion or addition).
  • the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding capacity) of the protein or polypeptide prior to alteration.
  • the substitutions can be conservative substitutions.
  • a part of the amino acid sequence of the antigen binding protein may be homologous to the corresponding amino acid sequence in an antibody from a specific species, or belong to a specific class.
  • both the variable and constant portions of an antibody can be derived from the variable and constant regions of an antibody of an animal species (eg, human).
  • the homologue may be at least about 85% (eg, having at least about 85%) the amino acid sequence of the protein and/or the polypeptide (eg, the antigen binding protein described herein). %, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology protein or polypeptide.
  • the homology generally refers to the similarity, similarity or relatedness between two or more sequences.
  • Perfect sequence homology can be calculated by comparing the two sequences to be aligned in a comparison window to determine the presence of identical nucleic acid bases (eg, A, T, C, G) in the two sequences or position of the same amino acid residue (eg, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) To obtain the number of matched positions, divide the number of matched positions by the total number of positions in the comparison window (ie, the window size), and multiply the result by 100 to yield the percent sequence homology.
  • Alignment to determine percent sequence homology can be accomplished in a variety of ways known in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length sequences being compared or within the region of the sequence of interest. The homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in W.R. Pearson and D.J. Lipman, "Improved Tools for Biological Sequence Comparison", Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D.J.
  • Antigen binding proteins eg, TIGIT antibodies
  • Antigen-binding proteins eg, antibodies
  • Antigen-binding proteins can typically bind TIGIT with a KD value of about 1 nM or higher affinity (eg, 1 nM, 100 pM, 10 pM, 2 pM, or 1 pM), but not sequences lacking TIGIT of other proteins.
  • an antibody that "specifically binds" TIGIT does not bind human CD226, human CD155, and human CD112.
  • the antigen binding proteins (eg, antibodies) described herein are capable of specifically binding TIGIT antigens or labeled forms thereof (eg, fluorescently labeled TIGIT antigens), but not other proteins that lack TIGIT epitopes. Whether an antigen binding protein (eg, an antibody) binds a TIGIT antigen can be determined using any assay known in the art. Examples of assays known in the art to determine binding affinity include surface plasmon resonance (eg, BIACORE) or similar techniques (eg, KinExa or OCTET). In certain instances, the TIGIT antibodies described herein can also cross-react with monkey and/or murine TIGIT. For example, detected by flow cytometry and ELISA. As used herein, "cross-reactivity" refers to the ability of an antibody to react with homologous proteins from other species.
  • Antigen binding proteins eg, TIGIT antibodies
  • Blocking assays can be detected using competitive methods, for example, combining the antigen-binding protein (eg, TIGIT antibody) with the antigen (or, cells that can express the antigen) and the ligand of the antigen (or, cells that express the ligand) Mixing, the ability of the antigen-binding protein to compete with the antigen's ligand for binding to the antigen is reflected in the intensity (eg, fluorescence intensity or concentration) of the detectable label.
  • the intensity eg, fluorescence intensity or concentration
  • antigen binding proteins eg, TIGIT antibodies
  • TIGIT antibodies have IC50s of about 0.1 ⁇ g/ml-0.05 ⁇ g/ml for blocking TIGIT antigen binding to CD155 ligand, as detected using flow cytometry.
  • the application provides a multispecific antibody that can comprise a first targeting moiety capable of specifically binding to a TIGIT protein.
  • the first targeting portion of the multispecific antibody may comprise the isolated antigen binding protein described above in the present application.
  • the multispecific antibody may also include a second targeting moiety.
  • the second targeting moiety is capable of specifically binding a tumor-associated antigen.
  • the second targeting moiety can block the interaction of PD-L1 and PD-1.
  • the second targeting moiety can specifically bind to PD-L1 protein.
  • the second targeting moiety capable of binding to PD-L1 protein comprises an antibody or an antigen-binding fragment thereof.
  • the antigen-binding fragment capable of binding PD-L1 protein includes Fab, Fab', Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
  • the antibody capable of binding to PD-L1 protein is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies and fully human antibodies.
  • the second targeting moiety of the multispecific antibody may comprise an antibody or antigen-binding fragment thereof capable of specifically binding to PD-L1 protein.
  • the antibody capable of binding PD-L1 protein may comprise HCDR3, for example, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 102.
  • the antibody capable of binding PD-L1 protein may comprise HCDR2, for example, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 101.
  • the antibody capable of binding PD-L1 protein may comprise HCDR1, for example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 100.
  • the antibody capable of binding PD-L1 protein may comprise HCDR1, HCDR2 and HCDR3, for example, the HCDR1, HCDR2 and HCDR3 may comprise the amino acid sequences shown in SEQ ID NOs: 100, 101 and 102, respectively.
  • the antibody capable of binding PD-L1 protein may comprise VH, for example, the VH may comprise the amino acid sequence shown in SEQ ID NO: 106 or 108.
  • the antibody capable of binding PD-L1 protein may comprise LCDR3, for example, the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 105.
  • the antibody capable of binding PD-L1 protein may comprise LCDR2, for example, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 104.
  • the antibody capable of binding PD-L1 protein may comprise LCDR1, for example, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 103.
  • the antibody capable of binding to the PD-L1 protein may comprise LCDR1, LCDR2 and LCDR3, for example, the LCDR1, LCDR2 and LCDR3 may comprise the amino acid sequences shown in SEQ ID NOs: 103, 104 and 105, respectively.
  • the antibody capable of binding PD-L1 protein may comprise VL, for example, the VL may comprise the amino acid sequence shown in SEQ ID NO: 107 or 109.
  • the multispecific antibody may comprise a first polypeptide chain and a second polypeptide chain.
  • the first polypeptide chain may comprise the heavy chain variable region VH of the antibody capable of binding PD-L1 protein, the heavy chain variable region VH capable of binding TIGIT protein, and the heavy chain variable region VH capable of binding to the PD-L1 protein.
  • the light chain variable region VL of the TIGIT protein; and the second polypeptide chain may comprise the light chain variable region VL of an antibody capable of binding the PD-L1 protein.
  • the VH of the antibody capable of binding TIGIT protein and the VL of the antibody capable of binding TIGIT protein in the first polypeptide chain may constitute a scFv.
  • the VH of the antibody capable of binding TIGIT protein and the VL of the antibody capable of binding TIGIT protein may constitute a scFv through a linking peptide.
  • the linker peptide can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 74-77.
  • the sequence of the scFv capable of binding the TIGIT protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 97-99.
  • the scFv capable of binding the TIGIT protein may be located at the N-terminus or the C-terminus of the VH of the antibody capable of binding the PD-L1 protein.
  • the N-terminus of the VL of the antibody capable of binding the TIGIT protein can be optionally linked to the C-terminus of the VH of the antibody capable of binding the PD-L1 protein through a linking peptide
  • the C-terminus of the VL capable of binding the TIGIT protein may be linked to the N-terminus of the VH of the antibody capable of binding the TIGIT protein, optionally via a linking peptide.
  • the N-terminus of the VH capable of binding the TIGIT protein can be optionally linked to the C-terminus of the VH of the antibody capable of binding the PD-L1 protein through a linking peptide, so The C-terminus of the VH capable of binding the TIGIT protein may be optionally linked to the N-terminus of the VL of the antibody capable of binding the TIGIT protein through a linking peptide.
  • the first polypeptide chain may further comprise a constant region, such as a human IgG constant region, and the human IgG constant region may be located at the C-terminus of the VH of the antibody capable of binding the PD-L1 protein and located at the The N-terminus of the scFv of the antibody capable of binding the TIGIT protein.
  • the human IgG constant region may be located at the C-terminus of the VH of the antibody capable of binding the PD-L1 protein and at the N-terminus of the VL of the antibody capable of binding the TIGIT protein.
  • the human IgG constant region can be linked directly or indirectly to the N-terminus of the VL of the antibody capable of binding the TIGIT protein, eg, by a linking peptide.
  • the human IgG constant region can be located C-terminal to the VH of the antibody capable of binding the PD-L1 protein and N-terminal to the VH of the antibody capable of binding the TIGIT protein.
  • the constant region may comprise the amino acid sequence set forth in SEQ ID NO: 29 or 30.
  • the human IgG constant region may be linked directly or indirectly to the N-terminus of the VH of the antibody capable of binding the TIGIT protein, eg, by a linking peptide.
  • the linker peptide can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 74-77.
  • the first polypeptide chain can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 85-87.
  • the first polypeptide chain may comprise the heavy chain variable region VH of the antibody capable of binding PD-L1 protein; and the second polypeptide chain may comprise the PD-L1 protein capable of binding The light chain variable region VL of the antibody, the heavy chain variable region VH capable of binding TIGIT protein and the light chain variable region VL capable of binding TIGIT protein.
  • the VH of the antibody capable of binding the TIGIT protein in the second polypeptide chain may form a scFv with the VL of the antibody capable of binding the TIGIT protein. In some cases, the VH of the antibody capable of binding the TIGIT protein may form a scFv with the VL of the antibody capable of binding the TIGIT protein through a linker peptide.
  • the scFv capable of binding the TIGIT protein is located at the N-terminus or C-terminus of the VL of the antibody capable of binding the PD-L1 protein.
  • the first polypeptide chain comprises the heavy chain variable region VH of the antibody capable of binding TIGIT protein, the heavy chain variable region VH capable of binding PD-L1 protein, and the heavy chain variable region VH capable of binding PD-L1 protein.
  • the light chain variable region VL of the L1 protein; and the second polypeptide chain comprises the light chain variable region VL of an antibody capable of binding the TIGIT protein.
  • the VH of the antibody capable of binding PD-L1 protein and the VL of the antibody capable of binding PD-L1 protein in the first polypeptide chain constitute a scFv.
  • the VH of the antibody capable of binding the PD-L1 protein and the VL of the antibody capable of binding the PD-L1 protein constitute an scFv through a linking peptide.
  • the linker peptide may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 74-77.
  • the sequence of the scFv capable of binding PD-L1 protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 89-96.
  • the scFv capable of binding the PD-L1 protein is located at the N-terminus or C-terminus of the VH of the antibody capable of binding the TIGIT protein.
  • the N-terminus of the VL capable of binding the PD-L1 protein and the C-terminus of the VH of the antibody capable of binding the TIGIT protein are optionally linked by a linking peptide, which is capable of binding to the TIGIT protein.
  • the C-terminus of the VL binding to the PD-L1 protein and the N-terminus of the VH of the antibody capable of binding the PD-L1 protein are optionally linked by a linking peptide.
  • the N-terminus of the VH capable of binding the PD-L1 protein may be optionally linked to the C-terminus of the VH of the antibody capable of binding the TIGIT protein through a linking peptide, so The C-terminus of the VH capable of binding the PD-L1 protein may be optionally linked to the N-terminus of the VL of the antibody capable of binding the PD-L1 protein through a linking peptide.
  • the first polypeptide chain may further comprise a human IgG constant region, and the human IgG constant region may be located at the C-terminus of the VH of the antibody capable of binding TIGIT protein and at the TIGIT protein binding The N-terminus of the antibody scFv.
  • the human IgG constant region may be located at the C-terminus of the VH of the antibody capable of binding the TIGIT protein and at the N-terminus of the VL of the antibody capable of binding the PD-L1 protein.
  • the constant region may comprise the amino acid sequence set forth in SEQ ID NO: 29 or 30.
  • the human IgG constant region can be linked directly or indirectly to the N-terminus of the VL of the antibody capable of binding the PD-L1 protein, eg, by a linking peptide.
  • the human IgG constant region can be located C-terminal to the VH of the antibody capable of binding the TIGIT protein and N-terminal to the VH of the antibody capable of binding the PD-L1 protein.
  • the human IgG constant region may be linked directly or indirectly to the N-terminus of the VH of the antibody capable of binding the PD-L1 protein, eg, by a linking peptide.
  • the linker peptide can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 74-77.
  • the first polypeptide chain can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 78-83.
  • the first polypeptide chain may comprise the heavy chain variable region VH of the antibody capable of binding the TIGIT protein; and the second polypeptide chain may comprise the light weight of the antibody capable of binding the TIGIT protein chain variable region VL, the heavy chain variable region VH capable of binding PD-L1 protein and the light chain variable region VL capable of binding PD-L1 protein.
  • the VH of the antibody capable of binding PD-L1 protein in the second polypeptide chain may form a scFv with the VL of the antibody capable of binding PD-L1 protein. In some cases, the VH of the antibody capable of binding PD-L1 protein may form an scFv with the VL of the antibody capable of binding PD-L1 protein through a linker.
  • the scFv capable of binding the PD-L1 protein is located at the N-terminus or C-terminus of the VL of the antibody capable of binding the TIGIT protein.
  • the VH of the antibody capable of binding TIGIT protein (or PD-L1 protein) and the VL of the antibody capable of binding TIGIT protein (or PD-L1 protein) can be modified to facilitate the formation of scFv, such as , amino acid mutations can occur, but do not affect the specific binding and binding affinity of the antibody.
  • amino acid mutations can be made to form disulfide bonds, so that the structure of the scFv is more stable.
  • Said amino acid mutation may be those common in the art, for example, amino acid mutation in the framework region, another example, amino acid Q at position 100 of the light chain variable region is mutated to C, or amino acid R at position 44 of the heavy chain variable region or G is mutated to C.
  • the multispecific antibody may comprise the structure shown in Figure 7A, wherein the first polypeptide chain may comprise the amino acid sequence shown in any one of SEQ ID NOs: 78-83, And the second polypeptide chain can comprise the amino acid sequence shown in SEQ ID NO:84.
  • the multispecific antibody may comprise the structure shown in Figure 7B, wherein the first polypeptide chain may comprise the amino acid sequence shown in any one of SEQ ID NOs: 85-87, And the second polypeptide chain can comprise the amino acid sequence shown in SEQ ID NO:88.
  • the application also provides isolated one or more nucleic acid molecules.
  • the one or more nucleic acid molecules can encode the multispecific antibodies described herein.
  • each of the one or more nucleic acid molecules can encode the entire multispecific antibody, or a portion thereof (eg, a first targeting moiety, a second targeting moiety, one or more of HCDR1-3, LCDR1-3, VL, VH, light chain or heavy chain).
  • the nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (i) amplified in vitro, for example by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified either (iv) synthetic, eg by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
  • nucleic acids encoding the multispecific antibodies can be prepared by a variety of methods known in the art, including, but not limited to, using restriction fragment manipulation or using overlapping synthetic oligonucleotides For extension PCR, see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausube et al. Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York N.Y., 1993.
  • the application provides one or more vectors comprising one or more nucleic acid molecules described herein.
  • One or more of the nucleic acid molecules may be included in each vector.
  • other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that allow the correct expression of the coding region in an appropriate host.
  • control elements are well known to those of skill in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like.
  • the expression control sequence is a tunable element.
  • the specific structure of the expression control sequence may vary depending on species or cell type function, but typically comprises 5' untranslated and 5' and 3' untranslated sequences involved in transcription and translation initiation, respectively, such as the TATA box, plus Cap sequences, CAAT sequences, etc.
  • a 5' non-transcribed expression control sequence may comprise a promoter region, which may comprise a promoter sequence for transcriptional control of a functionally linked nucleic acid.
  • the expression control sequences may also include enhancer sequences or upstream activator sequences.
  • suitable promoters may include, for example, the promoters for SP6, T3 and T7 polymerases, the human U6 RNA promoter, the CMV promoter, and artificial hybrid promoters thereof (such as CMV), wherein the promoter's A portion may be fused to a portion of the gene promoter for other cellular proteins (eg, human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), which may or may not contain additional introns.
  • One or more nucleic acid molecules described herein can be operably linked to the expression control element.
  • the vector may include, for example, a plasmid, cosmid, virus, phage or other vectors commonly used, for example, in genetic engineering.
  • the vector is an expression vector.
  • the application provides host cells that may comprise one or more nucleic acid molecules described herein and/or one or more vectors described herein.
  • each or each host cell may comprise one or one nucleic acid molecule or vector described herein.
  • each or each host cell may comprise a plurality (eg, 2 or more) or more (eg, 2 or more) of the nucleic acid molecules or vectors described herein.
  • the vectors described herein can be introduced into such host cells, eg, eukaryotic cells, such as cells from plants, fungi or yeast cells, and the like.
  • the vectors described herein can be introduced into the host cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, and the like.
  • the present application provides methods of making the multispecific antibodies.
  • the method may comprise culturing the host cell described herein under conditions such that the multispecific antibody is expressed.
  • these methods can be understood by those of ordinary skill in the art by using an appropriate medium, appropriate temperature and incubation time, and the like.
  • Example 1 Exemplary methods of producing the anti-human TIGIT antibodies of the present application are described in Example 1.
  • Humanized antibodies can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA and IgE.
  • the antibody is an IgG antibody, and the IgG1 subtype is used. Optimization of the necessary constant domain sequences to produce the desired biological activity can be achieved by screening antibodies using the biological assays described in the Examples below.
  • any type of light chain can be used in the compounds and methods herein. In particular, kappa, lambda chains or variants thereof are useful in the compounds and methods of the present application.
  • Example 2 Exemplary methods of humanizing the anti-human TIGIT antibodies of the present application are described in Example 2.
  • sequences of the DNA molecules of the multispecific antibodies or fragments thereof of the present application can be obtained by conventional techniques, such as PCR amplification or genomic library screening.
  • the coding sequences for the light and heavy chains can be fused together to form single chain antibodies.
  • recombinant methods can be used to obtain the relevant sequences in bulk. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
  • nucleic acid molecule can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • the present application also relates to vectors comprising suitable nucleic acid molecules as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
  • animal cells can include (but are not limited to): CHO-S, CHO-K1, HEK-293 cells.
  • the steps of transforming host cells with recombinant DNA described in this application can be performed using techniques well known in the art.
  • the obtained transformants can be cultured by conventional methods, and the transformants express the polypeptides encoded by the nucleic acid molecules of the present application. Depending on the host cell used, it is cultured with conventional media under appropriate conditions. Typically, the transformed host cells are cultured under conditions suitable for expression of the multispecific antibodies of the present application.
  • the multispecific antibody of the present application can be obtained by conventional separation and purification methods well known to those skilled in the art.
  • the resulting monoclonal or multispecific antibodies can be identified by conventional means.
  • the binding specificity of monoclonal or multispecific antibodies can be determined by immunoprecipitation or in vitro binding assays such as flow cytometric sorting (FACS), radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA) Determination.
  • FACS flow cytometric sorting
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the present application also provides a composition.
  • the composition may be a pharmaceutical composition comprising the multispecific antibody of the present application, together with a pharmaceutically acceptable carrier.
  • these materials can be formulated in nontoxic, inert and pharmaceutically acceptable aqueous carrier media.
  • the formulated pharmaceutical compositions can be administered by conventional routes.
  • the pharmaceutical composition described in this application can be directly used to bind TIGIT protein molecules or PD-L1 protein molecules, and thus can be used to prevent and treat TIGIT-related, PD-L1-related or PD-1-related diseases.
  • the pharmaceutical composition of the present application may contain a safe and effective amount of the antigen-binding protein described in the present application and a pharmaceutically acceptable adjuvant (which may include a carrier or excipient).
  • the drug formulation should match the mode of administration.
  • the multispecific antibodies or pharmaceutical compositions described herein can be formulated, administered and administered in a manner consistent with good medical practice. Considerations in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to medical practitioners .
  • the present application provides the use of the multispecific antibody, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition in the preparation of a medicament.
  • the medicament is used to prevent, alleviate and/or treat a disease or disorder, such as a disease associated with abnormal TIGIT.
  • a disease or disorder such as a disease associated with abnormal TIGIT.
  • the disorder associated with abnormal TIGIT may be a T cell dysfunctional disorder.
  • T cell dysfunction is reflected in T cell depletion, which is to achieve treatment or delay or remission of disease by enhancing NK cells and activating T cells to enhance the body's immune activity.
  • the TIGIT-related disease can be a tumor, cancer, or an infectious disorder.
  • the disease associated with abnormal TIGIT can be CD155-positive or PVR-positive tumor, cancer, immune disease or infectious disorder, including tumor, cancer, immune disease or infectious disorder and the like.
  • the tumor can be a solid tumor or a non-solid tumor.
  • the tumor can be colon cancer.
  • the antigen binding proteins of the present application can inhibit tumor growth and/or inhibit tumor cell proliferation.
  • the tumor comprises colon cancer.
  • the tumor or cancer is a tumor or cancer with abnormal expression of TIGIT.
  • the antigen binding proteins described herein are able to slow tumor growth.
  • the present application also provides a method for inhibiting the binding of PD-L1 to PD-1, the method comprising administering the multispecific antibody.
  • ELISA and FACS methods were used to screen out polyclonal cell lines that specifically bind to human TIGIT and can block the binding of TIGIT-CD155, and were then monocloned. ELISA and FACS methods were used to screen specificity again. The combined monoclonal cell line was tested, and the binding ability to monkey and mouse TIGIT was detected. The monoclonal antibody that blocked the binding of TIGIT-CD155 and TIGIT-CD122 was screened by FACS. The monoclonal cell line was screened by affinity (Biacore), and finally a monoclonal hybridoma cell line expressing human TIGIT antibody was obtained for sequence analysis. The screening data are listed in Table 1.
  • the final monoclonal hybridoma cell line obtained has high affinity, can bind to both human and monkey, has blocking function for the two ligands CD155 and CD122 of TIGIT, and has biological activity.
  • the hybridoma cell lines in Table 1 above were humanized.
  • variable region gene-specific cDNAs for heavy and light chains were synthesized using the SMARTer 5'RACE Synthesis Kit (TAKARA, #634859) according to the manufacturer's instructions.
  • the 5' and 3' ends of the cDNA sequence were modified with PCR primers designed to add appropriate leader sequences to the heavy and light chain variable region cDNAs, respectively, so that the resulting PCR products could be cloned into The heavy chain carrier pHB-Fc and the light chain carrier pHB-CK are expressed on the existing recombinant antibody.
  • the pHB-Fc expression vector contains the human IgG1 heavy chain constant region gene sequence, and the L234A and L235A (Eu encoding) mutations with antibody ADCC knock out (KO) effect on CH 2 ;
  • the pHB-C ⁇ vector contains human kappa light chain Constant region gene sequence.
  • the heavy and light chain variable region PCR amplification products were cloned into an expression vector by In-fusion cloning reagent (TAKARA, #639650) and transformed into E.coli DH5 ⁇ E. coli competent cells (Probiotics, #FYE607 -80VL).
  • Antibody variable region sequences were obtained by selecting monoclonal colonies for Sanger sequencing.
  • the variable region sequence of the anti-TIGIT antibody expressed by B3/29F6 is as follows:
  • CDRs (according to the definition of IMGT, the sequences are as follows):
  • the IgG1 wild type is a chimeric antibody and the ADCC KO type chimeric antibody shares the same light chain carrier, the difference lies in the 234 and 235 amino acids of heavy chain CH 2 on the carrier containing the human heavy chain constant region sequence, ADCC KO type It is A234/A235, and the wild type is L234/L235.
  • the method of the IgG1 wild-type chimeric heavy chain expression vector is the same as the construction method of the expression vector described in 2.1, that is, the heavy chain variable region gene is amplified by PCR with primers containing a leader sequence, and then cloned into a human-
  • the construction of IgG1 wild-type chimeric expression vector was completed on the vector of heavy chain constant region sequence (CH2 is L234/L235).
  • the expression vectors obtained in 2.1 and 2.2 were amplified by Escherichia coli, and a sufficient amount of plasmid was prepared with an endotoxin-depleted plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., #DP117) for transient transfection and expression of chimeric antibodies. .
  • the host cells used for expression were CHO-S cells (Thermo Fisher, #R80007). By mixing the prepared two heavy chain carriers with the light chain carrier, respectively, and polyetherimide (PEI, Polysciences, #24765-1) to form liposome complexes, CHO-S cells were transfected and put into Incubate in the incubator for 5-7 days.
  • the cell culture supernatant was collected by centrifugation, purified by Protein A affinity chromatography column to obtain ADCC KO-type human-mouse chimeric antibody (No. 900424) and IgG1 wild-type human-mouse chimeric antibody (Protein No. 900445).
  • the chimeric antibody VH and VH sequences obtained according to the above method are as follows:
  • the heavy chain constant region amino acid sequences of 900424, 900423 and 900428 are all shown in SEQ ID NO:30.
  • the light chain constant regions were all human kappa light chain constant regions (SEQ ID NO:31).
  • the humanization of antibodies was carried out using the following methods.
  • the antibody variable region sequences were compared with the available sequences in the NCBI IgBlast database, and through identification and analysis, human framework regions (FR regions) suitable for the construction of CDR-grafted heavy and light chains were finally determined.
  • FR regions human framework regions
  • the transformation site is designed, and the variable regions of the heavy and light chains of the chimeric antibody are respectively designed for humanization mutation.
  • PCR technology to amplify and construct humanized point mutant antibody expression plasmid.
  • the humanized point mutant antibody expression plasmids were expressed in CHO-S cells respectively, and the humanized antibody protein was obtained after purification.
  • detection methods such as ELISA, Biacore and flow cytometry, the receptor binding ability, functional inhibitory activity and ADCC effect of the humanized antibodies were screened, and four humanized anti-TIGIT antibodies with excellent performance were obtained.
  • the VH and VL sequences of the obtained humanized anti-TIGIT antibodies are shown below:
  • the underlined regions are CDRs (as defined by IMGT), 900461, 900464, 900466 and 900476 are the four humanized antibody protein numbers, respectively, and the corresponding project numbers are HB0030, HB0031, HB0032 and HB0033.
  • the heavy chain constant region amino acid sequences of HB0030, HB0031, HB0032 and HB0033 are all as shown in SEQ ID NO: 29, and the light chain constant regions are all human kappa light chain constant regions (SEQ ID NO: 31).
  • chimeric antibodies 900324, 900423, 900424, 900428, and humanized antibodies HB0030, HB0031, HB0032, and HB0033 was detected.
  • All chimeric antibodies and humanized antibodies were diluted to 20 ⁇ g/ml with 1% BSA-containing PBS solution (1% BSA/PBS), 20 ⁇ L per well was added to 96-well U-plate, and negative control was set simultaneously (only adding 1% BSA/PBS).
  • the 96-well U-shaped plate after the reaction was resuspended with 1% BSA/PBS, centrifuged (300g ⁇ 3min) to discard the supernatant, washed once in this way, and 1:200 diluted PE-goat anti-human-Fc (Jackson ImmunoResearch, #109-115-098), react at room temperature for 15 minutes in the dark; resuspend the reacted 96-well U-plate with 1% BSA/PBS, centrifuge (300g ⁇ 3min) to discard the supernatant, and wash 3 times in this way, and finally use each The wells were resuspended in 100 ⁇ L of 1% BSA/PBS, and the fluorescence intensity of the PE channel was detected by a flow cytometer (BD, #CantoII).
  • BD flow cytometer
  • the binding activity results of chimeric antibodies are shown in Figure 1 and Table 3, and the binding activity results of humanized antibodies are shown in Figure 2 and Table 4.
  • 900324 is the anti-human TIGIT antibody Tiragolumab from Genentech transiently expressed in CHO cells. Its light and heavy chains can be The variable region sequence is identical to that of antibody 10A7 in patent application WO2015009856A2.
  • the results showed that both the chimeric antibody and the humanized antibody had good affinity with cells expressing human TIGIT (CHOK1-huTIGIT-2A3, Huabo Biotechnology), and the affinity was comparable.
  • Antibody 900324 HB0030 HB0031 HB0032 HB0033 EC 50 ( ⁇ g/mL) 0.08994 0.04661 0.05806 0.07847 0.0514
  • the antigen huCD155-moFc (ACROBiosystems, #CD5-H5254) was diluted to 40 ⁇ g/ml with 1% BSA in PBS (1% BSA/PBS), and 10 ⁇ L per well was added to a 96-well U-plate with serial dilutions of the antibody. TIGIT antibody was mixed evenly at a volume ratio of 1:1, and a positive control was set simultaneously (only CD155-moFc was added).
  • the 96-well U-shaped plate after the reaction was resuspended with 1% BSA/PBS, and the supernatant was discarded by centrifugation (300 g ⁇ 3 min), washed once, and Alexa488-goat anti-mouse-Fc (Jackson ImmunoResearch, 1:300 diluted) was added.
  • Antibody 900324 HB0030 HB0031 HB0032 HB0033 IC50 ( ⁇ g/mL) 0.1349 0.05733 0.06653 0.0919 0.05598
  • HBS-EP 10x
  • His-Tag human TIGIT protein
  • huTIGIT histidine-tagged human TIGIT protein
  • a pH 1.5 glycine solution GE, #BR100354
  • the sample was analyzed by the multi-cycle kinetic program of the capture method, and the corresponding analysis program was used to analyze the data to confirm that there was no obvious reference binding. Kinetics, 1:1 binding model was used, and the kinetic parameters of the sample were obtained by fitting analysis.
  • inoculation volume 5 ⁇ 10 5 /100 ⁇ L/mice
  • the mice were randomly divided into groups of 6 mice according to the tumor volume. The day of grouping is defined as D0 day, and administration is performed on D0, D3, D7, D10, D14, and D17. D23 tumor size was observed and mice were weighed.
  • the anti-TIGIT antibody HB0030 of the present application has a better tumor suppressing effect than the control antibody Tiragolumab.
  • mice In all groups of mice in this study, there was no significant change in the body weight of mice 24 days after inoculation.
  • the PDL1-TIGIT bispecific antibody was constructed using HB0030.
  • the structure pattern of the double antibody belongs to the Morrison pattern (IgG-scFv), that is, the scFv fragments of the two heavy chains of one IgG antibody are connected to the scFv fragment of the other antibody. As shown in Figures 7A-7B.
  • TIGIT-IgG-PD-L1-scFv this form of bispecific antibody is abbreviated as TIGIT-IgG-PD-L1-scFv, and its heavy chain and light chain main
  • Table 8 when PD-L1 is intact IgG and TIGIT is scFv, this format of bispecific antibody is called PD-L1-IgG-TIGIT-scFv, and its heavy and light chain main composition
  • Table 9 The design is as shown in Table 9 below.
  • the sequence of the 900339 antibody is derived from the protein 900339 of the patent WO2020199860A1.
  • 900339-VL refers to the light chain variable region of 900339
  • 900339-VH refers to the heavy chain variable region of 900339.
  • HB0030-VL refers to the light chain variable region of HB0030
  • HB0030-VH refers to the heavy chain variable region of HB0030.
  • Q100C refers to the mutation of amino acid Q at position 100 of the variable region of the light chain to C according to the antibody coding rules of Kabat; R44C or G44C refers to the modification of amino acid R at position 44 of the variable region of the heavy chain according to the antibody coding rules of Kabat. Or G mutated to C. After these two amino acids are mutated, a disulfide bond will be formed, which enhances the stability of the scFv region and increases the druggability of the double antibody.
  • Linker1 (G4S) 3 (SEQ ID NO: 74), amino acid sequence of Linker2: (G4S) 4 (SEQ ID NO: 75), amino acid sequence of Linker3: (G4S) 5 (SEQ ID NO: 75) 76), the amino acid sequence of Linker4: (G4S)6 (SEQ ID NO: 77).
  • the expression vector obtained in Example 5 was amplified by Escherichia coli, and a sufficient amount of plasmid was prepared with an endotoxin-removing plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., #DP117) for transient transfection and expression of bispecificity Antibody.
  • the host cells used for expression were CHO-S cells (Thermo Fisher, #R80007).
  • PEI Polyetherimide
  • CHO-S cells were transfected and put into Incubate in the incubator for 5-7 days.
  • the cell culture supernatant was collected by centrifugation, purified by Protein A affinity chromatography column to obtain a series of bispecific antibodies, and the aggregation of the protein was further judged by molecular sieve chromatography.
  • the SPR method was used to determine the binding kinetics and affinity of PDL1-TIGIT bispecific antibody, PDL1 parental mAb 900339, and TIGIT parental mAb HB0030 to antigen.
  • the test method is as follows:
  • Anti-Human Capture-CM5 chip (GE, #BR-1005-30) was prepared according to the coupling method of Human Antibody Captrue Kit (GE, #BR-1008-39) and Amino Coupling Kit (GE, BR-1000-50). ). Equilibrate the chip at room temperature for 20-30min, load it into the Biacore 8K instrument; release the antigen and antibody to the experimental working concentration with equilibration buffer; dilute the antigen to 50nM with equilibration buffer, and then 3-fold dilution to 7 concentration gradients, and set 2 zero concentrations (i.e.
  • the test results are shown in Table 11 and Table 12 below.
  • the results show that for the TIGIT-IgG-PD-L1-scFv bispecific antibody, the affinity of the double antibody to TIGIT is comparable to that of the parental HB0030, and the affinity of the double antibody to PD-L1 is slightly lower. Better than the parental 900339 mAb.
  • the affinity of diabodies 900691, 900692 and 900693 for PD-L1 was comparable to that of the parental monoclonal antibody 900339, and the affinity of 900691, 900692, 900693 for TIGIT was the same as the parental monoclonal antibody 900339.
  • the affinity is still within an order of magnitude.
  • the experimental steps to detect the inhibitory effect of PDL1-TIGIT bispecific antibody on PDL1 function are as follows: the bispecific antibody and 900339 parental monoclonal antibody were diluted with PBS solution to 10 ⁇ g/ml, and then diluted 2 times in 9 gradients. The target cells overexpressing PD-1, Jurkat-NFAT-PD-1-5B8, were counted, resuspended at 5 ⁇ 10E5/mL, and 30 ⁇ l per well was plated into a 96-well white bottom plate.
  • the culture plate was equilibrated at room temperature for at least 15 minutes, then 90 ⁇ L of BIO-Glo assay was added to each well, and the reaction was performed in the dark at room temperature for 5 minutes. Finally, the signal intensity was detected by the MD i3x microplate reader.
  • Fluorescence reporter enzyme detects the inhibitory effect of PD-L1-IgG-TIGIT-scFv bispecific antibody on PDL1 function
  • the experimental steps to detect the inhibitory effect of PDL1-TIGIT bispecific antibody on TIGIT function are as follows: Dilute the bispecific antibody and HB0030 parental monoclonal antibody to 10 ⁇ g/ml with PBS solution, and then dilute 9 gradients in 2-fold ratio.
  • the target cells overexpressing TIGIT, Jurkat-TIGIT-22G8, were counted, resuspended at 4 ⁇ 10E6/mL, and 50 ⁇ l per well was plated into a 96-well white bottom plate. Then, the diluted bispecific antibody and the parental control antibody were sequentially added to the 96-well white bottom plate with plated cells, 50 ⁇ l per well, and incubated at 37° C. for 10 min.
  • TIGIT-IgG-PD-L1-scFv bispecific antibody The detection results of TIGIT-IgG-PD-L1-scFv bispecific antibody are shown in Figure 11 and Table 14, the results show that the bispecific antibody in the form of TIGIT-IgG-PD-L1-scFv is compared with TIGIT parental monoclonal antibody HB0030 There was no significant difference in the inhibitory effect of TIGIT function.
  • IL2 secretion assay detects the inhibitory effect of TIGIT-IgG-PD-L1-scFv bispecific antibody on TIGIT function
  • the experimental procedure for the thermal stability of the PDL1-TIGIT bispecific antibody was as follows: Bispecific antibodies 900691, 900692 and 900692 in the form of PD-L1-IgG-TIGIT-scFv were detected using a protein stability analyzer (UNcle, UNCHAINED LABS, US).
  • the melting temperature (Tm) and aggregation temperature (Tagg) of 900693 the temperature rise range of Tm and Tagg is 25°C to 95°C, and the heating rate is 0.3°C.
  • the thermal stability test results of the bispecific antibodies in the form of PD-L1-IgG-TIGIT-scFv are shown in Table 15 below.
  • the stability data Tm and Tagg values were both in the range of 65°C to 70°C, indicating that the three bispecific antibodies exhibited good thermal stability.
  • B-hPD-1/hPD-L1/hTIGIT humanized mice were used to establish MC38-hPD-L1 colon cancer animal model and to test the efficacy of the tested antibodies.
  • the B-hPD-1/hPD-L1/hTIGIT humanized mouse MC38-hPD-L1 colon cancer animal model was established.
  • the tumor-forming mice were equally divided into 7 experimental groups according to the mouse tumor volume and body weight. Group 6, intraperitoneal injection, 2 times a week for a total of 6 times.
  • the specific dosing schedule is shown in Table 16 below:
  • 900201 is the IgG1-TNP isotype control
  • 900339 is the recombinant humanized anti-human PD-L1 monoclonal antibody
  • HB0030 is the recombinant humanized anti-human TIGIT monoclonal antibody
  • 900693, 900692 and 900691 are the recombinant humanized anti-human PD -L1/TIGIT monoclonal antibody fusion protein
  • a : BIW means dosing twice a week.
  • mice After grouping, the tumor volume was measured twice a week. At the end of the experiment, the mice were excised and weighed, and the tumor volume inhibition rate TGITV was calculated and statistically analyzed to evaluate the effect of the test drug on the model. Antitumor effect in mice;
  • Body weight and general clinical observation body weight was measured twice a week during the test, and general clinical observation was carried out once a day during the adaptive feeding period and the experimental period to evaluate the safety and tolerability of the tested drugs to the model mice.
  • the mouse colorectal cancer cell CT26-hPDL1 was subcutaneously transplanted into the humanized mouse BALB/C-hPD1/hPDL1/hTIGIT mouse model to evaluate the effect of bispecific antibodies in the treatment of tumors in vivo.
  • mice were randomly divided into 4 groups according to the tumor volume, with 6 mice in each group, 900201 (control antibody, 3 mg/kg), 900339 (hPDL1 antibody, 3 mg/kg), HB0030 ( TIGIT antibody, 3 mg/kg) and 900693 (bispecific antibody, 4.1 mg/kg) groups. After grouping, mice in each group were given intraperitoneal injection, twice a week, for a total of 6 times for 3 weeks. The tumor volume and body weight were measured every week, and the relationship between the body weight and tumor volume of tumor-bearing mice and the administration time was recorded.

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Abstract

本申请提供了一种多特异性抗体，包含能够特异性结合TIGIT蛋白的第一靶向部分和能够结合肿瘤相关抗原的第二靶向部分，该多特异性抗体能够治疗肿瘤。本申请还提供了该多特异性抗体作为药物的用途。

Description

双特异性抗体及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种抗PD-L1/TIGIT双特异性抗体及其用途。
背景技术
肿瘤根据新生物的细胞特性及对机体的危害性程度，可分为良性肿瘤和恶性肿瘤两大类；其中恶性肿瘤疾病是当今社会上危害人类健康的重大疾病，致死程度高居第二。实体瘤常规治疗手段包括外科手术、化学治疗、放射治疗和分子靶向药物治疗以及免疫疗法等。很多实体瘤患者发现时已经是晚期，已丧失了手术治疗的机会，并且多数患者体质较弱，常常难以承受放化疗治疗引起的强烈副作用。生物类治疗药物，尤其是以PD-1等免疫检查点抗体为代表的癌症免疫疗法，凭借其低毒性、高有效性优势，应用越来越广泛，特别是对于转移性、复发性、难治性或现有标准化疗方法无效的晚期实体瘤患者，可能是唯一选择。
目前上市在售的免疫检查点抗体药物多为单克隆抗体，多数是针对一个特异性的靶标。然而，病人接受单克隆抗体治疗可能产生耐药性或无应答，并且很多疾病在体内的影响因素是多方面的，包括不同的信号通路、不同的细胞因子和受体的调节机制等，单一靶点的免疫疗法并不足以摧毁肿瘤细胞。因此，开发多特异性抗体，例如，双特异性抗体(Bispecific antibody，BsAb)，或可解决单抗药物的脱靶毒性、低应答率和药物耐受等问题，扩大免疫治疗受益群体。
双特异性抗体虽然是抗体药物研发的热点方向，但面临诸多挑战，比如临床前评价模型、表达量低、稳定性差、工艺复杂、质控差异性大等问题，因此一直以来双特异性抗体的研发困难重重。因此，本领域迫切需要开发一种特异性佳、疗效好且易于制备的抗肿瘤药物。
发明内容
本申请提供了一种多特异性抗体，其具有以下一种或多种性质：(1)能够以较高的亲和力分别与人PD-L1、人TIGIT特异性结合；(2)能够同时结合PD-L1蛋白以及TIGIT蛋白；(3)能够保持多特异性抗体与结合靶标的结合构型不变，保持分子稳定；(4)在初步的培养条件中，多特异性抗体纯度均在90％以上；(5)能维持结合靶标的结合亲和力，相比单独的抗PD-L1抗体(如HB0023)和抗TIGIT抗体(如HB0030)，能够发挥双靶点的协同效应； 和(6)有效地杀伤肿瘤细胞(尤其是高表达PD-L1的肿瘤)，例如，实体瘤。本申请的多特异性抗体的制备方法简便可行，具有良好的应用前景。
一方面，本申请提供了一种多特异性抗体，其包含能够特异性结合TIGIT蛋白的第一靶向部分，其中所述第一靶向部分包含分离的抗原结合蛋白，所述分离的抗原结合蛋白包含重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2包含SEQ ID NO:4或42所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:5或43所示的氨基酸序列。
在某些实施方式中，所述的多特异性抗体包含轻链可变区VL，所述VL包含LCDR1，LCDR2和LCDR3，其中，所述LCDR1包含SEQ ID NO:6或52所示的氨基酸序列，所述LCDR2包含SEQ ID NO:7所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:8或53所示的氨基酸序列。
在某些实施方式中，所述的多特异性抗体包含重链可变区VH，其中所述VH包含SEQ ID NO:110、1、9、12、14和32中任一项所示的氨基酸序列。
在某些实施方式中，所述的多特异性抗体包含轻链可变区VL，其中所述VL包含SEQ ID NO:111、2、10、11、13、33和34中任一项所示的氨基酸序列。
在某些实施方式中，所述的多特异性抗体还包括第二靶向部分。
在某些实施方式中，所述第二靶向部分能够特异性结合肿瘤相关抗原。
在某些实施方式中，所述第二靶向部分能够特异性结合PD-L1蛋白。
在某些实施方式中，所述第二靶向部分包括抗体或抗原结合片段，所述能够结合PD-L1蛋白的抗体包含HCDR1，HCDR2和HCDR3，其中所述HCDR1，HCDR2和HCDR3分别依次包含SEQ ID NO:100、101和102所示的氨基酸序列。
在某些实施方式中，所述能够结合PD-L1蛋白的抗体包含LCDR1，LCDR2和LCDR3，其中所述LCDR1，LCDR2和LCDR3分别依次包含SEQ ID NO:103、104和105所示的氨基酸序列。
在某些实施方式中，所述的多特异性抗体包含第一多肽链和第二多肽链，其中，所述第一多肽链包含所述能够结合PD-L1蛋白的抗体的重链可变区VH，所述能够结合TIGIT蛋白的重链可变区VH和所述能够结合TIGIT蛋白的轻链可变区VL；且所述第二多肽链包含能够结合所述PD-L1蛋白的抗体的轻链可变区VL，或者，所述第一多肽链包含所述能够结合TIGIT蛋白的抗体的重链可变区VH，所述能够结合PD-L1蛋白的重链可变区VH和所述能够结合PD-L1蛋白的轻链可变区VL；且所述第二多肽链包含能够结合所述TIGIT蛋白的抗 体的轻链可变区VL。
在某些实施方式中，所述第一多肽链中所述能够结合TIGIT蛋白的抗体的VH与所述能够结合TIGIT蛋白的抗体的VL构成scFv，或者，所述第一多肽链中所述能够结合PD-L1蛋白的抗体的VH与所述能够结合PD-L1蛋白的抗体的VL构成scFv。
在某些实施方式中，所述第一多肽链包含选自下组中任意一项所示的氨基酸序列：SEQ ID NO:78-83和85-87。
在某些实施方式中，所述第二多肽链包含SEQ ID NO:84或88所示的氨基酸序列。
另一方面，本申请提供了分离的一种或多种核酸分子，其编码所述的多特异性抗体。
另一方面，本申请提供了载体，其包含所述的核酸分子。
另一方面，本申请提供了细胞，其包含所述的核酸分子或根据所述的载体。
另一方面，本申请提供了制备所述的多特异性抗体的方法，所述方法包括在使得所述的多特异性抗体表达的条件下，培养所述的细胞。
另一方面，本申请提供了药物组合物，其包含所述的多特异性抗体、所述的核酸分子、所述的载体和/或所述的细胞，以及任选地药学上可接受的佐剂。
另一方面，本申请提供了所述的多特异性抗体、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗疾病或病症。
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。对附图简要说明书如下：
图1显示的是TIGIT嵌合抗体对表达人TIGIT的细胞结合活性。
图2显示的是TIGIT人源化抗体对表达人TIGIT的细胞结合活性。
图3显示的是TIGIT嵌合抗体对人TIGIT与其配体CD155结合的阻断活性。
图4显示的是TIGIT人源化抗体对人TIGIT与其配体CD155结合的阻断活性。
图5显示的是TIGIT抗体在动物体内药效检测。
图6显示的是TIGIT抗体在动物体内与对照抗体Tiragolumab的药效检测比较。
图7A-7B显示的是本申请所述多特异性抗体的示例性结构。
图8显示的是本申请所述多特异性抗体TIGIT-IgG-PDL1-scFv的SDS-PAGE电泳图结果。
图9显示的是本申请所述多特异性抗体PDL1-IgG-TIGIT-scFv的SDS-PAGE电泳图结果。
图10显示的是本申请所述多特异性抗体PDL1-IgG-TIGIT-scFv对PDL1功能的抑制效果。
图11显示的是本申请所述多特异性抗体TIGIT-IgG-PDL1-scFv对TIGIT功能的抑制效果。
图12显示的是本申请所述多特异性抗体对小鼠肿瘤模型的治疗效果。
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的其他优点及效果。
术语定义
在本申请中，术语“TIGIT”称为“含Ig和ITIM结构域的T细胞免疫受体”，通常为免疫球蛋白的PVR(脊髓灰质炎病毒受体)家族的成员，其可结合PVR/CD155和Nectin-2/CD112。本申请的TIGIT可以指来自任何脊椎动物来源，包括哺乳动物，诸如灵长类(例如人、恒河猴和食蟹猴)和啮齿类(例如小鼠和大鼠)的TIGIT蛋白。该术语涵盖全长的TIGIT或其片段(诸如其缺乏信号肽的成熟片段)，未加工的TIGIT，因细胞中的加工所致的任何形式的TIGIT，以及人工合成的TIGIT。该术语还涵盖TIGIT的变体，例如剪接变体或等位变体。在某些情形中，TIGIT为人TIGIT，人TIGIT的氨基酸序列包含UniProt登录号Q495A1所示的氨基酸序列。在某些情形中，TIGIT为食蟹猴TIGIT，食蟹猴TIGIT的氨基酸序列包含UniProt登录号G7NXM4所示的氨基酸序列。在某些情形中，TIGIT为小鼠TIGIT，小鼠TIGIT的氨基酸序列包含UniProt登录号P86176所示的氨基酸序列。
在本申请中，术语“PD-L1”通常是指程序性细胞死亡1配体1，也可称为B7同源物1、B7-H1、分化簇274、(3)274或CD274，其与PD-1结合后下调T细胞活化和细胞因子分泌。“PD-L1”包括任何脊椎动物来源的任何天然PD-L1，所述任何脊椎动物来源包括哺乳动物，诸如灵长类(例如，人和食蟹猴)和啮齿类(例如，小鼠和大鼠)。所述术语涵盖“全长”、未加工的PD-L1以及由细胞加工所产生的任何形式的PD-L1。PD-L1可作为跨膜蛋白或作为可溶性蛋白存在。“PD-L1”包括完整的PD-L1及其片段，还包括PD-L1的功能性变体、同工型、 物种同源物、衍生物、类似物，以及具有至少一个与PD-L1共同表位的类似物。PD-L1的基本结构包括4个结构域：胞外Ig样V型结构域和Ig样C2型结构域、跨膜结构域以及细胞质结构域。示例性的人PD-L1氨基酸序列可在NCBI登录号NP_001254653或UniProt登录号Q9NZQ7下找到。
在本申请中，术语“抗原结合蛋白”通常是指包含结合抗原的部分的蛋白质，以及任选地允许结合抗原的部分采用促进抗原结合蛋白与抗原结合的构象的支架或骨架部分。抗原结合蛋白的实例包括但不限于抗体、抗原结合片段(Fab，Fab’，F(ab) 2，Fv片段，F(ab’) 2，scFv，di-scFv和/或dAb)、免疫缀合物、多特异性抗体(例如双特异性抗体)、抗体片段、抗体衍生物、抗体类似物或融合蛋白等，只要它们显示出所需的抗原结合活性即可。
在本申请中，术语“Fab”通常是指含有重链可变结构域和轻链可变结构域的片段，并且还含有轻链的恒定结构域和重链的第一恒定结构域(CH1)；术语“Fab’”通常是指在重链CH1结构域的羧基端添加少量残基(包括一个或多个来自抗体铰链区的半胱氨酸)而不同于Fab的片段；术语“F(ab') 2”通常是指Fab’的二聚体，包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。术语“Fv”通常是指含有完整抗原识别与结合位点的最小抗体片段。在某些情形中，该片段可以由一个重链可变区和一个轻链可变区以紧密非共价结合的二聚体组成；术语“dsFv”通常是指二硫键稳定的Fv片段，其单个轻链可变区与单个重链可变区之间的键是二硫键。术语“dAb片段”通常是指由VH结构域组成的抗体片段。在本申请中，术语“scFv”通常是指抗体的一个重链可变结构域和一个轻链可变结构域通过柔性肽连接子共价连接配对形成的单价分子；此类scFv分子可具有一般结构：NH 2-VL-连接子-VH-COOH或NH 2-VH-连接子-VL-COOH。
在本申请中，术语“抗体”其以最广泛意义使用，且具体涵盖，但不限于，单克隆抗体(包括包含两条轻链和两条重链的全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、完全人类抗体、嵌合抗体和骆驼化单结构域抗体。“抗体”通常可以包含通过二硫键互相连接的至少两条重链(HC)和两条轻链(LC)的蛋白，或其抗原结合片段。每条重链包含重链可变区(VH)和重链恒定区。在某些天然存在的IgG、IgD和IgA抗体中，重链恒定区包含三个结构域，CH1、CH2和CH3。在某些天然存在的抗体中，各轻链包含轻链可变区(VL)和轻链恒定区。轻链恒定区包含一个结构域，CL。VH和VL区可进一步细分为超变性的区域，称为互补决定区(CDR)，其与称为框架区(FR)的较保守的区域交替。各VH和VL包含三个CDR和四个框架区(FR)，从氨基端至羧基端按以下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3和FR4。天然重链和轻链的可变结构域各自包 含四个FR区(H-FR1，H-FR2，H-FR3，H-FR4，L-FR1，L-FR2，L-FR3，L-FR4)，大部分采用β-折叠构型，通过三个CDRs连接，形成环连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDRs通过FR区紧密靠近在一起，并与来自另一条链的CDR一起形成抗体的抗原结合位点。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(Clq)结合。
在本申请中，术语“可变”通常是指这样的事实，即抗体的可变结构域的序列的某些部分变化强烈，它形成各种特定抗体对其特定抗原的结合和特异性。然而，变异性并非均匀地分布在抗体的整个可变区中。它集中在轻链和重链可变区中的三个区段，被称为互补决定区(CDR)或高变区(HVR)。可变域中更高度保守的部分被称为框架(FR)。在本领域中，可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))、基于结构环区域位置的Chothia定义规则(参见，A1-Lazikani等人，JMol Biol 273:927-48,1997)和基于IMGT本体论(IMGT-ONTOLOGY)的概念和IMGT Scientific图表规则的IMGT定义规则。
IMGT指国际ImMunoGeneTics信息系统，一种免疫遗传学和免疫信息学的全球参考数据库(http://www.imgt.org)。IMGT专门研究来自人类和其他脊椎动物的免疫球蛋白(IG)或抗体、T细胞受体(TR)、主要组织相容性(MH)，以及来自脊椎动物和非脊椎动物的免疫球蛋白超家族(IgSF)、MH超家族(MhSF)和免疫系统相关蛋白(RPI)。
在本申请中，术语“kabat的抗体编码规则”通常是指一种在抗体的重链和轻链可变区或其抗原结合部分中氨基酸残基的编号系统(Kabat等(1971)Ann.NY Acad.Sci.190：382-391和Kabat，E.A.等(1991)Sequence of Proteins of Immunological Interest，第五版，美国人类健康服务部，NIH公告第91-3242号)。对于重链可变区而言，CDR1为重链可变区的第31-35位氨基酸，CDR2为重链可变区的第50-65位氨基酸，CDR3为重链可变区的的第95-102位氨基酸。对于轻链可变区而言，CDR1为轻链可变区的第24-34位氨基酸、CDR21为轻链可变区的第50-56位氨基酸和CDR3的第89-97位氨基酸。
在本申请中，术语“分离的”抗原结合蛋白通常是指已经从其产生环境(例如，天然的或重组的)的组分中识别，分离和/或回收的抗原结合蛋白。其产生环境的污染组分通常是干扰其研究、诊断或治疗用途的物质，可以包括酶、激素和其他蛋白质或非蛋白质溶质。分离的抗原结合蛋白或抗体通常将通过至少一个纯化步骤来制备。
在本申请中，术语“多特异性抗体”通常是指一种具有识别一种或多种抗原上的一个以上 表位的可变区的抗体。多特异性抗体包括，但不限于，全长抗体、具有两个或更多VL和VH结构域的抗体、抗体片段，如Fab、Fv、dsFv、scFv、双抗体、双特异性双抗体和三抗体、已经共价或非共价连接的抗体片段。在某些实施方案中，多特异性抗体可以是识别相同或不同抗原上的两种不同表位的“双特异性抗体”。
在本申请中，术语“单克隆抗体”通常是指从一群基本上同质的抗体获得的抗体，即集群中的个别抗体是相同的，除了可能存在的少量的自然突变。单克隆抗体通常针对单个抗原位点具有高度特异性。而且，与常规多克隆抗体制剂(通常具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性之外，单克隆抗体的优点在于它们可以通过杂交瘤培养合成，不受其他免疫球蛋白污染。修饰语“单克隆”表示从基本上同质的抗体群体获得的抗体的特征，并且不被解释为需要通过任何特定方法产生抗体。例如，本申请使用的单克隆抗体可以在杂交瘤细胞中制备，或者可以通过重组DNA方法制备。
在本申请中，术语“嵌合抗体”通常是指其中可变区源自一个物种，而恒定区源自另一个物种的抗体。通常，可变区源自实验动物诸如啮齿动物的抗体(“亲本抗体”)，且恒定区源自人类抗体，使得所得嵌合抗体与亲本(例如小鼠来源)抗体相比，在人类个体中引发不良免疫反应的可能性降低。
在本申请中，术语“人源化抗体”通常是指非人抗体(例如小鼠抗体)的CDR区以外的部分或全部有的氨基酸被源自人免疫球蛋白的相应的氨基酸置换的抗体。在CDR区中，氨基酸的小的添加、缺失、插入、置换或修饰也可以是允许的，只要它们仍保留抗体结合特定抗原的能力。人源化抗体可任选地包含人类免疫球蛋白恒定区的至少一部分。“人源化抗体”保留类似于原始抗体的抗原特异性。非人(例如鼠)抗体的“人源化”形式可以最低限度地包含衍生自非人免疫球蛋白的序列的嵌合抗体。在某些情形中，可以将人免疫球蛋白(受体抗体)中的CDR区残基用具有所期望性质、亲和力和/或能力的非人物种(供体抗体)(诸如小鼠，大鼠，家兔或非人灵长类动物)的CDR区残基替换。在某些情形中，可以将人免疫球蛋白的FR区残基用相应的非人残基替换。此外，人源化抗体可包含在受体抗体中或在供体抗体中没有的氨基酸修饰。进行这些修饰可以是为了进一步改进抗体的性能，诸如结合亲和力。
在本申请中，术语“全人源抗体”通常是指将人类编码抗体的基因转移至基因工程改造的抗体基因缺失动物中，使动物表达的抗体。抗体所有部分(包括抗体的可变区和恒定区)均由人类来源的基因所编码。全人源抗体可以大大减少异源抗体对人体造成的免疫副反应。本领域获得全人源抗体的方法可以有噬菌体展示技术、转基因小鼠技术、核糖体展示技术和 RNA-多肽技术等。
在本申请中，术语“结合”、“特异性结合”或“对…特异性的”通常是指可测量且可再现的相互作用，诸如抗原和抗体之间的结合，其可以确定在存在分子(包括生物学分子)的异质群体的情况中靶物的存在。例如，抗体通过其抗原结合域与表位结合，并且该结合需要抗原结合域和表位之间的一些互补性。例如，特异性结合靶物(其可以是表位)的抗体是以比其结合其它靶物更大的亲和力、亲合力、更容易和/或以更大的持续时间结合此靶物的抗体。当抗体相比于其将结合随机的、不相关的表位而言更容易通过其抗原结合域与表位结合时，抗体被称为“特异性结合”该抗原。“表位”是指抗原上与抗原结合蛋白(如抗体)结合的特定的原子基团(例如，糖侧链、磷酰基、磺酰基)或氨基酸。
在本申请中，术语“KD”、“K D”可互换地使用，通常是指平衡解离常数，“KD”是解离速率常数(kdis，也称为“解离率(off-rate)(koff)”或“kd”)与结合速率常数(kon，也称为“结合率(kon)”或“ka”)的比值。可使用结合速率常数(kon)、解离速率常数(kdis)和平衡解离常数(KD)表示抗原结合蛋白(例如抗体)对抗原的结合亲和力。确定结合和解离速率常数的方法为本领域熟知，包括但不限于生物膜干涉技术(BLI)、放射免疫法(RIA)、平衡透析法、表面等离子共振(SPR)、荧光共振能量迁移(FRET)、免疫共沉淀(Co-IP)以及蛋白质芯片技术。如果在不同的条件(例如盐浓度、pH)下测量，则所测得的某种特定蛋白-蛋白相互作用的亲和力可不同。
在本申请中，术语“参比抗体”通常是指本申请所述抗原结合蛋白与之竞争结合抗原TIGIT的抗体。
在本申请中，术语“ADCC”或“抗体依赖性细胞介导的细胞毒性”通常是指这样一种细胞毒性形式，一些分泌型免疫球蛋白结合到某些细胞毒性效应细胞(例如，NK细胞、嗜中性粒细胞和巨噬细胞)上的Fc受体(FcR)上，使得这些细胞毒性效应细胞能够特异性结合携带抗原的靶细胞，随后用细胞毒素杀死靶细胞。介导ADCC的主要细胞(例如NK细胞)只表达FcγRIII，而单核细胞表达FcγRI、FcγRII和FeγRIII(可参见Ravetch and Kinet,Annu.Rev.Immunol.9:457-92(1991)第464页表3)。可以进行体外和/或体内细胞毒性测定法以评估目的分子的ADCC活性，例如，可进行体外ADCC测定法，可参见美国专利号No.5,500,362或No.5,821,337或美国专利No.6,737,056(Presta)中所记载的。可用于此类测定法的效应细胞包括PBMC和NK细胞。或者/另外，可在体内评估目的分子的ADCC活性，例如在动物模型中，诸如Clynes et al.，PNAS(USA)95:652-656(1998)中所披露的。例如，可以进行Fc受体(FcR)结合测定法以确保抗体缺乏FcγR结合(因此有可能缺乏ADCC活性)，但是保留 FcRn结合能力。
ADCC活性可通过修饰Fc区而减少。在某些情形中，影响与Fc受体结合的位点可被除去，例如，除去并非补救受体结合位点的位点。在某些情形中，Fc区可经修饰以除去ADCC位点。ADCC位点是本领域已知的，关于IgG1的ADCC位点，参见例如，Sarmay et al.(1992)Molec.Immunol.29(5):633-9。
在本申请中，术语“在……之间”通常是指某种氨基酸片段的C端与第一氨基酸片段的N端直接或间接连接，并且其N端与第二氨基酸片段的C端直接或间接连接。在轻链中，例如，所述L-FR2的N末端与所述LCDR1的C末端直接或间接相连，且所述L-FR2的C末端与所述LCDR2的N末端直接或间接相连。又例如，所述L-FR3的N末端与所述LCDR2的C末端直接或间接相连，且所述L-FR3的C末端与所述LCDR3的N末端直接或间接相连。在重链中，例如，所述H-FR2的N末端与所述HCDR1的C末端直接或间接相连，且所述H-FR2的C末端与所述HCDR2的N末端直接或间接相连。又例如，所述H-FR3的N末端与所述HCDR2的C末端直接或间接相连，且所述H-FR3的C末端与所述HCDR3的N末端直接或间接相连。在本申请中，“第一氨基酸片段”和“第二氨基酸片段”可以为相同或不同的任意一段氨基酸片段。
在本申请中，术语“分离的”抗原结合蛋白通常是指已经从其产生环境(例如，天然的或重组的)的组分中识别，分离和/或回收的抗原结合蛋白。其产生环境的污染组分通常是干扰其研究、诊断或治疗用途的物质，可以包括酶、激素和其他蛋白质或非蛋白质溶质。分离的抗原结合蛋白或抗体通常将通过至少一个纯化步骤来制备。本申请所述的分离的抗原结合蛋白通常不结合非TIGIT抗原的抗原。
在本申请中，术语“分离的核酸分子”或“分离的多核苷酸”通产是指基因组、mRNA、cDNA或合成来源的DNA或RNA或其一定组合，其不与在自然界中发现的多核苷酸的全部或一部分缔合，或连接至其在自然界中不连接的多核苷酸。
在本申请中，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，其将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。
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抗体或其抗原结合片段的个体细胞、细胞系或细胞培养物。所述细胞可以包括单个宿主细胞的子代。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抗体或其抗原结合片段即可。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)。在某些情形中，所述细胞可以是哺乳动物细胞。例如，所述哺乳动物细胞可以是CHO-K1细胞。在本申请中，术语“重组细胞”通常是指在其中引入了重组表达载体的细胞。所述重组宿主细胞不仅包括某种特定的细胞，还包括这些细胞的后代。
在本申请中，术语“药学上可接受的佐剂”通常包括药剂学可接受的载体、赋形剂或稳定剂，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物是无毒的。通常，生理学可接受的载体是PH缓冲水溶液。生理学可接受载体的例子可包括缓冲剂，诸如磷酸盐、柠檬酸盐和其它有机酸；抗氧化剂，包括抗坏血酸；低分子量(少于约10个残基)多肽，蛋白质，诸如血清清蛋白，明胶或免疫球蛋白；亲水性聚合物，诸如聚乙烯吡咯烷酮；氨基酸，诸如甘氨酸，谷氨酰胺，天冬酰胺，精氨酸或赖氨酸；单糖，二糖和其它碳水化合物，包括葡萄糖，甘露糖或糊精；螯合剂，诸如EDTA；糖醇，诸如甘露醇或山梨醇；成盐反荷离子，诸如钠；和/或非离子表面活性剂，诸如TWEEN TM，聚乙二醇(PEG)和PLURONICS TM。
如本文所用，术语“给予”和“处理”是指外源性药物、治疗剂、诊断剂或组合物应用于动物、人、受试者、细胞、组织、器官或生物流体。“给予”和“处理”可以指治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触、以及试剂与流体的接触、流体与细胞的接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理。“处理”当应用于人、动物或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断；包括TIGIT结合物与人或动物、受试者、细胞、组织、生理区室或生理流体的接触。
如本文所用，术语“治疗”指给予患者内用或外用治疗剂，包含本申请的任何一种TIGIT抗原结合蛋白及其组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，以有效缓解一种或多种疾病症状的治疗剂的量(治疗有效量)给予患者。治疗的期望效果包括降低疾病进展速率，改善或减轻疾病状态，和消退或改善的预后。例如，若一种或多种与癌症有关的症状是减轻或消除的，包括但不限于，降低(或破坏)癌细胞增殖，减少源自疾病的症状，提高那些患有疾病的个体的生命质量，降低治疗疾病需要 的其它药物的剂量，延迟疾病的进展，和/或延长个体存活，则个体得到成功“治疗”。
在本申请中，术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，以及所有癌前(pre-cancerous)和癌性细胞和组织。
如本文所用，术语“任选”或“任选地”意味着随后所描述的事件或情况可以发生但不是必须发生。
在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
TIGIT抗原结合蛋白
一方面，本申请提供了一种分离的抗原结合蛋白，其可以包含重链可变区VH中的至少一个CDR，所述VH可包含SEQ ID NO:55所示的氨基酸序列。
一方面，本申请提供了一种分离的抗原结合蛋白，其可以包含重链可变区VH中的至少一个CDR，所述VH可包含SEQ ID NO:54所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的VH可包含HCDR1、HCDR2和HCDR3。
本申请所述的抗原结合蛋白可以包含HCDR1，其中所述HCDR1可包含SEQ ID NO:3所示的氨基酸序列：GYSITSDYA。例如，该序列可以是根据IMGT定义规则确定的序列。
本申请所述的抗原结合蛋白可以包含HCDR2，其中所述HCDR2可包含SEQ ID NO:56所示的氨基酸序列：IX 2X 3SGX 6X 7，其中，X 2为S或T，X 3为S或Y，X 6为A或S，且X 7为P或T。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述HCDR2可以包含SEQ ID NO:4和42中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含HCDR2，其中所述HCDR2可包含SEQ ID NO:4所示的氨基酸序列：ITSSGST。例如，该序列可以是根据IMGT定义规则确定的序列。
本申请所述的抗原结合蛋白可以包含HCDR3，其中所述HCDR3可包含SEQ ID NO:57所示的氨基酸序列：AX 2LX 4X 5X 6X 7YX 9X 10AMDY，其中，X 2为R或S，X 4为D或G，X 5为F或T，X 6为D或G，X 7为N或Y，X 9为G或不存在，X 10为G或不存在。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述HCDR3可以包含SEQ ID NO:5和43中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含HCDR3，其中所述HCDR3可包含SEQ ID NO:5所示的氨基酸序列：ARLDFGNYGGAMDY。例如，该序列可以是根据IMGT定义规则确定的序列。
本申请所述的抗原结合蛋白还可以包含框架区H-FR1、H-FR2、H-FR3和H-FR4。
本申请所述的抗原结合蛋白可以包含框架区H-FR1，其中所述H-FR1可包含SEQ ID NO:58所示的氨基酸序列：X 1VQLQESGPGLVKPSX 16X 17LSLTCTVX 25，其中，X 1为D或Q，X 16为E或Q，X 17为S或T，且X 25为S或T。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述H-FR1可以包含SEQ ID NO:23和35中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区H-FR1，其中所述H-FR1可包含SEQ ID NO:23所示的氨基酸序列：QVQLQESGPGLVKPSETLSLTCTVS。例如，该序列可以是根据IMGT定义规则确定的序列。
本申请所述的抗原结合蛋白可以包含框架区H-FR2，其中所述H-FR2可包含SEQ ID NO:60所示的氨基酸序列：WX 2WIRQX 7PGX 10X 11X 12EWX 15GY；其中，X 2为I或N，X 7为F或P，X 10为K或N，X 11为G、R或K，X 12为L或V，且，X 15为I或M。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述H-FR2可包含SEQ ID NO:24-26和36-37中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区H-FR2，其中所述H-FR2可包含SEQ ID NO:59所示的氨基酸序列：WIWIRQPPGX 10X 11LEWIGY；其中，X 10为K或N，X 11为G或K。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述H-FR2可包含SEQ ID NO:24-26中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区H-FR3，其中所述H-FR3可包含SEQ ID NO:61所示的氨基酸序列：X 1YNPSLKSRX 10X 11X 12X 13X 14DTSKNQFX 22LX 24LX 26X 27VTX 30X 31DTATYYC，其中，X 1为R、S或Y，X 10为I或V，X 11为S或T，X 12为F或I，X 13为S或T，X 14为R或V，X 22为F或S，X 24为K或Q，X 26为S或T，X 27为F或S，X 30为A或T，且，X 31为A或E。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述H-FR3可包含SEQ ID NO:27、38和39中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区H-FR3，其中所述H-FR3可包含SEQ ID NO: 27所示的氨基酸序列：YYNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYC。例如，该序列可以是根据IMGT定义规则确定的序列。
本申请所述的抗原结合蛋白可以包含框架区H-FR4，其中所述H-FR4可包含SEQ ID NO:62所示的氨基酸序列：WGQGTX 6VX 8VSS，其中，X 6为L或S，且X 8为I或T。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述H-FR4可包含SEQ ID NO:28、40和41中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区H-FR4，其中所述H-FR4可包含SEQ ID NO:28所示的氨基酸序列：WGQGTLVTVSS。例如，该序列可以是根据IMGT定义规则确定的序列。
本申请所述的抗原结合蛋白可以包含重链可变区VH，其中所述VH可包含SEQ ID NO:55所示的氨基酸序列：X 1VQLQESGPGLVKPSX 16X 17LSLTCTVX 25GYSITSDYAWX 36WIRQX 41PGX 44X 45X 46EWX 49GYIX 53X 54SGX 57X 58X 59YNPSLKSRX 68X 69X 70X 71X 72DTSKNQFX 80LX 82LX 84X 85VTX 88X 89DTATYYCAX 98LX 100X 101X 102X 103YX 105X 106AMDYWGQGTX 116VX 118VSS，其中X 1为D或Q，X 16为E或Q，X 17为S或T，X 25为S或T，X 36为I或N，X 41为F或P，X 44为N或K，X 45为G、K或R，X 46为L或V，X 49为I或M，X 53为S或T，X 54为S或Y，X 57为A或S，X 58为P或T，X 59为R、S或Y，X 68为I或V，X 69为S或T，X 70为F或I，X 71为S或T，X 72为R或V，X 80为F或S，X 82为K或Q，X 84为S或T，X 85为F或S，X 88为A或T，X 89为A或E，X 98为R或S，X 100为D或G，X 101为F或T，X 102为D或G，X 103为N或Y，X 105为G或不存在，X 106为G或不存在，X 116为L或S，且X 118为I或T。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述的抗原结合蛋白可以包含重链可变区VH，其中所述VH可包含SEQ ID NO:1、9、12、14和32中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含重链可变区VH，其中所述VH可包含SEQ ID NO:54所示的氨基酸序列：QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWIWIRQPPGX 44X 45LEWIGYITSSGSTYYNPSLKSRVTFSVDTSKNQFSLKLSSVTAADTATYYCARLDFGNYGGAMDYWGQGTLVTVSS，其中X 44为K或N，且，X 45为G或K。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述的抗原结合蛋白可以包含重链可变区VH，其中所述VH可包含SEQ ID NO:9、 12和14中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含重链恒定区，所述重链恒定区可以来自人IgG，例如，人IgG1。在某些情形中，可以对人IgG1的Fc区进行修饰以达到所期望的性质(例如，ADCC KO)。所述修饰可以是氨基酸突变。在某些情形中，对IgG1的修饰可以为L234A/L235A，即，按照EU编号，第234位和第235位的氨基酸分别由亮氨酸(L)突变为丙氨酸(A)。在某些情形中，本申请所述的抗原结合蛋白的重链恒定区可以来自野生型的人IgG1。
例如，本申请所述的抗原结合蛋白可以包含重链恒定区，其可包含SEQ ID NO:35-36中任一项所示的氨基酸序列。
本申请所述的分离的抗原结合蛋白，其可以包含轻链可变区VL中的至少一个CDR，所述VL可包含SEQ ID NO:64所示的氨基酸序列。
本申请所述的分离的抗原结合蛋白，其可以包含轻链可变区VL中的至少一个CDR，所述VL可包含SEQ ID NO:63所示的氨基酸序列。
在本申请中，所述抗原结合蛋白的VL可包含LCDR1、LCDR2和LCDR3。
本申请所述的抗原结合蛋白可以包含LCDR1，其中所述LCDR1可包含SEQ ID NO:65所示的氨基酸序列：QHVSX 5A，其中，X 5为N或T。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述LCDR1可包含SEQ ID NO:6和52中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含LCDR1，其中所述LCDR1可包含SEQ ID NO:6所示的氨基酸序列：QHVSTA。例如，该序列可以是根据IMGT定义规则确定的序列。
本申请所述的抗原结合蛋白可以包含LCDR2，其中所述LCDR2可包含SEQ ID NO:7所示的氨基酸序列：SAS。例如，该序列可以是根据IMGT定义规则确定的序列。
本申请所述的抗原结合蛋白可以包含LCDR3，其中所述LCDR3可包含SEQ ID NO:66所示的氨基酸序列：QQX 3YX 5X 6PX 8T，其中，X 3为H或Y，X 5为I或S，X 6为L或T，且X 8为W或Y。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述LCDR3可包含SEQ ID NO:8和53中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含LCDR3，其中所述LCDR3可包含SEQ ID NO:8所示的氨基酸序列：QQHYITPYT。例如，该序列可以是根据IMGT定义规则确定的序列。
本申请所述的抗原结合蛋白还可以包含框架区L-FR1、L-FR2、L-FR3和L-FR4。
本申请所述的抗原结合蛋白可以包含框架区L-FR1，其中所述L-FR1可包含SEQ ID NO:68所示的氨基酸序列：DIX 3MTQSX 8X 9X 10X 11X 12X 13SX 15GDRVX 20ITCX 24AS；其中，X 3为 Q或V，X 8为H或P，X 9为K或S，X 10为F或S，X 11为L或M，X 12为F或S，X 13为A或T，X 15为I或V，X 20为S或T，X 24为K或R。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述L-FR1可包含SEQ ID NO:15、16、44和45中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区L-FR1，其中所述L-FR1可包含SEQ ID NO:67所示的氨基酸序列：DIQMTQSPSSLSASVGDRVTITCX 24AS；其中，X 24为K或R。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述L-FR1可包含SEQ ID NO:15和16中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区L-FR2，其中所述L-FR2可包含SEQ ID NO:70所示的氨基酸序列：X 1X 2WYQQKPGX 10X 11PKLLIX 17；其中，X 1为L或V，X 2为A或N，X 10为K或Q，X 11为A或S，且，X 17为H或Y。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述L-FR2可包含SEQ ID NO:17、18、46和47中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区L-FR2，其中所述L-FR2可包含SEQ ID NO:69所示的氨基酸序列：X 1X 2WYQQKPGKAPKLLIY；其中，X 1为L或V，且X 2为A或N。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述L-FR2可包含SEQ ID NO:17和18中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区L-FR3，其中所述L-FR3可包含SEQ ID NO:72所示的氨基酸序列：YX 2X 3X 4GVPX8RFX 11GX 13X 14SGTDFTX 21TIX 24SX 26QX 28EDX 31AX 33YYC；其中，X 2为L或R，X 3为Q或Y，X 4为S或T，X 8为D或S，X 11为I、S或T，X 13为R或S，X 14为G或R，X 21为F或L，X 24为N或S，X 26为L或V，X 28为A或P，X 33为F或L，且X 14为T或V。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述L-FR3可包含SEQ ID NO:19-21和48-50中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区L-FR3，其中所述L-FR3可包含SEQ ID NO:71所示的氨基酸序列：YX 2X 3SGVPSRFSGSX 14SGTDFTLTISSLQPEDFATYYC；其中，X 2为L或R，X 3为Q或Y，且X 14为G或R。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述L-FR3可包含SEQ ID NO:19-21中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区L-FR4，其中所述L-FR4可包含SEQ ID NO: 73所示的氨基酸序列：FGX 3GTKLEIK，其中，X 3为G或Q。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述L-FR4可包括SEQ ID NO:22和51中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含框架区L-FR4，其中所述L-FR4可包含SEQ ID NO:22所示的氨基酸序列：FGQGTKLEIK。例如，该序列可以是根据IMGT定义规则确定的序列。
本申请所述的抗原结合蛋白可以包含轻链可变区VL，其中所述VL可包含SEQ ID NO:64所示的氨基酸序列：DIX 3MTQSX 8X 9X 10X 11X 12X1 3SX 15GDRVX 20ITCX 24ASQHVSX 31AX 33X 34WYQQKPGX 42X 43PKLLIX 49SASYX 54X 55X 56GVPX 60RFX 63GX 65X 66SGTDFTX 73TIX 76SX 78QX 80EDX 83AX 85YYCQQX 91YX 93X 94PX 96TFGX 100GTKLEIK，其中，X 3为Q或V，X 8为H或P，X 9为K或S，X 10为F或S，且，X 11为L或M，X 12为F或S，X 13为A或T，X 15为I或V，X 20为S或T，X 24为K或R，X 31为N或T，X 33为L或V，X 34为A或N，X 42为K或Q，X 43为A或S，X 49为H或Y，X 54为L或R，X 55为Q或Y，X 56为S或T，X 60为D或S，X 63为I、S或T，X 65为S或R，X 66为G或R，X 73为L或F，X 76为S或N，X 78为L或V，X 80为A或P，X 83为F或L，X 85为T或V，X 91为H或Y，X 93为I或S，X 94为L或T，X 96为W或Y，且，X 100为Q或G。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述的抗原结合蛋白可以包含轻链可变区VL，其中所述VL可包含SEQ ID NO:2、10、11、13、33和34中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含轻链可变区VL，其中所述VL可包含SEQ ID NO:63所示的氨基酸序列：DIQMTQSPSSLSASVGDRVTITCX 24ASQHVSTAX 33X 34WYQQKPGKAPKLLIYSASYX 54X 55SGVPSRFSGSX 66SGTDFTLTISSLQPEDFATYYCQQHYITPYTFGQGTKLEIK，其中，X 24为K或R，X 33为L或V，X 34为A或N，X 54为L或R，X 55为Q或Y，且，X 66为G或R。例如，该序列可以是根据IMGT定义规则确定的序列。
例如，所述的抗原结合蛋白可以包含轻链可变区VL，其中所述VL可包含SEQ ID NO:10、11和13中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可以包含轻链恒定区，所述轻链恒定区可以包含人轻链恒定区序列，例如，可以包含人κ轻链恒定区。例如，本申请所述分离的抗原结合蛋白的轻链恒定区可包含SEQ ID NO:31所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:56所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:57所示的氨基酸序列。
例如，在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:4和42中任一项所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:5和43中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体重链可变区CDR——HCDR1、HCDR2和HCDR3，所述HCDR1、所述HCDR2和所述HCDR3可分别依次包含SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:5所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体轻链可变区CDR——LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:65所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:7所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:66所示的氨基酸序列。
例如，在本申请中，所述分离的抗原结合蛋白可包含抗体轻链可变区CDR——LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:6和52中任一项所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:7所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:8和53中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含抗体轻链可变区CDR——LCDR1、LCDR2和LCDR3，所述LCDR1、所述LCDR2和所述LCDR3可分别依次包含SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:56所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:57所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:65所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:7所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:66所示的氨基酸序列。
例如，在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:3所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:4和42中任一项所示的氨基酸序列，所述HCDR3可包含SEQ ID NO:5和43中任一项所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:6和52中任一项所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:7所示的氨基酸序列，且所述LCDR3可包含SEQ  ID NO:8和53中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1、所述HCDR2、所述HCDR3、所述LCDR1、所述LCDR2和所述LCDR3可分别依次包含SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7和SEQ ID NO:8所示的氨基酸序列。
在某些情形中，本申请所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1、所述HCDR2、所述HCDR3、所述LCDR1、所述LCDR2和所述LCDR3可分别依次包含SEQ ID NO:3、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:52、SEQ ID NO:7和SEQ ID NO:53所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:55所示的氨基酸序列，且所述VL可包含SEQ ID NO:64所示的氨基酸序列。
在某些情形中，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:110、SEQ ID NO:1、SEQ ID NO:9、SEQ ID NO:12、SEQ ID NO:32和SEQ ID NO:14中任一项所示的氨基酸序列，且所述VL可包含SEQ ID NO:111、SEQ ID NO:2、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:33、SEQ ID NO:34和SEQ ID NO:13中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:54所示的氨基酸序列，且所述VL可包含SEQ ID NO:63所示的氨基酸序列。
在某些情形中，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:110、SEQ ID NO:9、SEQ ID NO:12和SEQ ID NO:14中任一项所示的氨基酸序列，且所述VL可包含SEQ ID NO:111、SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:13中任一项所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:9所示的氨基酸序列，且所述VL可包含SEQ ID NO:10所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:9所示的氨基酸序列，且所述VL可包含SEQ ID NO:11所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:12所示的氨基酸序列，且所述VL可包含SEQ ID NO:13所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:14所示的氨基酸序列，且所述VL可包含SEQ ID NO:11所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:1所示的氨基酸序列，且所述VL可包含SEQ ID NO:2所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:32所示的氨基酸序列，且所述VL可包含SEQ ID NO:34所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:32所示的氨基酸序列，且所述VL可包含SEQ ID NO:33所示的氨基酸序列。
本申请所述分离的抗原结合蛋白还可包含重链恒定区和轻链恒定区，所述重链恒定区可以来自人IgG1的恒定区，所述轻链恒定区可以来自人κ轻链恒定区。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:9所示的氨基酸序列，且所述VL可包含SEQ ID NO:10所示的氨基酸序列；且，所述分离的抗原结合蛋白可包含重链恒定区和轻链恒定区，所述重链恒定区可包含SEQ ID NO:29所示的氨基酸序列，且所述轻链恒定区可包含SEQ ID NO:31所示的氨基酸序列，该分离的抗原结合蛋白可称为HB0030。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:9所示的氨基酸序列，且所述VL可包含SEQ ID NO:11所示的氨基酸序列；且，所述分离的抗原结合蛋白可包含重链恒定区和轻链恒定区，所述重链恒定区可包含SEQ ID NO:29所示的氨基酸序列，且所述轻链恒定区可包含SEQ ID NO:31所示的氨基酸序列该分离的抗原结合蛋白可称为HB0031。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:12所示的氨基酸序列，且所述VL可包含SEQ ID NO:13所示的氨基酸序列；且，所述分离的抗原结合蛋白可包含重链恒定区和轻链恒定区，所述重链恒定区可包含SEQ ID NO:29所示的氨基酸序列，且所述轻链恒定区可包含SEQ ID NO:31所示的氨基酸序列，该分离的抗原结合蛋白可称为HB0032。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:14所示的氨基酸序列，且所述VL可包含SEQ ID NO:11所示的氨基酸序列；且，所述分离的抗原结合蛋白可包含重链恒定区和轻链恒定区，所述重链恒定区可包含SEQ ID NO:29所示的氨基酸序列，且所述轻链恒定区可包含SEQ ID NO:31所示的氨基酸序列，该分离的抗原结合蛋白可称为HB0033。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:1所示的氨基酸序列，且所述VL可包含SEQ ID NO:2所示的氨基酸序列；且，所述分离的抗原结合蛋白可包含重链恒定区和轻链恒定区，所述重链恒定区可包含SEQ ID NO:30所示的氨基酸序列，且所述轻链恒定区可包含SEQ ID NO:31所示的氨基酸序列该分离的抗原结合蛋白可称为900424。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:32所示的氨基酸序列，且所述VL可包含SEQ ID NO:34所示的氨基酸序列；且，所述分离的抗原结合蛋白可包含重链恒定区和轻链恒定区，所述重链恒定区可包含SEQ ID NO:30所示的氨基酸序列，且所述轻链恒定区可包含SEQ ID NO:31所示的氨基酸序列，该分离的抗原结合蛋白可称为900423。
例如，所述分离的抗原结合蛋白可包含重链可变区VH和轻链可变区VL，所述VH可包含SEQ ID NO:32所示的氨基酸序列，且所述VL可包含SEQ ID NO:33所示的氨基酸序列；且，所述分离的抗原结合蛋白可包含重链恒定区和轻链恒定区，所述重链恒定区可包含SEQ ID NO:30所示的氨基酸序列，且所述轻链恒定区可包含SEQ ID NO:31所示的氨基酸序列，该分离的抗原结合蛋白可称为900428。
本申请所述的分离的抗原结合蛋白，能够与参比抗体竞争结合所述TIGIT蛋白，其中所述参比抗体可包含重链可变区和轻链可变区，所述参比抗体的重链可变区可以包含HCDR1、HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:3所示的氨基酸序列；所述HCDR2可以包含SEQ ID NO:56所示的氨基酸序列；所述HCDR3可以包含SEQ ID NO:57所示的氨基酸序列，所述参比抗体的轻链可变区可以包含LCDR1、LCDR2和LCDR3，所述LCDR1可以包含SEQ ID NO:65所示的氨基酸序列；所述LCDR2可以包含SEQ ID NO:7所示的氨基酸序列；所述LCDR3可以包含SEQ ID NO:66所示的氨基酸序列。
本申请所述的分离的抗原结合蛋白，能够与参比抗体竞争结合所述TIGIT蛋白，其中所述参比抗体可包含重链可变区和轻链可变区，所述参比抗体的重链可变区可以包含HCDR1、HCDR2和HCDR3，所述HCDR1可以包含SEQ ID NO:3所示的氨基酸序列；所述HCDR2可以包含SEQ ID NO:4所示的氨基酸序列；所述HCDR3可以包含SEQ ID NO:5所示的氨基酸序列，所述参比抗体的轻链可变区可以包含LCDR1、LCDR2和LCDR3，所述LCDR1可以包含SEQ ID NO:6所示的氨基酸序列；所述LCDR2可以包含SEQ ID NO:7所示的氨基酸序列；所述LCDR3可以包含SEQ ID NO:8所示的氨基酸序列。
在本申请中涉及的蛋白质、多肽和/或氨基酸序列，还应理解为至少包含以下的范围：与 该所述蛋白质或多肽具备相同或类似功能的变体或同源物。
在本申请中，所述变体可以为，在所述蛋白质和/或所述多肽(例如，本申请所述的抗原结合蛋白)的氨基酸序列中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽。例如，所述功能性变体可包含已经通过至少1个，例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的蛋白质或多肽。所述功能性变体可基本上保持改变(例如取代、缺失或添加)之前的所述蛋白质或所述多肽的生物学特性。例如，所述功能性变体可保持改变之前的所述蛋白质或所述多肽的至少60％，70％，80％，90％，或100％的生物学活性(例如抗原结合能力)。例如，所述取代可以为保守取代。
在本申请中，所述抗原结合蛋白的氨基酸序列的一部分可以与来自特定物种的抗体中相应的氨基酸序列同源，或者属于特定的类别。例如，抗体的可变区及恒定部分均可以来自一个动物物种(如人)的抗体的可变区及恒定区。在本申请中，所述同源物可以为，与所述蛋白质和/或所述多肽(例如，本申请所述的抗原结合蛋白)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)序列同源性的蛋白质或多肽。
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。可以通过以下方式计算“序列同源性百分比”：将两条待比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同源性百分比。为了确定序列同源性百分数而进行的比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。所述同源性也可以通过以下的方法测定：FASTA和BLAST。对FASTA算法的描述可以参见W.R.Pearson和D.J.Lipman的“用于生物学序列比较的改进的工具”，美国国家科学院院刊(Proc.Natl.Acad.Sci.)，85：2444-2448，1988；和D.J.Lipman和W.R.Pearson的“快速灵敏的蛋白质相似性搜索”，Science，227：1435-1441，1989。对BLAST算法的描述可参见S.Altschul、W.Gish、W.Miller、E.W.Myers和D.Lipman的“一种基本的局部对比(alignment)搜索工具”，分子生物学杂志，215：403-410，1990。
本申请所述的抗原结合蛋白(例如，TIGIT抗体)能够特异性结合TIGIT抗原。“特异性 结合”TIGIT抗原的抗原结合蛋白(例如，抗体)通常可以以约1nM的KD值或更高亲和力(例如，1nM、100pM、10pM、2pM或1pM)结合TIGIT，但不结合缺乏TIGIT序列的其它蛋白。例如，“特异性结合”TIGIT的抗体不结合人CD226、人CD155和人CD112。本申请所述的抗原结合蛋白(例如，抗体)能够特异性结合TIGIT抗原或其标记形式(例如，荧光标记的TIGIT抗原)，但不会结合缺乏TIGIT表位的其它蛋白。抗原结合蛋白(例如，抗体)是否结合TIGIT抗原可使用本领域中已知的任何测定法确定。本领域中已知测定结合亲和力的分析的实例包括表面等离子共振(例如，BIACORE)或类似技术(例如，KinExa或OCTET)。在某些情形中，本申请所述的TIGIT抗体还可以与猴和/或鼠的TIGIT交叉反应。例如，通过流式分析技术和酶联免疫反应所检测的。如本文所用，“交叉反应性”是指抗体与来自其它物种的同源蛋白反应的能力。
本申请所述的抗原结合蛋白(例如，TIGIT抗体)能够抑制TIGIT与CD155配体的结合。阻断实验可以使用竞争法进行检测，例如，将所述的抗原结合蛋白(例如，TIGIT抗体)与抗原(或，可表达抗原的细胞)和抗原的配体(或，表达配体的细胞)混合，根据可检测标记的的强度(例如，荧光强度或浓度)反应抗原结合蛋白与抗原的配体竞争性结合抗原的能力。例如，如使用流式细胞仪所检测的，本申请所述的抗原结合蛋白(例如，TIGIT抗体)阻断TIGIT抗原与CD155配体结合的IC50为约0.1μg/ml-0.05μg/ml。
多特异性抗体
另一方面，本申请提供了一种多特异性抗体，其可包含能够特异性结合TIGIT蛋白的第一靶向部分。在本申请中，所述多特异性抗体的所述第一靶向部分可以包含本申请上述的分离的抗原结合蛋白。
在某些情形中，所述多特异性抗体还可包括第二靶向部分。例如，所述第二靶向部分能够特异性结合肿瘤相关抗原。又例如，所述第二靶向部分能够阻断PD-L1和PD-1的相互作用。又例如，所述第二靶向部分能够特异性结合PD-L1蛋白。又例如，所述能够结合PD-L1蛋白的第二靶向部分包括抗体或其抗原结合片段。又例如，所述能够结合PD-L1蛋白的抗原结合片段包括Fab，Fab’，Fv片段，F(ab’) 2，scFv，di-scFv和/或dAb。又例如，所述能够结合PD-L1蛋白的抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
在本申请中，所述多特异性抗体的第二靶向部分可包含能够特异性结合PD-L1蛋白的抗体或其抗原结合片段。
在本申请中，所述能够结合PD-L1蛋白的抗体可包含HCDR3，例如，所述HCDR3可包含SEQ ID NO:102所示的氨基酸序列。
在本申请中，所述能够结合PD-L1蛋白的抗体可包含HCDR2，例如，所述HCDR2可包含SEQ ID NO:101所示的氨基酸序列。
在本申请中，所述能够结合PD-L1蛋白的抗体可包含HCDR1，例如，所述HCDR1可包含SEQ ID NO:100所示的氨基酸序列。
在本申请中，所述能够结合PD-L1蛋白的抗体可包含HCDR1、HCDR2和HCDR3，例如，所述HCDR1、HCDR2和HCDR3可分别包含SEQ ID NO:100、101和102所示的氨基酸序列。
在本申请中，所述能够结合PD-L1蛋白的抗体可包含VH，例如，所述VH可包含SEQ ID NO:106或108所示的氨基酸序列。
在本申请中，所述能够结合PD-L1蛋白的抗体可包含LCDR3，例如，所述LCDR3可包含SEQ ID NO:105所示的氨基酸序列。
在本申请中，所述能够结合PD-L1蛋白的抗体可包含LCDR2，例如，所述LCDR2可包含SEQ ID NO:104所示的氨基酸序列。
在本申请中，所述能够结合PD-L1蛋白的抗体可包含LCDR1，例如，所述LCDR1可包含SEQ ID NO:103所示的氨基酸序列。
在本申请中，所述能够结合PD-L1蛋白的抗体可包含LCDR1、LCDR2和LCDR3，例如，所述LCDR1、LCDR2和LCDR3可分别包含SEQ ID NO:103、104和105所示的氨基酸序列。
在本申请中，所述能够结合PD-L1蛋白的抗体可包含VL，例如，所述VL可包含SEQ ID NO:107或109所示的氨基酸序列。
在本申请中，所述多特异性抗体可包含第一多肽链和第二多肽链。
在本申请中，所述第一多肽链可以包含所述能够结合PD-L1蛋白的抗体的重链可变区VH，所述能够结合TIGIT蛋白的重链可变区VH和所述能够结合TIGIT蛋白的轻链可变区VL；且所述第二多肽链可以包含能够结合所述PD-L1蛋白的抗体的轻链可变区VL。
在某些情形中，所述第一多肽链中所述能够结合TIGIT蛋白的抗体的VH与所述能够结合TIGIT蛋白的抗体的VL可以构成scFv。在某些情形中，所述能够结合TIGIT蛋白的抗体的VH与所述能够结合TIGIT蛋白的抗体的VL可以通过连接肽构成scFv。例如，所述连接肽可以包括如SEQ ID NO:74-77中任一项所示的氨基酸序列。例如，所述能够结合TIGIT蛋白的scFv的序列可以包含SEQ ID NO:97-99中任一项所示的氨基酸序列。
在某些情形中，在所述第一多肽链中，所述能够结合TIGIT蛋白的scFv可以位于所述能够结合PD-L1蛋白的抗体的VH的N端或C端。
例如，在所述第一多肽链中，所述能够结合TIGIT蛋白的抗体的VL的N端可以与所述能够结合PD-L1蛋白的抗体的VH的C端可选地通过连接肽连接，所述能够结合TIGIT蛋白的VL的C端可以与所述能够结合TIGIT蛋白的抗体的VH的N端可选地通过连接肽连接。
又例如，在所述第一多肽链中，所述能够结合TIGIT蛋白的VH的N端可以与所述能够结合PD-L1蛋白的抗体的VH的C端可选地通过连接肽连接，所述能够结合TIGIT蛋白的VH的C端可以与所述能够结合TIGIT蛋白的抗体的VL的N端可选地通过连接肽连接。
在本申请中，所述第一多肽链中还可包含恒定区，例如人IgG恒定区，所述人IgG恒定区可位于所述能够结合PD-L1蛋白的抗体的VH的C端且位于所述能够结合TIGIT蛋白的抗体的scFv的N端。在本申请中，所述人IgG恒定区可位于所述能够结合PD-L1蛋白的抗体的VH的C端且位于所述能够结合TIGIT蛋白的抗体的VL的N端。
在某些情形中，所述人IgG恒定区可与所述能够结合TIGIT蛋白的抗体的VL的N端直接或间接连接，例如，可通过连接肽连接。在另一些情形中，所述人IgG恒定区可位于所述能够结合PD-L1蛋白的抗体的VH的C端且位于所述能够结合TIGIT蛋白的抗体的VH的N端。例如，所述恒定区可包含SEQ ID NO:29或30所示的氨基酸序列。
在某些情形中，所述人IgG恒定区可与所述能够结合TIGIT蛋白的抗体的VH的N端直接或间接连接，例如，可通过连接肽连接。例如，所述连接肽可以包括如SEQ ID NO:74-77中任一项所示的氨基酸序列。
例如，所述第一多肽链可包含SEQ ID NO:85-87中任一项所示的氨基酸序列。
在本申请中，所述第一多肽链可包含所述能够结合PD-L1蛋白的抗体的重链可变区VH；且所述第二多肽链可包含能够结合所述PD-L1蛋白的抗体的轻链可变区VL，所述能够结合TIGIT蛋白的重链可变区VH和所述能够结合TIGIT蛋白的轻链可变区VL。
在某些情形中，所述第二多肽链中所述能够结合TIGIT蛋白的抗体的VH可与所述能够结合TIGIT蛋白的抗体的VL构成scFv。在某些情形中，所述能够结合TIGIT蛋白的抗体的VH可与所述能够结合TIGIT蛋白的抗体的VL通过连接肽构成scFv。
在某些情形中，在所述第二多肽链中，所述能够结合TIGIT蛋白的scFv位于所述能够结合PD-L1蛋白的抗体的VL的N端或C端。
在本申请中，所述第一多肽链包含所述能够结合TIGIT蛋白的抗体的重链可变区VH，所述能够结合PD-L1蛋白的重链可变区VH和所述能够结合PD-L1蛋白的轻链可变区VL；且所述第二多肽链包含能够结合所述TIGIT蛋白的抗体的轻链可变区VL。
在某些情形中，所述第一多肽链中所述能够结合PD-L1蛋白的抗体的VH与所述能够结 合PD-L1蛋白的抗体的VL构成scFv。在某些情形中，所述能够结合PD-L1蛋白的抗体的VH与所述能够结合PD-L1蛋白的抗体的VL通过连接肽构成scFv。例如，所述连接肽可包括如SEQ ID NO:74-77中任一项所示的氨基酸序列。例如，所述能够结合PD-L1蛋白的scFv的序列可以包含SEQ ID NO:89-96中任一项所示的氨基酸序列。
在某些情形中，在所述第一多肽链中，所述能够结合PD-L1蛋白的scFv位于所述能够结合TIGIT蛋白的抗体的VH的N端或C端。
例如，在所述第一多肽链中，所述能够结合PD-L1蛋白的VL的N端与所述能够结合TIGIT蛋白的抗体的VH的C端可选地通过连接肽连接，所述能够结合PD-L1蛋白的VL的C端与所述能够结合PD-L1蛋白的抗体的VH的N端可选地通过连接肽连接。
又例如，在所述第一多肽链中，所述能够结合PD-L1蛋白的VH的N端可以与所述能够结合TIGIT蛋白的抗体的VH的C端可选地通过连接肽连接，所述能够结合PD-L1蛋白的VH的C端可以与所述能够结合PD-L1蛋白的抗体的VL的N端可选地通过连接肽连接。
在本申请中，所述第一多肽链中还可包含人IgG恒定区，所述人IgG恒定区可位于所述能够结合TIGIT蛋白的抗体的VH的C端且位于所述能够结合TIGIT蛋白的抗体的scFv的N端。在本申请中，所述人IgG恒定区可位于所述能够结合TIGIT蛋白的抗体的VH的C端且位于所述能够结合PD-L1蛋白的抗体的VL的N端。例如，所述恒定区可包含SEQ ID NO:29或30所示的氨基酸序列。
在某些情形中，所述人IgG恒定区可与所述能够结合PD-L1蛋白的抗体的VL的N端直接或间接连接，例如，可通过连接肽连接。在另一些情形中，所述人IgG恒定区可位于所述能够结合TIGIT蛋白的抗体的VH的C端且位于所述能够结合PD-L1蛋白的抗体的VH的N端。
在某些情形中，所述人IgG恒定区可与所述能够结合PD-L1蛋白的抗体的VH的N端直接或间接连接，例如，可通过连接肽连接。例如，所述连接肽可以包括如SEQ ID NO:74-77中任一项所示的氨基酸序列。
例如，所述第一多肽链可包含SEQ ID NO:78-83中任一项所示的氨基酸序列。
在本申请中，所述第一多肽链可包含所述能够结合TIGIT蛋白的抗体的重链可变区VH；且所述第二多肽链可包含能够结合所述TIGIT蛋白的抗体的轻链可变区VL，所述能够结合PD-L1蛋白的重链可变区VH和所述能够结合PD-L1蛋白的轻链可变区VL。
在某些情形中，所述第二多肽链中所述能够结合PD-L1蛋白的抗体的VH可与所述能够结合PD-L1蛋白的抗体的VL构成scFv。在某些情形中，所述能够结合PD-L1蛋白的抗体的 VH可与所述能够结合PD-L1蛋白的抗体的VL通过连接子构成scFv。
在某些情形中，在所述第二多肽链中，所述能够结合PD-L1蛋白的scFv位于所述能够结合TIGIT蛋白的抗体的VL的N端或C端。
在本申请中，所述能够结合TIGIT蛋白(或PD-L1蛋白)的抗体的VH与所述能够结合TIGIT蛋白(或PD-L1蛋白)的抗体的VL可以被修饰以有利于形成scFv，例如，可以发生氨基酸突变，但是不影响所述抗体的特异性结合性和结合亲和能力。例如，可以进行氨基酸突变以形成二硫键，是的scFv的结构更加稳定。所述氨基酸突变可以是本领域常见的那些，例如，在框架区的氨基酸突变，又例如，轻链可变区的100位氨基酸Q突变为C，或重链可变区的44位氨基酸R或G突变为C。这些突变前和突变后的VH、VL也在本申请保护的范围内。
例如，在本申请中，所述多特异性抗体可包含如图7A所示的结构，其中所述第一多肽链可包含SEQ ID NO:78-83中任一项所示的氨基酸序列，且所述第二多肽链可包含SEQ ID NO:84所示的氨基酸序列。
例如，在本申请中，所述多特异性抗体可包含如图7B所示的结构，其中所述第一多肽链可包含SEQ ID NO:85-87中任一项所示的氨基酸序列，且所述第二多肽链可包含SEQ ID NO:88所示的氨基酸序列。
核酸、载体和细胞
在另一个方面，本申请还提供了分离的一种或多种核酸分子。所述一种或多种核酸分子可编码本申请所述的的多特异性抗体。例如，所述一种或多种核酸分子中的每一个核酸分子可以编码完整的所述的多特异性抗体，也可以编码其中的一部分(例如，第一靶向部分、第二靶向部分、HCDR1-3、LCDR1-3、VL、VH、轻链或重链中的一种或多种)。
本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。
在本申请中，可以通过本领域已知的多种方法来制备编码所述的多特异性抗体的核酸，这些方法包括但不限于，采用限制性片段操作或采用合成性寡核苷酸的重叠延伸PCR，具体操作可参见Sambrook等人，Molecular Cloning，A Laboratory Manual，Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.，1989；和Ausube等人Current Protocols in Molecular Biology，Greene Publishing and Wiley-Interscience，New York N.Y.，1993。
在另一个方面，本申请提供了一种或多种载体，其包含本申请所述的一种或多种核酸分子。每种载体中可包含一种或多种所述核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。在某些实施方式中，所述表达控制序列为可调的元件。所述表达控制序列的具体结构可根据物种或细胞类型的功能而变化，但通常包含分别参与转录和翻译起始的5’非转录序列和5’及3’非翻译序列，例如TATA盒、加帽序列、CAAT序列等。例如，5’非转录表达控制序列可包含启动子区，启动子区可包含用于转录控制功能性连接核酸的启动子序列。所述表达控制序列还可包括增强子序列或上游活化子序列。在本申请中，适当的启动子可包括，例如用于SP6、T3和T7聚合酶的启动子、人U6RNA启动子、CMV启动子及其人工杂合启动子(如CMV)，其中启动子的某部分可与其他细胞蛋白(如人GAPDH，甘油醛-3-磷酸脱氢酶)基因启动子的某部分融合，其可包含或不包含另外的内含子。本申请所述的一种或多种核酸分子可以与所述表达控制元件可操作地连接。所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。例如，所述载体为表达载体。
在另一个方面，本申请提供了宿主细胞，所述宿主细胞可包含本申请所述的一种或多种核酸分子和/或本申请所述的一种或多种载体。在某些实施方式中，每种或每个宿主细胞可包含一个或一种本申请所述的核酸分子或载体。在某些实施方式中，每种或每个宿主细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体。例如，可将本申请所述的载体引入所述宿主细胞中，例如真核细胞，如来自植物的细胞、真菌或酵母细胞等。可通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。
制备方法
在另一个方面，本申请提供了制备所述的多特异性抗体的方法。所述方法可包括，在使得所述多特异性抗体表达的条件下，培养所述本申请所述的宿主细胞。例如，可通过使用适当的培养基、适当的温度和培养时间等，这些方法是本领域普通技术人员所了解的。
生产本申请的抗人TIGIT抗体的示例性方法描述于实施例1。
人源化抗体可以选自任何种类的免疫球蛋白，包括IgM、IgD、IgG、IgA和IgE。在本申请中，抗体是IgG抗体，使用IgG1亚型。可以通过用下文实施例中描述的生物学测定筛选抗 体实现必需恒定结构域序列的优化，以产生所需生物学活性。同样，任一类轻链都可以在本文的化合物和方法中使用。具体地说，κ、λ链或其变体在本申请的化合物和方法中是可以用的。
人源化本申请的抗人TIGIT抗体的示例性方法描述于实施例2。
本申请的多特异性抗体或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。然后可将该核酸分子引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本申请还涉及包含上述的适当核酸分子以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。例如，动物细胞可以包括(但并不限于)：CHO-S、CHO-K1、HEK-293细胞。
本申请中所述的用重组DNA转化宿主细胞的步骤可用本领域熟知的技术进行。获得的转化子可用常规方法培养，转化子表达本申请的核酸分子所编码的多肽。根据所用的宿主细胞，用常规培养基在合适的条件下培养。通常，在适合本申请多特异性抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本申请的多特异性抗体。
所得单克隆抗体或多特异性抗体可用常规手段来鉴定。比如，单克隆抗体或多特异性抗体的结合特异性可用免疫沉淀或体外结合试验(如流式细胞分选技术(FACS)、放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。
药物组合物
另一方面，本申请还提供了一种组合物。在某些情形中，所述的组合物可以是药物组合物，它含有本申请的多特异性抗体，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中。配制好的药物组合物可以通过常规途径进行给药。
本申请所述的药物组合物可直接用于结合TIGIT蛋白分子或PD-L1蛋白分子，因而可用于预防和治疗TIGIT相关、PD-L1相关或PD-1相关的疾病。本申请的药物组合物可以含有安全有效量的本申请所述的抗原结合蛋白以及药学上可接受的佐剂(可包括载体或赋形剂)。药物制剂应与给药方式相匹配。
本文所述的多特异性抗体或药物组合物可以符合良好医疗实践的方式配制、给药和施用。在此情形下的考虑因素包括所治疗的特定病症、所治疗的特定哺乳动物、单个患者的临床病状、病症的病因、药剂递送部位、施用方法、施用排程和医学从业者已知的其他因素。
方法和用途
另一方面，本申请提供了所述多特异性抗体、所述核酸分子、所述载体、所述细胞和/或所述药物组合物在制备药物中的用途。所述药物用于预防、缓解和/或治疗疾病或病症，例如TIGIT异常相关的疾病。在某些情形中，所述TIGIT异常相关的疾病可以是T细胞功能障碍性病症。T细胞功能障碍体现在T细胞耗尽，是通过增强NK细胞和激活T细胞，增强机体免疫活性实现对疾病的治疗或延迟或缓解。例如，所述TIGIT相关的疾病可以是肿瘤、癌症或感染性病症。例如，所述TIGIT异常相关的疾病可以是CD155阳性或PVR阳性的肿瘤、癌症、免疫性疾病或感染性病症，包括肿瘤、癌症、免疫性疾病或感染性病症等。在某些情形中，所述肿瘤可以为实体瘤或非实体瘤。例如，所述肿瘤可以为结肠癌。
本申请的抗原结合蛋白可以抑制肿瘤生长和/或抑制肿瘤细胞增殖。在某些实施方式中，所述肿瘤包含结肠癌。在某些实施方式中，所述肿瘤或癌症为TIGIT表达异常的肿瘤或癌症。
例如，在结肠癌小鼠模型中，本申请所述的抗原结合蛋白(例如，TIGIT抗体)能够减缓肿瘤生长。
另一方面，本申请还提供了抑制CD155与TIGIT结合的方法，所述方法包括施用所述的多特异性抗体。
另一方面，本申请还提供了抑制PD-L1与PD-1结合的方法，所述方法包括施用所述的多特异性抗体。
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抗原结合蛋白、制备方法和用途等，而不用于限制本申请的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例
实施例1抗人TIGIT的小鼠单克隆抗体——原始抗体的制备方法
1.1制备产生鼠源单克隆抗体的杂交瘤细胞
制备鼠源单克隆抗体的方法采用Kohler和Milstein 1975年发明的杂交瘤制备技术(Nature，1975，256：495-497)。首先将人TIGIT带有鼠Fc标签蛋白(ACRO，#TIT-H5253)与弗氏佐剂乳化，然后对BALB/c、CD1、C57BL/6每个品系各5只小鼠进行多点皮下免疫。三轮免疫后取血清用ELISA法检测效价，FACS检测结合活性及功能活性，挑选最佳小鼠取脾细胞与SP2/0骨髓瘤细胞进行融合。经过HAT筛选杂交瘤多克隆细胞，采用ELISA、FACS方法，筛选出特异性结合人TIGIT且可以阻断TIGIT-CD155结合的多克隆细胞株后进行单克隆化，再次使用ELISA、FACS方法筛选特异性结合的单克隆细胞株，并检测了与猴、小鼠TIGIT结合力，通过FACS筛选出阻断TIGIT-CD155与TIGIT-CD122结合的单克隆抗体，后续进行细胞功能学活性检测，并对筛选出的单克隆细胞株进行亲和力(Biacore)筛选，最终得到表达人TIGIT抗体的单克隆杂交瘤细胞株进行序列分析，筛选数据列举见表1。
表1.杂交瘤筛选数据
Figure PCTCN2021133003-appb-000001
Figure PCTCN2021133003-appb-000002
经过高通量筛选，最终得到的单克隆杂交瘤细胞株具有高亲和力，可以与人和猴都具有结合活性，对TIGIT的两个配体CD155及CD122同时具有阻断功能，并且具有生物活性。将以上表1中的杂交瘤细胞株进行人源化改造。
实施例2抗TIGIT抗体可变区基因序列克隆以及人源化
2.1杂交瘤细胞中抗体的可变区基因克隆
基于TAKARA的5’RACE技术原理，克隆出由杂交瘤细胞株表达的小鼠抗体可变区的cDNA序列。简言之，用SMARTer 5’RACE合成试剂盒(TAKARA，#634859)按说明书合成重链和轻链的可变区基因特异性cDNA。用PCR引物修饰cDNA序列的5’和3’端，所述引物设计成为分别在重链和轻链可变区cDNA上增加合适的前导序列，使所得PCR产物能够通过无缝克隆的方法克隆到现有重组抗体表达的重链载体pHB-Fc和轻链载体pHB-Cκ上。pHB-Fc表达载体上含有人IgG1重链恒定区基因序列，其中CH 2上带有抗体ADCC knock out(KO)效应的L234A和L235A(Eu编码)突变；pHB-Cκ载体上含有人κ轻链恒定区基因序列。将重链和轻链可变区PCR扩增产物通过In-fusion克隆试剂(TAKARA,#639650)克隆到表达载体上，并转化到E.coli DH5α大肠杆菌感受态细胞(益生生技，#FYE607-80VL)中。通过挑选单克隆菌落进行Sanger测序，经分析获得抗体可变区序列。其中B3/29F6表达的抗TIGIT抗体可变区序列如下：
B3/29F6 VH SEQ ID NO:1
Figure PCTCN2021133003-appb-000003
B3/29F6 VL SEQ ID NO:2
Figure PCTCN2021133003-appb-000004
其中，下划线区为CDRs(按照IMGT定义，序列分别如下)：
表2.鼠源抗TIGIT抗体CDR序列
Figure PCTCN2021133003-appb-000005
Figure PCTCN2021133003-appb-000006
2.2 IgG1野生型嵌合表达载体构建
IgG1野生型为嵌合抗体和ADCC KO型嵌合抗体共用相同的轻链载体，不同之处在于所用含有人重链恒定区序列的载体上重链CH 2的234和235位氨基酸，ADCC KO型为A234/A235，野生型为L234/L235。IgG1野生型嵌合重链表达载体的方法与2.1中所述表达载体构建方法原理相同，即通过含有前导序列的引物PCR扩增重链可变区基因，再用无缝连接方法克隆到含有人重链恒定区序列(CH2为L234/L235)的载体上，完成IgG1野生型嵌合表达载体的构建。
2.3嵌合抗体的表达
2.1和2.2中所得表达载体经过大肠杆菌扩增，用去内毒素质粒抽提试剂盒(天根生化科技(北京)有限公司，#DP117)制备足量质粒，用于瞬时转染表达嵌合抗体。表达所用的宿主细胞为CHO-S细胞(赛默飞，#R80007)。通过将制备所得的两种重链载体分别和轻链载体一起，与聚醚酰亚胺(PEI，Polysciences，#24765-1)混合形成脂质体复合物后，转染CHO-S细胞，放入培养箱中培养5-7天。离心收集细胞培养液上清，通过Protein A亲和层析柱纯化得到ADCC KO型人鼠嵌合抗体(编号为900424)和IgG1野生型人鼠嵌合抗体(蛋白编号900445)。
按照上述方法得到的嵌合抗体VH和VH序列分别如下：
900423和900428 VH SEQ ID NO:32
Figure PCTCN2021133003-appb-000007
900424 VH SEQ ID NO:1
Figure PCTCN2021133003-appb-000008
900424 VL SEQ ID NO:2
Figure PCTCN2021133003-appb-000009
900423 VL SEQ ID NO:34
Figure PCTCN2021133003-appb-000010
900428 VL SEQ ID NO:33
Figure PCTCN2021133003-appb-000011
其中，900424、900423和900428的重链恒定区氨基酸序列均如SEQ ID NO:30所示。轻链恒定区均为人κ轻链恒定区(SEQ ID NO:31)。
2.4鼠源抗人TIGIT抗体的人源化——人源化抗体的制备方法
抗体的人源化采用以下方法。将抗体的可变区序列与NCBI IgBlast数据库中的可用序列比较，通过鉴定和分析，最终确定了适合在其上构建CDR移植重链和轻链的人源构架区(FR区)。
改造时，根据人抗体FR区保守的氨基酸残基以及抗体FR区中重要的氨基酸残基，设计改造位点，对嵌合抗体的重轻链的可变区分别进行人源化突变设计，利用PCR技术扩增并构建人源化点突变抗体表达质粒。将人源化点突变抗体表达质粒分别经CHO-S细胞表达，纯化后得到人源化抗体蛋白。利用ELISA，Biacore和流式细胞术等检测方法，对人源化抗体的受体结合能力，功能抑制活性和ADCC效应等指标进行筛选，获得了四个性能优异的人源化抗TIGIT抗体。所获得人源化抗TIGIT抗体的VH和VL序列下所示：
900461和900464 VH SEQ ID NO:9
Figure PCTCN2021133003-appb-000012
900461 VL SEQ ID NO:10
Figure PCTCN2021133003-appb-000013
900464和900476 VL SEQ ID NO:11
Figure PCTCN2021133003-appb-000014
900466 VH SEQ ID NO:12
Figure PCTCN2021133003-appb-000015
Figure PCTCN2021133003-appb-000016
900466 VL SEQ ID NO:13
Figure PCTCN2021133003-appb-000017
900476VH SEQ ID NO:14
Figure PCTCN2021133003-appb-000018
其中，下划线区为CDRs(按照IMGT定义)，900461、900464、900466和900476分别为四个人源化抗体蛋白编号，对应项目编号为HB0030、HB0031、HB0032和HB0033。
其中，HB0030、HB0031、HB0032和HB0033的重链恒定区氨基酸序列均如SEQ ID NO:29所示，轻链恒定区均为人κ轻链恒定区(SEQ ID NO:31)。
实施例3嵌合抗体及人源化抗体的检测
3.1测试TIGIT抗体对表达人TIGIT的细胞结合活性
嵌合抗体900324、900423、900424、900428，人源化抗体HB0030、HB0031、HB0032、HB0033与表达人TIGIT的细胞(CHOK1-huTIGIT-2A3，华博生物)的结合活性检测。
所有嵌合抗体、人源化抗体均用含1％BSA的PBS溶液(1％BSA/PBS)稀释至20μg/ml，每孔20μL加入96孔U型板中，同步设定阴性对照(只加1％BSA/PBS)。取对数生长期内表达人TIGIT的细胞(CHOK1-huTIGIT-2A3，华博生物)悬液，离心(1000rpm×5min)弃培养液，用1％BSA/PBS重悬至活细胞密度为1×10 6/mL，每孔20μL(2×10 4个细胞)加入已有抗TIGIT抗体的96孔U型板中室温反应30min。将反应后的96孔U型板用1％BSA/PBS重悬，离心(300g×3min)弃上层液，如此洗涤1遍，加入1:200稀释的PE-羊抗人-Fc(Jackson ImmunoResearch，#109-115-098)，室温避光反应15min；将反应后的96孔U型板用1％BSA/PBS重悬，离心(300g×3min)弃上层液，如此洗涤3遍，最终用每孔100μL 1％BSA/PBS重悬，用流式细胞仪(BD，#CantoⅡ)检测PE通道的荧光强度。
嵌合抗体结合活力结果见图1及表3，人源化抗体结合活力结果见图2及表4，其中900324为CHO细胞瞬时表达的来自基因泰克公司的抗人TIGIT抗体Tiragolumab，其轻重链可变区序列与专利申请WO2015009856A2中的抗体10A7的序列相同。结果表明嵌合抗体与人源化抗体均与表达人TIGIT的细胞(CHOK1-huTIGIT-2A3，华博生物)有很好的亲和力，且亲和 力相当。
表3.抗TIGIT嵌合抗体对人TIGIT结合活性
抗体 900428 900424 900423 900324
EC 50(μg/mL) 0.075 0.053 0.06 0.124
表4.抗TIGIT人源化抗体对人TIGIT结合活性
抗体 900324 HB0030 HB0031 HB0032 HB0033
EC 50(μg/mL) 0.08994 0.04661 0.05806 0.07847 0.0514
3.2测试TIGIT抗体阻断TIGIT抗原和TIGIT分子结合的实验(竞争法)
将抗原huCD155-moFc(ACROBiosystems，#CD5-H5254)用含1％BSA的PBS溶液(1％BSA/PBS)稀释至40μg/ml，每孔10μL加入96孔U型板中，与系列稀释的抗TIGIT抗体按体积比1:1混合均匀，同步设定阳性对照(只加CD155-moFc)。取对数生长期内表达人TIGIT的细胞(CHOK1-huTIGIT-2A3，华博生物)悬液，离心(1000rpm×5min)弃培养液，用1％BSA/PBS重悬至活细胞密度为1×10 6/mL，每孔20μL(2×10 4个细胞)加入CD155-moFc与抗TIGIT抗体混匀的96孔U型板中室温反应30min。将反应后的96孔U型板用1％BSA/PBS重悬，离心(300g×3min)弃上层液，如此洗涤1遍，加入1:300稀释的Alexa488-羊抗鼠-Fc(Jackson ImmunoResearch，#115-545-071)，室温避光反应15min；将反应后的96孔U型板用1％BSA/PBS重悬，离心(300g×3min)弃上层液，如此洗涤3遍，最终用每孔100μL1％BSA/PBS重悬，用流式细胞仪(BD，#CantoⅡ)检测FITC通道的荧光强度。
嵌合抗体结合活力结果见图3及表5，人源化抗体结合活力结果见图4及表6。结果表明嵌合抗体和人源化抗体对人的CD155具有显著的阻断作用，且阻断作用力相当。
表5.抗TIGIT嵌合抗体对人TIGIT与其配体CD155结合的阻断活性
抗体 900428 900424 900423 900324
IC 50(μg/mL) 0.069 0.062 0.065 0.174
表6.抗TIGIT人源化抗体对人TIGIT与其配体CD155结合的阻断活性
抗体 900324 HB0030 HB0031 HB0032 HB0033
IC 50(μg/mL) 0.1349 0.05733 0.06653 0.0919 0.05598
3.3人源化单克隆抗体的亲和性检测
本试验使用SPR方法测定抗原-抗体结合动力学及亲和力。采用BIOCORE(GE，#Biacore 8K)。Sereis S Sensor Chip ProteinA芯片(GE，#29-1275-56)室温平衡20～30min，将芯片装入仪器。用平衡缓冲液将抗体样品稀释至实验工作浓度，2～8℃密封备用。用平衡缓冲液HBS-EP(10x)(GE，#BR-1006-69)稀释抗原，组氨酸标记(His-Tag)的人TIGIT蛋白(huTIGIT， ACRO Biosystems，#TIT-H52H3)，稀释抗原20nM起始2.5倍稀释度5个浓度梯度，并设置2个零浓度(即平衡缓冲液)和最低浓度重复。pH1.5甘氨酸溶液(GE，#BR100354)再生芯片。采用捕获法多循环动力学程序分析样品，选用对应的分析程序对数据分析，确认无明显reference binding，选用Kinetics，1:1binding modle，拟合分析，获得样品的动力学参数。
检测结果如表7所示。与huTIGIT的亲和力常数(KD(M))结果显示，本申请的人源化单克隆抗体HB0030的亲和力接近10 -11数量级，具有极强的亲和力。
表7.人源化Mab-TIGIT抗体与hu TIGIT亲和力检测结果
抗体 Ka(1/Ms) Kd(1/s) KD(M)
HB0030 3.17E+06 1.85E-04 5.83E-11
实施例4动物体内药效试验
本实验研究了抗人TIGIT抗体HB0030、HB0031、HB0032、HB0033在免疫检测点人源化小鼠BALB/c-hPD1/hTIGIT皮下接种CT26.WT结肠癌肿瘤模型中的药效。
本实验取用小鼠结肠癌细胞CT26.WT细胞，收集对数生长期的CT26.WT细胞，去除培养液并用PBS洗两次后接种(荷瘤前、荷瘤后CT26.WT细胞存活率分别为：98.57％及98.28％)，接种量：5×10 5/100μL/只。接种后第11天，平均肿瘤体积达到94.02mm 3时，小鼠根据肿瘤体积随机分组，每组8只。分组当天定义为D0天，并于D0、D3、D7、D10、D14、D17进行给药，开始给药后，于D0、D2、D4、D6、D8、D10、D13、D15、D17、D20、D23观测肿瘤大小并称量小鼠体重。瘤体积计算方式为：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)。结果如图5所示。
观察图5实验结果，可见本申请的抗TIGIT人源化单克隆抗体HB0030、HB0031、HB0032、HB0033在单独给药10mg/kg时具有明显的肿瘤抑制效果。
进一步的研究了HB0030与对照抗体Tiragolumab在10mg/kg的剂量下，对免疫检测点人源化小鼠BALB/c-hPD1/hTIGIT皮下接种CT26.WT结肠癌肿瘤模型中的药效。
本实验取用小鼠结肠癌细胞CT26细胞，收集对数生长期的CT26细胞，去除培养液并用PBS洗两次后接种(荷瘤前、荷瘤后CT26细胞存活率分别为：98.51％及97.250％)，接种量：5×10 5/100μL/只，接种第12天，平均肿瘤体积达到80.33mm 3时，小鼠根据肿瘤体积随机分组，每组6只。分组当天定义为D0天，并于D0、D3、D7、D10、D14、D17进行给药，开始给药后，于D0、D2、D4、D6、D8、D10、D13、D15、D17、D20、D23观测肿瘤大小并称量小鼠体重。瘤体积计算方式为：肿瘤体积(mm 3)＝0.5×(肿瘤长径×肿瘤短径 2)。结果如图6所示。
观察图6结果可知，本申请的抗TIGIT抗体HB0030与对照抗体Tiragolumab比较，表现出更好的肿瘤抑制效果。
本研究中所有组小鼠在接种后24天，小鼠体重均无明显变化。
实施例5双特异性抗体的重链和轻链的序列设计
使用HB0030构建PDL1-TIGIT双特异性抗体，双抗的结构模式属于Morrison模式(IgG-scFv)，即在一个IgG抗体的两条重链的C端均连接另一个抗体的scFv片段，其构型如图7A-7B所示。其中，为了区分不同的抗体形式，当TIGIT为完整IgG，PD-L1为scFv时，这种形式的双特异性抗体简称为TIGIT-IgG-PD-L1-scFv，其重链和轻链的主要组成设计如下面的表8；当PD-L1为完整IgG，TIGIT为scFv时，这种形式的双特异性抗体称为PD-L1-IgG-TIGIT-scFv，其重链和轻链的主要组成设计如下面的表9。
表8 TIGIT-IgG-PD-L1-scFv重链和轻链的主要组成设计
Figure PCTCN2021133003-appb-000019
表9 PD-L1-IgG-TIGIT-scFv重链和轻链的主要组成设计
Figure PCTCN2021133003-appb-000020
在表8和表9中：
1、900339抗体的序列来源于专利WO2020199860A1的蛋白900339。900339-VL是指900339的轻链可变区，900339-VH是指900339的重链可变区。HB0030-VL是指HB0030的轻链可变区，HB0030-VH是指HB0030的重链可变区。
2、Q100C是指根据kabat的抗体编码规则，将轻链可变区的100位氨基酸Q突变为C；R44C或G44C是指根据kabat的抗体编码规则，将重链可变区的44位氨基酸R或G突变为C。这两个氨基酸突变之后会形成二硫键，增强scFv区域的稳定性，增加双抗的成药性。
3、Linker1的氨基酸序列：(G4S)3(SEQ ID NO：74)，Linker2的氨基酸序列：(G4S)4(SEQ ID NO：75)，Linker3的氨基酸序列：(G4S)5(SEQ ID NO：76)，Linker4的氨基酸序列：(G4S)6(SEQ ID NO：77)。
实施例6双特异性抗体的表达与纯化
实施例5中所得表达载体经过大肠杆菌扩增，用去内毒素质粒抽提试剂盒(天根生化科技(北京)有限公司，#DP117)制备足量质粒，用于瞬时转染表达双特异性抗体。表达所用的宿主细胞为CHO-S细胞(赛默飞，#R80007)。通过将制备所得的两种重链载体分别和轻链载体一起，与聚醚酰亚胺(PEI，Polysciences，#24765-1)混合形成脂质体复合物后，转染CHO-S细胞，放入培养箱中培养5-7天。离心收集细胞培养液上清，通过Protein A亲和层析柱纯化得到系列双特异性抗体，并通过过分子筛层析进一步判断蛋白的聚合情况。
双特异抗体的表达纯化以及检测情况如图8、图9以及表10所示，检测结果表明对于 TIGIT-IgG-PD-L1-scFv形式的双特异性抗体而言，900683的SEC纯度好；对于PD-L1-IgG-TIGIT-scFv形式的双特异性抗体而言，900693的SEC纯度好。还原和非还原SDS-PAGE图显示无论是对于TIGIT-IgG-PD-L1-scFv形式的双特异性抗体还是PD-L1-IgG-TIGIT-scFv形式的双特异性抗体，都没有明显的降解，表明蛋白质稳定性好。
表10 PDL1-TIGIT双特异性抗体的SEC纯度数据
Figure PCTCN2021133003-appb-000021
实施例7双特异性抗体的动力学参数测定
本试验使用SPR方法测定PDL1-TIGIT双特异性抗体，PDL1亲本单抗900339，TIGIT亲本单抗HB0030与抗原的结合动力学及亲和力。
试验方法如下：
按照Human Antibody Captrue Kit(GE，#BR-1008-39)以及氨基偶联试剂盒(GE，BR-1000-50)偶联法准备Anti-Human Capture-CM5芯片(GE，#BR-1005-30)。将芯片置室温平衡20～30min，装入Biacore 8K仪器；用平衡缓冲液将抗原和抗体释至实验工作浓度；抗原用平衡缓冲液稀释至50nM，再3倍稀释度7个浓度梯度，并设置2个零浓度(即平衡缓冲液)和一个重复浓度(一般为最低浓度重复)；按照抗体，抗原，再生的次序，循环往复对10个抗原浓度(2个零浓度，7个梯度浓度及1个重复浓度)进行实验分析，抗原进样流速30μL/分钟，结合时间120秒，解离时间600秒；分析完成后，选用对应的分析程序分析数据，确认无明显reference binding，选用Kinetics，1:1 binding model，拟合数据，获得双特异性抗体和单抗亲本抗体的动力学相关参数Ka，Kd和KD值。
检测结果如下表11和表12所示，结果表明对于TIGIT-IgG-PD-L1-scFv双特异性抗体而言，双抗对TIGIT的亲和力与亲本HB0030相当，双抗对PD-L1的亲和力略优于亲本900339单抗。对于PD-L1-IgG-TIGIT-scFv双特异性抗体而言，双抗900691、900692和900693对PD-L1的亲和力和亲本单抗900339亲和力相当，900691、900692、900693对TIGIT的亲和力和亲本单抗HB0030相比，亲和力依然在一个数量级内。
表11 TIGIT-IgG-PD-L1-scFv双特异性抗体对抗原的亲和力检测
配体 分析物 ka(1/Ms) kd(1/s) KD(M)
900339 人PD-L1 3.00E+05 2.00E-04 6.71E-9
900681 人PD-L1 2.90E+05 4.27E-04 1.47E-9
900683 人PD-L1 4.69E+05 4.92E-04 1.05E-9
900684 人PD-L1 2.91E+05 3.76E-04 1.29E-9
900685 人PD-L1 1.91E+06 5.27E-04 2.76E-9
900686 人PD-L1 4.69E+05 4.19E-04 8.93E-10
900687 人PD-L1 2.78E+05 3.80E-05 1.37E-9
HB0030 人TIGIT 5.07E+06 1.75E-04 3.46E-11
900681 人TIGIT 3.53E+06 2.45E-04 6.93E-11
900683 人TIGIT 4.10E+06 1.96E-04 4.78E-11
900684 人TIGIT 3.67E+06 1.84E-04 5.02E-11
900685 人TIGIT 4.89E+06 2.10E-04 4.28E-11
900686 人TIGIT 4.19E+06 2.00E-04 4.76E-11
900687 人TIGIT 3.96E+06 2.04E-04 5.15E-11
表12 PD-L1-IgG-TIGIT-scFv双特异性抗体对抗原的亲和力检测
配体 分析物 ka(1/Ms) kd(1/s) KD(M)
900339 人PD-L1 1.99E+05 5.53E-04 2.77E-9
900691 人PD-L1 2.14E+05 4.81E-04 2.24E-9
900692 人PD-L1 2.04E+05 5.70E-04 2.79E-9
900693 人PD-L1 2.19E+05 4.98E-04 2.27E-9
HB0030 人TIGIT 4.82E+06 1.67E-04 3.45E-11
900691 人TIGIT 9.87E+05 9.13E-05 9.25E-11
900692 人TIGIT 1.21E+06 9.11E-05 7.51E-11
900693 人TIGIT 9.30E+05 8.03E-05 8.63E-11
实施例8双特异性抗体的生物学活性测定
8.1 PDL1-TIGIT双特异性抗体对PDL1功能的抑制效果
检测PDL1-TIGIT双特异性抗体对PDL1功能的抑制效果的实验步骤如下：将双特异性抗体以及900339亲本单抗用PBS溶液稀释到10μg/ml，再2倍比例稀释9个梯度。对过表达PD-1的靶细胞Jurkat-NFAT-PD-1-5B8进行计数，重悬为5×10E5/mL，每孔30μl铺到96孔白底板中。对过表达PD-L1的效应细胞CHO-K1-OS8-PD-L1-8D6进行计数，重悬为5×10E5/mL，每孔30μl加入到前面铺有Jurkat-NFAT-PD-1-5B8的96孔白底板中。然后将稀释好的双特异性抗体和亲本对照抗体依次加入到铺好细胞的96孔白底板中，每孔30μl，37℃孵育6h。孵育结束后培养板室温平衡至少15min，然后每孔加入90μL BIO-Glo assay，室温避光反应5min，最后MD i3x酶标仪读值检测信号强度。
PD-L1-IgG-TIGIT-scFv双特异性抗体检测结果如图10以及表13所示，结果表明900691、900692、900693双特异性抗体与PD-L1亲本单抗900339对PD-L1功能的抑制效果相当。
表13荧光报告素酶检测PD-L1-IgG-TIGIT-scFv双特异性抗体对PDL1功能的抑制效果
抗体 900339 900691 900692 900693
EC50(nM) 1.794 1.841 2.091 1.980
8.2 PDL1-TIGIT双特异性抗体对TIGIT功能的抑制效果
检测PDL1-TIGIT双特异性抗体对TIGIT功能的抑制效果的实验步骤如下：将双特异性抗体以及HB0030亲本单抗用PBS溶液稀释到10μg/ml，再2倍比例稀释9个梯度。对过表达TIGIT的靶细胞Jurkat-TIGIT-22G8进行计数，重悬为4×10E6/mL，每孔50μl铺到96孔白底板中。然后将稀释好的双特异性抗体和亲本对照抗体依次加入到铺好细胞的96孔白底板中，每孔50μl，37℃孵育10min。然后加入50μl浓度为20μg/ml PHA和50μl浓度为8ug/ml CD155，混匀，培养箱培养24h。孵育结束后，1500rpm×5min离心去上清，用ELISA试剂盒检测IL-2 的含量。
TIGIT-IgG-PD-L1-scFv双特异性抗体检测结果如图11以及表14所示，结果表明TIGIT-IgG-PD-L1-scFv形式的双特异性抗体与TIGIT亲本单抗HB0030相比对TIGIT功能的抑制效果并无显著差别。
表14 IL2分泌assay检测TIGIT-IgG-PD-L1-scFv双特异性抗体对TIGIT功能的抑制效果
抗体 HB0030 900681 900683 900684 900685 900686 900687
EC50(nM) 5.817 8.959 8.645 11.06 12.09 12.88 11.02
实施例9 PDL1-TIGIT双特异性抗体的热稳定性的检测
PDL1-TIGIT双特异性抗体的热稳定性的实验步骤如下：使用蛋白稳定性分析仪(UNcle,UNCHAINED LABS,US)检测PD-L1-IgG-TIGIT-scFv形式的双特异性抗体900691、900692和900693的熔解温度(Tm)、聚集温度(Tagg)，Tm以及Tagg的升温范围是25℃到95℃，升温速率是0.3℃。
PD-L1-IgG-TIGIT-scFv形式的双特异性抗体的热稳定性检测结果如下表15所示，结果表明PD-L1-IgG-TIGIT-scFv形式的双特异性抗体900691、900692和900693热稳定性数据Tm和Tagg值都处于65℃到70℃范围内，说明这三个双特异性抗体的热稳定性表现好。
表15 PD-L1-IgG-TIGIT-scFv形式的双特异性抗体的热稳定性数据Tm，Tagg值检测
抗体 Tm值(℃) Tagg值(℃)
900691 69.2 66.5
900692 69.4 67.1
900693 70.1 67.5
实施例10双特异性抗体的体内治疗肿瘤的效果检测
本实验利用B-hPD-1/hPD-L1/hTIGIT人源化小鼠建立MC38-hPD-L1结肠癌动物模型并测试受试抗体的药效。首先，建立B-hPD-1/hPD-L1/hTIGIT人源化小鼠MC38-hPD-L1结肠癌动物模型，成瘤小鼠根据小鼠肿瘤体积和体重平均分配到7个实验组中，每组6只，腹腔注射给药，每周给药2次共给药6次。具体给药方案见下表16：
表16给药方案
Figure PCTCN2021133003-appb-000022
Figure PCTCN2021133003-appb-000023
注：900201为IgG1-TNP同型对照；900339为重组人源化抗人PD-L1单克隆抗体；HB0030重组人源化抗人TIGIT单克隆抗体；900693、900692和900691为重组人源化抗人PD-L1/TIGIT单克隆抗体融合蛋白； a：BIW指每周给药2次。
肿瘤体积及肿瘤重量测量：分组后对肿瘤体积进行每周2次测量，实验结束时，剥取小鼠肿瘤称重，计算肿瘤体积抑制率TGITV并进行统计学分析，评价受试药物对模型小鼠的抑瘤作用；
体重及一般临床观察：试验期间进行每周2次体重测量，适应性饲养期和实验期间进行每天1次一般临床观察，评价受试药物对模型小鼠的安全性及耐受性。
结果显示，本申请所述的双特异性抗体900693/900692/900691能抑制MC38-hPD-L1结肠癌小鼠的肿瘤体积和肿瘤重量增长，且具有一定的安全性及耐受性。
实施例11双特异性抗体的体内治疗肿瘤的效果检测
本实验利用小鼠结直肠癌细胞CT26-hPDL1皮下移植人源化小鼠BALB/C-hPD1/hPDL1/hTIGIT小鼠模型评价双特异性抗体在体内治疗肿瘤的效果。
将小鼠结直肠癌CT26-hPDL1细胞接种于雌性人源化BALB/C-hPD1/hPDL1/hTIGIT小鼠右侧背部皮下。当荷瘤鼠肿瘤生长平均达到70.03mm 3时，根据肿瘤体积随机分成4组，每组6只，分别为900201(对照抗体，3mg/kg)、900339(hPDL1抗体，3mg/kg)、HB0030(TIGIT抗体，3mg/kg)和900693(双特异性抗体,4.1mg/kg)组。分组后，各组小鼠均腹腔注射给药，每周2次，给药3周共6次。每周测量肿瘤体积及体重，记录荷瘤鼠体重和肿瘤体积变化与给药时间的关系。
结果如图12所示(其中G1-G4分别依次表示900201、900458、HB0030和900693的施用结果)，图12的结果说明，本申请的双特异性抗体在小鼠结直肠癌细胞CT26-hPDL1皮下 移植人源化小鼠BALB/C-hPD1/hPDL1/hTIGIT中表现显著的抗肿瘤作用，同时优于900458和HB0030，表现潜在的临床前景。

Claims (94)

  1. 多特异性抗体，其包含能够特异性结合TIGIT蛋白的第一靶向部分，其中所述第一靶向部分包含分离的抗原结合蛋白，所述分离的抗原结合蛋白具有下述性质中的一种或多种：
    1)能够以1×10 -10M或更低的KD值结合TIGIT蛋白，其中所述KD值通过表面等离子体共振法测定；
    2)在FACS测定中，能够阻断CD155与TIGIT的结合；和
    3)能够抑制肿瘤生长和/或肿瘤细胞增殖。
  2. 根据权利要求1所述的多特异性抗体，其中所述能够结合TIGIT蛋白的抗原结合蛋白包含重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:55所示的氨基酸序列。
  3. 根据权利要求1-2中任一项所述的多特异性抗体，其中所述能够结合TIGIT蛋白的抗原结合蛋白包含轻链可变区VL中的至少一个CDR，所述VL包含SEQ ID NO:64所示的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的多特异性抗体，其中所述能够结合TIGIT蛋白的抗原结合蛋白包括抗体或其抗原结合片段。
  5. 根据权利要求4所述的多特异性抗体，其中所述抗原结合片段包括Fab，Fab’，Fv片段，F(ab’)2，scFv，di-scFv和/或dAb。
  6. 根据权利要求4-5中任一项所述的多特异性抗体，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。
  7. 根据权利要求1-6中任一项所述的多特异性抗体，其中所述能够结合TIGIT蛋白的抗原结合蛋白与参比抗体竞争结合所述TIGIT蛋白，其中所述参比抗体包含重链可变区和轻链可变区，所述参比抗体的重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:3所示的氨基酸序列；所述HCDR2包含SEQ ID NO:4或42所示的氨基酸序列；所述HCDR3包含SEQ ID NO:5或43所示的氨基酸序列，所述参比抗体的轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:6或52所示的氨基酸序列；所述LCDR2包含SEQ ID NO:7所示的氨基酸序列；所述LCDR3包含SEQ ID NO:8或53所示的氨基酸序列。
  8. 根据权利要求1-7中任一项所述的多特异性抗体，其中所述能够结合TIGIT蛋白的抗原结合蛋白包含HCDR1，HCDR2和HCDR3，其中所述HCDR3包含SEQ ID NO:57所示的氨基酸序列。
  9. 根据权利要求8所述的多特异性抗体，其中所述HCDR3包含SEQ ID NO:5或43所示的氨基酸序列。
  10. 根据权利要求8-9中任一项所述的多特异性抗体，其中所述HCDR2包含SEQ ID NO:56所示的氨基酸序列。
  11. 根据权利要求8-10中任一项所述的多特异性抗体，其中所述HCDR2包含SEQ ID NO:4或42所示的氨基酸序列。
  12. 根据权利要求8-11中任一项所述的多特异性抗体，其中所述HCDR1包含SEQ ID NO:3所示的氨基酸序列。
  13. 根据权利要求1-12中任一项所述的多特异性抗体，其中所述能够结合TIGIT蛋白的抗原结合蛋白包括框架区H-FR1，H-FR2，H-FR3和H-FR4。
  14. 根据权利要求13所述的多特异性抗体，其中所述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:58所示的氨基酸序列。
  15. 根据权利要求13或14所述的多特异性抗体，其中所述H-FR1包含SEQ ID NO:23和35中任一项所示的氨基酸序列。
  16. 根据权利要求13-15中任一项所述的多特异性抗体，其中所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:60所示的氨基酸序列。
  17. 根据权利要求13-16中任一项所述的多特异性抗体，其中所述H-FR2包含SEQ ID NO:24、25、26、36和37中任一项所示的氨基酸序列。
  18. 根据权利要求13-17中任一项所述的多特异性抗体，其中所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:61所示的氨基酸序列。
  19. 根据权利要求13-18中任一项所述的多特异性抗体，其中所述H-FR3包含SEQ ID NO:27、38和39所示的氨基酸序列。
  20. 根据权利要求13-19中任一项所述的多特异性抗体，其中所述H-FR4的N末端与所述HCDR3的C末端相连，且包含SEQ ID NO:62所示的氨基酸序列。
  21. 根据权利要求13-20中任一项所述的多特异性抗体，其中所述H-FR4包含SEQ ID NO:28、40和41中任一项所示的氨基酸序列。
  22. 根据权利要求1-21中任一项所述的多特异性抗体，其中所述能够结合TIGIT蛋白的抗原结合蛋白包含VH，且所述VH包含SEQ ID NO:55所示的氨基酸序列。
  23. 根据权利要求22所述的多特异性抗体，其中所述VH包含SEQ ID NO:110、1、9、12、14和32中任一项所示的氨基酸序列。
  24. 根据权利要求1-23中任一项所述的多特异性抗体，其中所述能够结合TIGIT蛋白的抗原结合蛋白包括抗体重链恒定区。
  25. 根据权利要求24所述的多特异性抗体，其中所述抗体重链恒定区源自人IgG恒定区。
  26. 根据权利要求1-25中任一项所述的多特异性抗体，其中所述能够结合TIGIT蛋白的抗原结合蛋白包含LCDR1，LCDR2和LCDR3，其中所述LCDR1包含SEQ ID NO:65所示的氨基酸序列。
  27. 根据权利要求26所述的多特异性抗体，其中所述LCDR1包含SEQ ID NO:6和52中任一项所示的氨基酸序列。
  28. 根据权利要求26或27所述的多特异性抗体，其中所述LCDR2包含SEQ ID NO:7所示的氨基酸序列。
  29. 根据权利要求26-28中任一项所述的多特异性抗体，其中所述LCDR3包含SEQ ID NO:66所示的氨基酸序列。
  30. 根据权利要求26-29中任一项所述的多特异性抗体，其中所述LCDR3包含SEQ ID NO:8或53所示的氨基酸序列。
  31. 根据权利要求1-30中任一项所述的多特异性抗体，其中所述能够结合TIGIT蛋白的抗原结合蛋白包括框架区L-FR1，L-FR2，L-FR3和L-FR4。
  32. 根据权利要求31所述的多特异性抗体，其中所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:68所示的氨基酸序列。
  33. 根据权利要求31-32中任一项所述的多特异性抗体，其中所述L-FR1包含SEQ ID NO:15、16、44和45中任一项所示的氨基酸序列。
  34. 根据权利要求31-33中任一项所述的多特异性抗体，其中所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:70所示的氨基酸序列。
  35. 根据权利要求31-34中任一项所述的多特异性抗体，其中所述L-FR2包含SEQ ID NO:17、18、46和47中任一项所示的氨基酸序列。
  36. 根据权利要求31-35中任一项所述的多特异性抗体，其中所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:72所示的氨基酸序列。
  37. 根据权利要求31-36中任一项所述的多特异性抗体，其中所述L-FR3包含SEQ ID NO:19-21和48-50中任一项所示的氨基酸序列。
  38. 根据权利要求31-37中任一项所述的多特异性抗体，其中所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4包含SEQ ID NO:73所示的氨基酸序列。
  39. 根据权利要求31-38中任一项所述的多特异性抗体，其中所述L-FR4包含SEQ ID NO:22或51中任一项所示的氨基酸序列。
  40. 根据权利要求1-39中任一项所述的多特异性抗体，其中所述能够结合TIGIT蛋白的抗原结合蛋白包括VL，且所述VL包含SEQ ID NO:64所示的氨基酸序列。
  41. 根据权利要求40所述的多特异性抗体，其中所述VL包含SEQ ID NO:111、2、10、11、13、33和34中任一项所示的氨基酸序列。
  42. 根据权利要求1-41中任一项所述的多特异性抗体，其中所述能够结合TIGIT蛋白的抗原结合蛋白包括抗体轻链恒定区。
  43. 根据权利要求42所述的多特异性抗体，其中所述抗体轻链恒定区来自人κ轻链恒定区。
  44. 根据权利要求1-43中任一项所述的多特异性抗体，其还包括第二靶向部分。
  45. 根据权利要求44所述的多特异性抗体，其中所述第二靶向部分能够特异性结合肿瘤相关抗原。
  46. 根据权利要求44或45所述的多特异性抗体，其中所述第二靶向部分能够阻断PD-L1和PD-1的相互作用。
  47. 根据权利要求44-46中任一项所述的多特异性抗体，其中所述第二靶向部分能够特异性结合PD-L1蛋白。
  48. 根据权利要求47所述的多特异性抗体，其中所述能够结合PD-L1蛋白的第二靶向部分包括抗体或其抗原结合片段。
  49. 根据权利要求48所述的多特异性抗体，其中所述能够结合PD-L1蛋白的抗原结合片段包括Fab，Fab’，Fv片段，F(ab’)2，scFv，di-scFv和/或dAb。
  50. 根据权利要求48-49中任一项所述的多特异性抗体，其中所述能够结合PD-L1蛋白的抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
  51. 根据权利要求48-50中任一项所述的多特异性抗体，其中所述能够结合PD-L1蛋白的抗体包含HCDR1，HCDR2和HCDR3，其中所述HCDR3包含SEQ ID NO:102所示的氨基酸序列。
  52. 根据权利要求51所述的多特异性抗体，其中所述HCDR2包含SEQ ID NO:101所示的氨基酸序列。
  53. 根据权利要求51-52中任一项所述的多特异性抗体，其中所述HCDR1包含SEQ ID NO:100所示的氨基酸序列。
  54. 根据权利要求48-53中任一项所述的多特异性抗体，其中所述能够结合PD-L1蛋白的抗体包括重链可变区VH，所述VH包含SEQ ID NO:108或110所示的氨基酸序列。
  55. 根据权利要求48-54中任一项所述的多特异性抗体，其中所述能够结合PD-L1蛋白的抗 体包含LCDR1，LCDR2和LCDR3，其中所述LCDR3包含SEQ ID NO:105所示的氨基酸序列。
  56. 根据权利要求55所述的多特异性抗体，其中所述LCDR2包含SEQ ID NO:104所示的氨基酸序列。
  57. 根据权利要求55-56中任一项所述的多特异性抗体，其中所述LCDR1包含SEQ ID NO:103所示的氨基酸序列。
  58. 根据权利要求48-57中任一项所述的多特异性抗体，其中所述能够结合PD-L1蛋白的抗体包括轻链可变区VL，所述VL包含SEQ ID NO:107或109所示的氨基酸序列。
  59. 根据权利要求1-58中任一项所述的多特异性抗体，其包含第一多肽链和第二多肽链，其中，所述第一多肽链包含所述能够结合PD-L1蛋白的抗体的重链可变区VH，所述能够结合TIGIT蛋白的重链可变区VH和所述能够结合TIGIT蛋白的轻链可变区VL；且所述第二多肽链包含能够结合所述PD-L1蛋白的抗体的轻链可变区VL。
  60. 根据权利要求59所述的多特异性抗体，所述第一多肽链中所述能够结合TIGIT蛋白的抗体的VH与所述能够结合TIGIT蛋白的抗体的VL构成scFv。
  61. 根据权利要求60所述的多特异性抗体，其中在所述第一多肽链中，所述能够结合TIGIT蛋白的scFv位于所述能够结合PD-L1蛋白的抗体的VH的N端或C端。
  62. 根据权利要求59-61中任一项所述的多特异性抗体，其中所述第一多肽链中还包含人IgG恒定区，所述人IgG恒定区位于所述能够结合PD-L1蛋白的抗体的VH的C端且位于所述能够结合TIGIT蛋白的抗体的VL的N端。
  63. 根据权利要求60-62中任一项所述的多特异性抗体，其中所述scFv包含连接肽。
  64. 根据权利要求63所述的多特异性抗体，其中所述连接肽包含SEQ ID NO:74-77中任一项所示的氨基酸序列。
  65. 根据权利要求60-64中任一项所述的多特异性抗体，其中所述scFv的序列包含SEQ ID NO:97-99中任一项所示的氨基酸序列。
  66. 根据权利要求59-65中任一项所述的多特异性抗体，其中所述第一多肽链包含选自下组中任意一项所示的氨基酸序列：SEQ ID NO:85-87。
  67. 根据权利要求59-66中任一项所述的多特异性抗体，其中所述第二多肽链包含SEQ ID NO:88所示的氨基酸序列。
  68. 根据权利要求1-67中任一项所述的多特异性抗体，其包含第一多肽链和第二多肽链，其中，所述第一多肽链包含所述能够结合TIGIT蛋白的抗体的重链可变区VH，所述能够结合 PD-L1蛋白的重链可变区VH和所述能够结合PD-L1蛋白的轻链可变区VL；且所述第二多肽链包含能够结合所述TIGIT蛋白的抗体的轻链可变区VL。
  69. 根据权利要求68所述的多特异性抗体，所述第一多肽链中所述能够结合PD-L1蛋白的抗体的VH与所述能够结合PD-L1蛋白的抗体的VL构成scFv。
  70. 根据权利要求69所述的多特异性抗体，其中在所述第一多肽链中，所述能够结合PD-L1蛋白的scFv位于所述能够结合TIGIT蛋白的抗体的VH的N端或C端。
  71. 根据权利要求68-70中任一项所述的多特异性抗体，其中所述第一多肽链中还包含人IgG恒定区，所述人IgG恒定区位于所述能够结合TIGIT蛋白的抗体的VH的C端且位于所述能够结合PD-L1蛋白的抗体的VL的N端。
  72. 根据权利要求69-71中任一项所述的多特异性抗体，其中所述scFv包含连接肽。
  73. 根据权利要求72所述的多特异性抗体，其中所述连接肽包含SEQ ID NO:74-77中任一项所示的氨基酸序列。
  74. 根据权利要求69-73中任一项所述的多特异性抗体，其中所述scFv的序列包含SEQ ID NO:89-96中任一项所示的氨基酸序列。
  75. 根据权利要求68-74中任一项所述的多特异性抗体，其中所述第一多肽链包含选自下组中任意一项所示的氨基酸序列：SEQ ID NO:78-83。
  76. 根据权利要求59-75中任一项所述的多特异性抗体，其中所述第二多肽链包含SEQ ID NO:84所示的氨基酸序列。
  77. 根据权利要求1-76中任一项所述的多特异性抗体，其为同二聚体。
  78. 分离的一种或多种核酸分子，其编码权利要求1-77中任一项所述的多特异性抗体。
  79. 载体，其包含根据权利要求78所述的核酸分子。
  80. 细胞，其包含根据权利要求78所述的核酸分子或根据权利要求79所述的载体。
  81. 制备权利要求1-77中任一项所述的多特异性抗体的方法，所述方法包括在使得权利要求1-77中任一项所述的多特异性抗体表达的条件下，培养根据权利要求80所述的细胞。
  82. 药物组合物，其包含权利要求1-77中任一项所述的多特异性抗体、权利要求78所述的核酸分子、权利要求79所述的载体和/或权利要求80所述的细胞，以及任选地药学上可接受的佐剂。
  83. 权利要求1-77中任一项所述的多特异性抗体、权利要求78所述的核酸分子、权利要求79所述的载体、权利要求80所述的细胞和/或权利要求82所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗疾病或病症。
  84. 根据权利要求83所述的用途，所述疾病或病症包括TIGIT相关疾病。
  85. 根据权利要求83-84中任一项所述的用途，所述TIGIT相关疾病为T细胞功能障碍性病症。
  86. 根据权利要求83-85中任一项所述的用途，所述疾病或病症为肿瘤。
  87. 根据权利要求86所述的用途，所述肿瘤为结肠癌。
  88. 预防、缓解或治疗疾病或病症的方法，所述方法包括向有需要的受试者施用权利要求1-77中任一项所述的多特异性抗体。
  89. 根据权利要求88所述的方法，所述疾病或病症包括TIGIT相关疾病。
  90. 根据权利要求88-89中任一项所述的方法，所述TIGIT相关疾病为T细胞功能障碍性病症。
  91. 根据权利要求88-90中任一项所述的方法，所述疾病或病症为肿瘤。
  92. 根据权利要求91所述的方法，所述肿瘤为结肠癌。
  93. 抑制CD155与TIGIT结合的方法，所述方法包括施用权利要求1-77中任一项所述的多特异性抗体。
  94. 抑制PD-L1与PD-1结合的方法，所述方法包括施用权利要求46-77中任一项所述的多特异性抗体。
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