WO2022222530A1 - Anticorps anti-tigit et son utilisation - Google Patents

Anticorps anti-tigit et son utilisation Download PDF

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WO2022222530A1
WO2022222530A1 PCT/CN2021/141417 CN2021141417W WO2022222530A1 WO 2022222530 A1 WO2022222530 A1 WO 2022222530A1 CN 2021141417 W CN2021141417 W CN 2021141417W WO 2022222530 A1 WO2022222530 A1 WO 2022222530A1
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antibody
seq
antigen
amino acid
acid sequence
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PCT/CN2021/141417
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Chinese (zh)
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吴振华
聂磊
王晓泽
李娜
王海彬
陈娟
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浙江博锐生物制药有限公司
海正生物制药有限公司
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Publication of WO2022222530A1 publication Critical patent/WO2022222530A1/fr

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • 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
    • 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
    • A61K2039/507—Comprising a combination of two or more separate 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
    • C07K2317/50—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
    • C07K2317/70—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
    • C—CHEMISTRY; METALLURGY
    • 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
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biology and relates to an anti-TIGIT antibody.
  • Malignant tumors are one of the major diseases that seriously affect the health of the Chinese population, and the morbidity and mortality of malignant tumors have been on the rise in recent decades.
  • the treatment methods for tumors are still surgery, radiotherapy and chemotherapy.
  • Immunotherapy has completely changed the clinical treatment of advanced malignant tumors, and has also greatly reversed the current status of tumor treatment methods.
  • Immunotherapy represented by immune checkpoint inhibitors such as PD-1/PD-L1 and CTLA-4 can block immunosuppressive signals, thereby promoting the activation of T cells and the immune response to tumors.
  • current immunotherapy suffers from low response rates and drug resistance, suggesting that other immune tolerance mechanisms exist to help tumors escape immune surveillance.
  • TIGIT is an immune co-suppressive receptor, a member of the immunoglobulin superfamily, mainly expressed in effector T cells, memory T cells, regulatory T cells (Treg), and NK cells.
  • the known ligands of TIGIT are CD155 and CD112.
  • CD155 and CD112 are mainly expressed on the surface of immune cells such as DC cells, macrophages, and T cells.
  • CD155 and CD112 are highly expressed on many tumor cells.
  • the TIGIT signaling pathway plays an important role in tumor cells escaping immune surveillance. The binding of TIGIT to CD155 or CD112 can attenuate the antigen-presenting function of DC cells, or directly transmit inhibitory signals to T cells and NK cells.
  • TIGIT signaling pathway When TIGIT is expressed on regulatory T cells, the TIGIT signaling pathway enhances the suppressive function of regulatory T cells, thereby inhibiting the activity of a variety of immune cells. Studies have shown that blocking the TIGIT signaling pathway can enhance the activity of effector T cells and NK cells, and promote the expression of IFN ⁇ by immune cells. Targeting TIGIT mAbs can slow or inhibit tumor growth in mouse tumor models, and clinically, the combination of anti-TIGIT mAbs with other immune checkpoint inhibitors can improve patient response rates and reduce disease risk. Therefore, specific anti-TIGIT mAb can enhance the anti-tumor immune response and is an effective strategy to realize tumor immunotherapy.
  • the purpose of the present invention is to provide a novel anti-TIGIT antibody, which can specifically bind to TIGIT and has great potential for tumor therapy.
  • the present invention provides an anti-TIGIT antibody or antigen-binding fragment thereof that binds TIGIT or a fragment thereof, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising Three CDRs, respectively VH CDR1, VH CDR2 and VH CDR3, the light chain variable region includes three CDRs, respectively VL CDR1, VL CDR2 and VL CDR3; wherein,
  • VH CDR1 The amino acid sequence of VH CDR1 is shown in SEQ ID NO: 3, or has more than 95% sequence identity with SEQ ID NO: 3, such as 96%, 97%, 98%, 99%;
  • VH CDR2 The amino acid sequence of VH CDR2 is shown in SEQ ID NO: 4, or more than 95%, such as 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 4;
  • VH CDR3 The amino acid sequence of VH CDR3 is shown in SEQ ID NO: 5, or more than 95%, such as 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 5;
  • VL CDR1 The amino acid sequence of VL CDR1 is shown in SEQ ID NO: 8, or more than 95%, such as 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 8;
  • VL CDR2 The amino acid sequence of VL CDR2 is shown in SEQ ID NO: 9, or more than 95%, such as 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 9;
  • VL CDR3 The amino acid sequence of VL CDR3 is shown in SEQ ID NO: 10, or more than 95%, such as 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 10.
  • amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 1, or at least 90%, at least 95%, at least 96%, at least 97%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO:6, or at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
  • the three CDRs contained in the heavy chain variable region, and/or the three CDRs contained in the light chain variable region are defined by the Kabat or Chothia numbering system.
  • the antibody or antigen-binding fragment thereof of the present invention may further comprise one or more of a heavy chain constant region, a light chain constant region.
  • the light chain constant region is a lambda chain or kappa chain constant region.
  • the antibody or antigen-binding fragment thereof is of type IgGl, IgG2, IgG3 or IgG4.
  • the antibody or antigen-binding fragment thereof is a chimeric antibody or a humanized antibody or antigen-binding fragment thereof.
  • an antibody of the invention includes a heavy chain and a light chain, the heavy chain having an amino acid sequence as set forth in SEQ ID NO:11, or at least 90%, at least 95%, at least 96% identical to SEQ ID NO:11 , at least 97%, at least 98% or at least 99% sequence identity; the amino acid sequence of the light chain is shown in SEQ ID NO: 12, or has at least 90%, at least 95%, at least 96% with SEQ ID NO: 12 %, at least 97%, at least 98%, or at least 99% sequence identity.
  • nucleic acid molecule comprising nucleotides encoding an antibody or antigen-binding fragment thereof of the present invention.
  • the nucleic acid molecule encodes a heavy chain variable region and/or a light chain variable region of the antibody or antigen-binding fragment thereof.
  • the nucleic acid molecule encodes a heavy chain variable region, the nucleotide sequence of which is set forth in SEQ ID NO:2, or at least 90%, at least 95%, at least 96% identical to SEQ ID NO:2 %, at least 97%, at least 98%, or at least 99% sequence identity.
  • the nucleic acid molecule encodes a light chain variable region, the nucleotide sequence of which is as shown in SEQ ID NO:7, or at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
  • the present invention provides a biological material, which is:
  • a vector, host cell or microorganism, etc. comprising the nucleic acid molecule of the present invention.
  • the present invention provides a composition comprising the antibody or antigen-binding fragment thereof of the present invention; preferably, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier.
  • a method for preparing an antibody or antigen-binding fragment thereof of the present invention comprising: culturing the above-described host cell to express the antibody or antigen-binding fragment, and isolating the antibody or antigen-binding fragment from the host cell.
  • a pharmaceutical combination comprising an antibody or antigen-binding fragment thereof of the invention and one or more other therapeutic agents; preferably, the other therapeutic agent is another antibody, such as an anti-PD-L1 antibody .
  • an antibody of the present invention or an antigen-binding fragment thereof or a nucleic acid molecule of the present invention or a biological material of the present invention or a composition of the present invention or a pharmaceutical combination of the present invention is provided in preparation for blocking the binding of TIGIT to its ligand.
  • the present invention provides a method of stimulating an immune response or enhancing immune cell activity in an individual, comprising administering to an individual in need thereof an anti-TIGIT antibody or antigen-binding fragment thereof of the present invention, or a composition of the present invention, or the present invention drug combination.
  • the present invention provides a method of treating a tumor or blocking the binding of TIGIT to CD155 or CD112, comprising administering to an individual in need thereof an anti-TIGIT antibody or antigen-binding fragment thereof of the present invention, or a composition of the present invention, or the present invention drug combination.
  • the tumor is leukemia, lymphoma, bladder cancer, breast cancer, head and neck cancer, gastric cancer, melanoma, pancreatic cancer, colorectal cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, prostate cancer, Ovarian cancer, thyroid cancer, glioma, cervical cancer, nasopharyngeal cancer.
  • the anti-TIGIT antibody and the antigen-binding fragment thereof of the present invention are blocking antibodies, which can effectively block the binding of TIGIT and CD155 or CD122, thereby effectively promoting the activation of T cells and enhancing the anti-tumor immune response, and have good drug prospects.
  • Figure 1 shows binding of murine antibodies to TIGIT protein.
  • Figure 2 shows binding of murine antibodies to TIGIT on cell membranes.
  • Figure 3 shows the blocking effect of murine antibodies on the binding of TIGIT to CD155.
  • FIG. 4 shows the blocking effect of murine antibodies on the binding of TIGIT to CD112.
  • Figure 5 shows the binding of chimeric antibody c7D3 to TIGIT protein.
  • Figure 6 shows the blocking effect of the chimeric antibody c7D3 on the binding of TIGIT to CD155.
  • Figure 7 shows the blocking effect of chimeric antibody c7D3 on binding of TIGIT to CD112.
  • Figure 8 shows that the chimeric antibody c7D3 promotes the expression of T-cell activation-related reporter genes in the TIGIT/CD155 reporter gene assay.
  • Figure 9 shows that chimeric antibody c7D3 activates PBMC cells to release IL-2 cytokine.
  • Figure 10 shows that chimeric antibody c7D3 activates PBMC cells to release IFN- ⁇ cytokines.
  • an antibody refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or fully synthetically (eg recombinantly) produced, including at least the retained full length of a portion of the variable region of an immunoglobulin molecule Any fragment of the binding specificity of an immunoglobulin.
  • an antibody includes any protein having a binding domain that is homologous or substantially homologous to the antigen binding domain of an immunoglobulin (the antibody binding site).
  • Antibodies encompass a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), antibody fragments, synthetic antibodies, recombinantly produced antibodies, human antibodies, non-human antibodies (such as murine antibodies), humanized antibodies, chimeric antibodies, intrabodies, and antibody fragments such as, but not limited to, Fab fragments, Fab' fragments, F(ab') 2 fragments, Fv fragments, Fd fragments, Fd' fragments , a single-chain Fv (scFv), a single-chain Fab (scFab), or an antigen-binding fragment of any of the foregoing antibodies.
  • Fab fragments fragments, Fab' fragments, F(ab') 2 fragments, Fv fragments, Fd fragments, Fd' fragments , a single-chain Fv (scFv), a single-chain Fab (scFab), or an antigen-binding fragment of any of the
  • Antibodies provided herein include any immunoglobulin class (eg, IgG, IgM, IgD, IgE, IgA, and IgY), any class (eg, IgGI, IgG2, IgG3, IgG4, I.A1, and IgA2) or subclass (eg, IgGI, IgG2, IgG3, IgG4, I.A1, and IgA2) , IgG2a and IgG2b).
  • the antibodies of the invention are murine or chimeric antibodies.
  • an "antibody fragment” or “antigen-binding fragment” of an antibody refers to any portion of a full-length antibody that is less than full-length, but which comprises at least a portion of the variable region (eg, one or more of the variable regions of said antibody that binds an antigen) CDRs and/or one or more antibody binding sites), and thus retain binding specificity and at least part of the specific binding capacity of the full-length antibody.
  • an antigen-binding fragment refers to an antibody fragment comprising an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment is derived.
  • Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, eg, recombinantly produced derivatives.
  • Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments and other fragments, including modified fragments (see, For example Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov).
  • the fragments may comprise multiple chains linked together, eg, by disulfide bonds and/or by peptide linkers.
  • Antibody fragments generally comprise at least or about 50 amino acids, and typically at least or about 200 amino acids.
  • Antigen-binding fragments include any antibody fragment that, when inserted into the antibody framework (eg, by substituting the corresponding regions), results in an antibody that immunospecifically binds an antigen.
  • inventional antibody refers to an antibody comprising two heavy chains (which may be designated H and H') and two light chains (which may be designated L and L') and two antigen binding sites, wherein Each heavy chain can be a full-length immunoglobulin heavy chain or any functional region thereof that retains antigen-binding capacity (eg, heavy chains include, but are not limited to, VH chains, VH-CH1 chains, and VH-CH1-CH2-CH3 chains), and Each light chain can be a full-length light chain or any functional region (eg, light chains include, but are not limited to, VL chains and VL-CL chains). Each heavy chain (H and H') is paired with a light chain (L and L', respectively).
  • a full-length antibody is an antibody having two full-length heavy chains (eg, VH-CH1-CH2-CH3) and two full-length light chains (VL-CL) and a hinge region, eg, by antibody-secreting B cells natively produced antibodies as well as synthetically produced antibodies with the same domains.
  • dsFv refers to Fvs with engineered intermolecular disulfide bonds that stabilize the VH-VL pair.
  • a Fab fragment is an antibody fragment obtained by papain digestion of a full-length immunoglobulin, or a fragment of the same structure produced synthetically, for example, by recombinant methods.
  • a Fab fragment comprises a light chain (comprising VL and CL) and another chain comprising the variable domain (VH) of the heavy chain and one constant region domain (CH1) of the heavy chain.
  • an F(ab')2 fragment is an antibody fragment obtained by pepsin digestion of immunoglobulin at pH 4.0-4.5, or a fragment of the same structure produced synthetically, for example, by recombinant methods.
  • F(ab')2 fragments essentially comprise two Fab fragments, each of which contains several additional amino acids in the heavy chain portion, including cysteines that form a disulfide bond linking the two fragments.
  • a Fab' fragment is a fragment comprising half of an F(ab') 2 fragment (one heavy chain and one light chain).
  • a ScFv fragment refers to an antibody fragment comprising a variable light chain (VL) and a variable heavy chain (VH) covalently linked in any order by a polypeptide linker.
  • variable domain or variable region is a domain of an antibody heavy or light chain that recognizes and specifically binds an antigen and comprises amino acid sequences that vary between different antibodies.
  • Each light chain and each heavy chain have one variable region domain, VL and VH, respectively.
  • the variable domains provide antigen specificity and are therefore responsible for antigen recognition.
  • Each variable region comprises CDRs and framework regions (FRs), the CDRs being part of the antigen binding site domain.
  • antigen binding domain and “antigen binding site” are used synonymously to refer to a domain within an antibody that recognizes and physically interacts with the same antigen.
  • Natural conventional full-length antibody molecules have two conventional antigen binding sites, each comprising a heavy chain variable region portion and a light chain variable region portion.
  • Conventional antigen binding sites comprise loops linking antiparallel beta strands within the variable region domains.
  • the antigen binding site may comprise other portions of the variable region domains.
  • Each conventional antigen binding site contains 3 hypervariable regions from the heavy chain and 3 hypervariable regions from the light chain. Hypervariable regions are also known as complementarity determining regions (CDRs).
  • variable region domain contains 3 CDRs, designated CDR1, CDR2 and CDR3.
  • the light chain variable region domain contains 3 CDRs, named VL CDR1, VL CDR2 and VL CDR3;
  • the heavy chain variable region domain contains 3 CDRs, named H CDRI (or VH CDR1), H CDR2 ( or VH CDR2) and H CDR3 (or VH CDR3).
  • the three CDRs in the variable region are discontinuous along the linear amino acid sequence, but are close together in the folded polypeptide.
  • the CDRs are located within loops of parallel strands connecting the beta sheets of the variable domains.
  • those skilled in the art know and can identify CDRs based on Kabat or Chotnia numbering (see e.g. Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Tnterest, Fifth Edition, U.S. Department of Health and Human Services, AIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917).
  • framework regions are domains within the variable region domains of antibodies that lie within the beta sheet; FR regions are relatively more conserved than hypervariable regions in terms of amino acid sequence.
  • a "constant region” domain is a domain in an antibody heavy or light chain that comprises a relatively more conserved amino acid sequence than the amino acid sequence of a variable region domain.
  • each light chain has a single light chain constant region (CL) domain
  • each heavy chain contains one or more heavy chain constant region (CH) domains, including (CH1, CH2, CH3 and CH4).
  • Antibody constant regions can serve effector functions such as, but not limited to, clearance of antigens, pathogens, and toxins to which the antibody specifically binds, such as by interacting with various cells, biomolecules, and tissues.
  • a functional region of a VH domain is at least a portion of an intact VH domain that retains at least a portion of the binding specificity of an intact VH domain (eg, by retaining one or more CDRs of an intact VH domain) such that the VH domain
  • a functional region of a domain binds antigen alone or in combination with another antibody domain (eg, a VL domain) or regions thereof.
  • An exemplary functional region of a VH domain is the region comprising the CDR1, CDR2 and/or CDR3 of the VH domain.
  • a functional region of a VL domain is at least a portion of an intact VL domain that retains at least a portion of the binding specificity of an intact VL domain (eg, by retaining one or more CDRs of an intact VL domain), such that the VL A functional region of a domain binds antigen alone or in combination with another antibody domain (eg, a VH domain) or regions thereof.
  • An exemplary functional region of a VL domain is the region comprising the CDRI, CDR2 and/or CDR3 of the VL domain.
  • polynucleotide and “nucleic acid molecule” refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including usually linked together by phosphodiester bonds Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • an isolated nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule.
  • An "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized.
  • Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen-binding fragments.
  • Sequence "identity” has an art-recognized meaning, and the percent sequence identity between two nucleic acid or peptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along regions of the molecule.
  • identity is well known to the skilled artisan (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988) ).
  • expression refers to the process by which a polypeptide is produced by transcription and translation of a polynucleotide. Expression levels of a polypeptide can be assessed using any method known in the art, including, for example, methods that determine the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, Coomassie blue staining followed by gel electrophoresis, Lowry protein assay, and Bradford protein assay.
  • a "host cell” is a cell used to receive, maintain, replicate and amplify a vector. Host cells can also be used to express the polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. Host cells can be eukaryotic cells or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, HeLa cells, HEK cells such as HEK 293 cells.
  • a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when transformed into an appropriate host cell.
  • References to vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction digestion and ligation. References to vectors also include those that contain nucleic acid encoding a polypeptide. Vectors are used to introduce nucleic acid encoding a polypeptide into a host cell, to amplify the nucleic acid, or to express/display the polypeptide encoded by the nucleic acid. Vectors generally remain episomal, but can be designed to integrate the gene or portion thereof into the chromosome of the genome. Also contemplated are artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles is well known to those skilled in the art.
  • an "expression vector” includes a vector capable of expressing DNA operably linked to regulatory sequences, such as promoter regions, capable of affecting the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and optionally, one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, which, when introduced into an appropriate host cell, results in the expression of cloned DNA. Appropriate expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic and/or prokaryotic cells as well as those that remain episomal or that integrate into the host cell genome.
  • treating an individual with a disease or condition means that the individual's symptoms are partially or completely alleviated, or remain unchanged following treatment.
  • treatment includes prevention, treatment and/or cure.
  • Prevention refers to preventing an underlying disease and/or preventing the worsening of symptoms or the development of a disease.
  • Treatment also includes any provided antibodies or antigen-binding fragments thereof and any pharmaceutical uses of the compositions provided herein.
  • therapeutic effect refers to an effect resulting from treatment of an individual that alters, generally ameliorates or ameliorates the symptoms of a disease or condition, or cures the disease or condition.
  • a “therapeutically effective amount” or “therapeutically effective dose” refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, retard or partially retard the symptoms of a disease or disorder.
  • a prophylactically effective amount or “prophylactically effective dose” refers to an amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, eg, prevent or delay disease or the occurrence or recurrence of symptoms, reducing the likelihood of the occurrence or recurrence of disease or symptoms.
  • a fully prophylactically effective dose need not occur by administering one dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.
  • the term "individual” refers to a mammal, such as a human.
  • the present invention provides an antibody against TIGIT, namely an anti-TIGIT antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically recognizes and binds to TIGIT.
  • the antibodies or antigen-binding fragments thereof of the present invention can specifically bind to TIGIT and block the binding of TIGIT to its ligands (including CD155 and CD122), induce T cell activation, and promote anti-tumor immune responses.
  • tumors targeted include, but are not limited to, those described below for neoplastic diseases.
  • the antibodies or antigen-binding fragments thereof of the invention are capable of inhibiting tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50% %.
  • the present invention provides an antibody against TIGIT or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising three CDRs, are respectively VH CDR1, VH CDR2 and VH CDR3, and the light chain variable region includes three CDRs, respectively VL CDR1, VL CDR2 and VL CDR3; wherein,
  • VH CDR1 The amino acid sequence of VH CDR1 is shown in SEQ ID NO: 3, or has more than 95% sequence identity with SEQ ID NO: 3, such as 96%, 97%, 98%, 99%;
  • VH CDR2 The amino acid sequence of VH CDR2 is shown in SEQ ID NO: 4, or more than 95%, such as 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 4;
  • VH CDR3 The amino acid sequence of VH CDR3 is shown in SEQ ID NO: 5, or more than 95%, such as 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 5;
  • VL CDR1 The amino acid sequence of VL CDR1 is shown in SEQ ID NO: 8, or more than 95%, such as 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 8;
  • VL CDR2 The amino acid sequence of VL CDR2 is shown in SEQ ID NO: 9, or more than 95%, such as 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 9;
  • VL CDR3 The amino acid sequence of VL CDR3 is shown in SEQ ID NO: 10, or more than 95%, such as 96%, 97%, 98%, 99% sequence identity with SEQ ID NO: 10.
  • amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 1, or at least 90%, at least 95%, at least 96%, at least 97%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO:6, or at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
  • amino acid modifications which may be one or more, can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc variants.
  • Fc variants may comprise human Fc region sequences comprising amino acid modifications at one or more amino acid positions.
  • Antibody and antigen-binding fragments thereof suitable for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, multispecific, recombinant, heterologous, chimeric, humanized, deimmunized Antibodies, or Fab fragments, Fab' fragments, F(ab') 2 fragments, single chain antibodies, Nanobodies and epitope binding fragments of any of the above.
  • the antibodies of the invention may be monospecific, bispecific or multispecific.
  • An anti-TIGIT antibody can be linked to another antibody or antibody fragment to generate a bispecific or multispecific antibody with a second or more binding specificity.
  • the antibody may be further modified to add functional components, moieties suitable for antibody derivatization include, but are not limited to, the following examples, PEG, dextran, proteins, lipids, therapeutic agents, or toxins.
  • Antibodies can be modified by phosphorylation, acetylation, glycosylation, pegylation, amidation, or linkage to other proteins, and the like.
  • Blockade of TIGIT by the antibodies of the present invention can enhance the patient's immune response to tumor cells. Binding of TIGIT to its ligands CD155 or CD112 inhibits the activation of immune cells, which in turn helps tumor cells evade T cell killing. Targeting TIGIT produced favorable antitumor effects, enhanced the activity of effector T cells and NK cells, and reduced the activity of immunosuppressive regulatory T cells.
  • the antibodies or antigen-binding fragments thereof of the present invention can be used to treat neoplastic diseases.
  • Preferred neoplastic diseases (or cancers) that can be prevented and/or treated using the antibodies or antigen-binding fragments thereof of the invention include cancers that are generally responsive to immunotherapy.
  • Non-limiting examples of treatable cancers include, but are not limited to, leukemia, lymphoma, bladder cancer, breast cancer, head and neck cancer, stomach cancer, melanoma, pancreatic cancer, colorectal cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, Prostate cancer, ovarian cancer, thyroid cancer, glioma, cervical cancer, nasopharyngeal cancer.
  • the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence region encoding the aforementioned antibody or antigen-binding fragment thereof of the present invention.
  • the nucleotide sequence of the nucleotide sequence region may be codon-optimized for the host cell used for expression.
  • the nucleotide sequence of the nucleotide sequence region includes SEQ ID NO: 2 and SEQ ID NO: 7, encoding the variable regions of the antibody heavy and light chains, respectively.
  • the present invention also provides an expression vector comprising at least one of the aforementioned nucleic acid molecules of the present invention.
  • One aspect of the present invention also provides a host cell transformed with at least one of the aforementioned nucleic acid molecules or expression vectors of the present invention.
  • Yet another aspect of the present invention also relates to a method of producing an anti-TIGIT antibody or antigen-binding fragment thereof, the method comprising: (i) culturing the host cell of the present invention under conditions suitable for expression of the nucleic acid molecule or expression vector, and (ii) isolating and purifying the antibody or antigen-binding fragment thereof expressed by the nucleic acid molecule or expression vector.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial, antifungal, absorption delaying agents and the like that are physiologically compatible.
  • the carrier is suitable for intraperitoneal, intravenous, subcutaneous, intranasal, intramuscular administration (eg, by injection or infusion).
  • the active compound ie, the antibody molecule, immunoconjugate
  • the active compound can be encapsulated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound.
  • compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • sterile aqueous solutions or dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • Conventional media or agents, except to any extent incompatible with the active compound, are possible in the pharmaceutical combinations of the present invention.
  • Supplementary active compounds can also be incorporated into the compositions.
  • compositions must generally be sterile and stable under the conditions of manufacture and storage.
  • the compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.
  • the carrier can be a solvent or dispersant containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, and the like), and suitable mixtures thereof.
  • the antibodies or antigen-binding fragments thereof of the invention or the pharmaceutical compositions of the invention can be administered by one or more routes of administration using one or more methods known in the art.
  • Preferred routes of administration for the antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion.
  • the antibodies of the invention or antigen-binding fragments thereof or the pharmaceutical compositions of the invention may also be administered by non-parenteral routes, such as topical, epidermal or mucosal routes, eg, intranasally, orally, sublingually.
  • the antibodies or antigen-binding fragments thereof of the invention in pharmaceutical compositions may also be conjugated to therapeutic moieties such as cytotoxins, radioisotopes, or biologically active proteins.
  • Cytotoxins include any agent that is detrimental to cells (eg, kills cells). Examples include, but are not limited to: paclitaxel, mitomycin, vincristine, colchicine, doxorubicin, daunorubicin, actinomycin D, ipecine and puromycin and their analogs or homologs thing.
  • Antibodies of the invention can also be conjugated to radioisotopes to produce cytotoxic radiopharmaceuticals, also known as radioantibody conjugates.
  • radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine 131, indium 111, yttrium 90. Methods for preparing radioantibody conjugates are well known in the art.
  • Antibodies of the present invention can also be conjugated to proteins having the desired biological activity and can be used to modify specific biological responses.
  • biologically active proteins include: ricin A, Pseudomonas exotoxin, diphtheriamycin, tumor necrosis factor, interferon gamma, INF gamma, lymphokines, interleukin-1, interleukin-2, interleukin-6, interleukin -10.
  • the present invention also provides combination therapy, comprising the anti-TIGIT antibody or pharmaceutical composition of the present invention in combination with at least one other therapeutic agent or agents, including but not limited to chemotherapeutic agents, radiotherapy agents and Biomacromolecules such as anti-PD-L1 antibodies.
  • chemotherapeutic agents including but not limited to chemotherapeutic agents, radiotherapy agents and Biomacromolecules such as anti-PD-L1 antibodies.
  • the antibody of the present invention and the chemotherapeutic agent, radiotherapeutic agent or biomacromolecule drug may be administered all at one time or separately. When administered separately (in the case of mutually different administration regimens), they may be administered continuously without interruption or at predetermined intervals.
  • Anti-TIGIT antibody sequences exemplified by the present invention are exemplified by the present invention.
  • Table 1 Amino acid sequence numbers of VH, VH-CDR1, VH-CDR2, VH-CDR3, VL, VL-CDR1, VL-CDR2, VL-CDR3 of the antibodies of the present invention
  • This example describes the preparation of mouse anti-human TIGIT monoclonal antibodies using hybridoma technology.
  • the TIGIT protein in the extracellular region (Uniprot: Q495A1) was expressed as an immunogen, and a 6xHis tag was added to the C-terminal of the amino acid sequence of the TIGIT protein in the extracellular region (Met22-Pro141), and then cloned into an expression vector to obtain the TIGIT protein in the extracellular region.
  • the expression vector was transiently transfected into 293 cells (source: ATCC), and the cell culture fluid was collected and purified 5 days later.
  • mice 4-week-old BAL B/c mice (provided by Viton Lever) were firstly immunized with 100 ⁇ g of TIGIT protein in the extracellular region. On the 14th and 28th days after the first immunization, the immunized mice were re-immunized with 50 ⁇ g of TIGIT protein in the extracellular region. The serum titer of the mice after immunization was detected by ELISA. The TIGIT protein in the extracellular region was diluted with PBS to a concentration of 0.5 ⁇ g/ml, and the microplate was coated overnight at 4°C. Then block with 5% BSA-PBST blocking solution at 37°C for 1 h.
  • mice with sufficient titers of anti-TIGIT antibodies were boosted with 50 ⁇ g of TIGIT protein in the extracellular region.
  • IMDM manufactured by Shanghai Yuanpei, product number: L610KJ
  • the fused cells were diluted into IMEM selective medium containing 15% fetal bovine serum (manufacturer: Zhejiang Tianhang Biology, product number: 11011-8611) and 1 ⁇ HAT (manufacturer: Sigma, product number: H0262-1VL). 200 microliters per well was added to a 96-well cell culture plate and placed in a 5% CO 2 , 37° C. incubator. After 10-14 days, the anti-TIGIT antibody in the hybridoma supernatant was detected by ELISA. Ten different hybridoma clones were identified, including 8G6, 8E11, 8D11, 8A11, 7D3, 7D1, 7A11, 7A1, 15B10, 15B11.
  • CD155 (manufacturer: Acrobiosystems, product number: CD5-H5251) was diluted to 3 ⁇ g/ml with PBS, coated on a 96-well microtiter plate and kept overnight at 4°C; after washing the plate, 2% BSA-PBST blocking solution was added, and the cells were blocked at 37°C for 1.5 h.
  • BSA-PBST blocking solution was added, and the cells were blocked at 37°C for 1.5 h.
  • TIT-H82E5 TIT-H82E5
  • HRP-labeled streptavidin manufactured by Abcam, product number: ab7403
  • TMB solution react at room temperature in the dark for 30 minutes, add 2N H 2 SO 4 to stop the reaction, and detect the absorbance at 450 nm wavelength (OD450) with a microplate reader.
  • IgG provided by Baiying Biotechnology
  • the inhibition rate of hybridoma supernatant on the binding of TIGIT to CD155 was calculated according to the following formula, wherein the minimum signal well was the blank solution group, and the maximum signal well was no hybridoma supernatant, only biotin-labeled TIGIT was added:
  • Human TIGIT protein (manufacturer: Acrobiosystems, product number: TIT-H52H3) was diluted into PBS at a concentration of 0.5 ⁇ g/ml, and coated on a microplate at 4°C. overnight. Then blocked with 5% BSA-PBST blocking solution at 37°C for 1h; after washing the plate with PBST, 7D3 was diluted to different concentrations (133.33nM starting, 4 times dilution, a total of 10 concentrations), added to the plate and incubated at 37°C for 1 hour.
  • the binding of murine mAb to TIGIT-expressing 293T-TIGIT cells was analyzed by flow cytometry (FACS).
  • the construction process of 293T-TIGIT cells is as follows: 293T cells were infected with the viral supernatant expressing TIGIT (sourced from Saiye Bio) by lentiviral transfection, and after 2 weeks of pressure screening, the expression of TIGIT was detected, and TIGIT-overexpressing cells were obtained. 293 cell line. 293T-TIGIT cells were reacted with different concentrations of 7D3 (the highest concentration was 33 nM, 5-fold dilution, 8 concentration points in total) at 4°C for 30 minutes.
  • Example 3 Blocking effect of murine anti-TIGIT antibody on binding of TIGIT to CD155
  • the blocking effect of anti-TIGIT antibody 7D3 on the binding of TIGIT to CD155 was detected by the competitive ELISA method described in Example 1.
  • the initial concentration of the antibody was 133.33 nM, and 4-fold gradient dilution was performed with 0.2% BSA-PBST to obtain a total of 10 gradients.
  • Use 22G2 as a positive control, according to the sequence disclosed in US10189902 (the heavy and light chain variable region sequences are respectively SEQ ID NO:7, SEQ ID NO:9, and the constant region sequences are respectively SEQ ID NO:45, SEQ ID NO:49) yields 22G2.
  • CD112 The binding of 7D3 to TIGIT and its other ligand CD112 was analyzed by competition ELISA.
  • CD112 (manufacturer: Acrobiosystems, product number: PV2-H5253) was diluted with PBS to 4 ⁇ g/ml and coated on a 96-well microtiter plate and overnight at 4°C; after washing the plate, 2% BSA-PBST blocking solution was added and blocked at 37°C for 1.5h .
  • 7D3 and positive antibody 22G2 were diluted to a concentration of 133.33nM, and then 4-fold serial dilution was performed with 0.2% BSA-PBST to obtain a total of 10 gradients, and biotin-labeled TIGIT (manufacturer: Acrobiosystems, product number: TIT-H82E5) was added to the plate on and incubated at 37°C for 1 hour. After washing the plate, add HRP-labeled streptavidin (manufacturer: Abcam, product number: ab7403), and react at 37°C for 1 hour.
  • the hybridoma cells expressing 7D3 were subjected to RNA extraction and reverse transcribed into cDNA according to conventional methods, and the sequence of the antibody gene was determined to obtain the heavy chain variable region and light chain variable region sequences of 7D3.
  • the amino acid sequences are respectively as SEQ ID NO: 1 and 6 are shown, and the nucleotide sequences are shown in SEQ ID NO: 2 and 7, respectively. Further, the amino acid sequence was analyzed, and the CDRs were determined with reference to the database of Kabat et al. (Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Tnterest, Fifth Edition, U.S. Department of Health and Human Services, AIH Publication No.
  • the heavy chain and light chain variable regions of 7D3 were respectively linked with the human antibody heavy chain IgG1 constant region and the human antibody light chain kappa constant region, and cloned into the vector pBRex-3.
  • the chimeric antibody is named c7D3, wherein the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 11, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 12; human antibody heavy chain IgG1 constant region, human antibody light
  • the nucleotide sequence of the chain kappa constant region is a conventional sequence.
  • TIGIT manufactured by Acrobiosystems, product number: TIT-H52H3
  • TIGIT-H52H3 was diluted into PBS at a concentration of 0.5 ⁇ g/ml, and coated on a microplate at 4°C overnight. Then block with 5% BSA-PBS blocking solution at 37°C for 1 h; after washing the plate with PBST, dilute c7D3 and control antibody 22G2 to different concentrations (133.33nM starting, 4-fold dilution, a total of 10 concentrations) added to the plate and 37°C Incubate for 1 hour.
  • Example 7 Blocking effect of chimeric antibodies on the binding of TIGIT to CD155
  • the blocking effect of anti-TIGIT chimeric antibody c7D3 on the binding of TIGIT to CD155 was detected by the competitive ELISA method described in Example 1.
  • the initial concentration of the antibody was 133.33 nM, and 2-fold gradient dilution was performed with 0.2% BSA-PBST to obtain a total of 10 gradients.
  • the results are shown in Figure 6, showing that c7D3 retains the blocking effect of the parent antibody on the binding of TIGIT to CD155.
  • the IC50 of c7D3 is 3.516nM, and the IC50 of 22G2 is 8.545nM, indicating that the blocking effect of c7D3 antibody on TIGIT and CD155 is stronger than that of 22G2.
  • Example 8 Blocking effect of chimeric antibodies on binding of TIGIT to CD112
  • the blocking effect of chimeric antibody c7D3 on the binding of TIGIT to CD112 was detected according to the method described in Example 4. The results are shown in Figure 7.
  • the IC50s of c7D3 and control antibody 22G2 were 18.13 nM and 43.58 nM, respectively, indicating that c7D3 can effectively block TIGIT and CD112.
  • the binding of CD112, c7D3 has a stronger blocking effect than the control antibody 22G2.
  • Example 9 Chimeric antibodies inhibit TIGIT/CD155 reporter gene activity
  • the biological activity of c7D3 at the cellular level was further analyzed by TIGIT/CD155 reporter gene assay.
  • the reporter gene experiment consists of two genetically engineered cell lines: TIGIT effector cells (source: GenScript) and CD155 aAPC/CHO-K1 cells (source: GenScript), where TIGIT effector cells express human TIGIT and Luciferase reporter gene Jurkat T cells, CD155 aAPC/CHO-K1 cells are CHO-K1 cells expressing human CD155 and TCR activating protein.
  • TIGIT effector cells express human TIGIT and Luciferase reporter gene Jurkat T cells
  • CD155 aAPC/CHO-K1 cells are CHO-K1 cells expressing human CD155 and TCR activating protein.
  • the antibody When an anti-TIGIT blocking antibody is introduced into the experimental system, the antibody binds to TIGIT expressed by effector cells, blocks the interaction between TIGIT and CD155, releases the inhibitory signal, and activates the T cell receptor (TCR) signaling pathway and IL2 promoter, which promotes the expression of luciferase. The latter catalyzes the substrate to generate a chemiluminescent signal.
  • TCR T cell receptor
  • the CD155 aAPC/CHO-K1 cells were seeded into a 96-well cell culture plate, incubated in the incubator for 16-20 hours, then TIGIT effector cells and c7D3 antibody were added, the plate was incubated in the incubator for about 6 hours, and luciferase was added.
  • Example 10 Chimeric Antibody Activates PBMC Cells to Release Cytokines
  • CD155 (manufacturer: Acrobiosystems, product number: CD5-H5251) was diluted with PBS to a concentration of 1 ⁇ g/ml, added to a 96-well cell culture plate, 100 ⁇ l per well, and coated overnight at 4°C; PBMC cells (derived from ALLcells) The density was adjusted to 2.5 ⁇ 10 6 cells/ml with RPMI 1640 medium, 40 ⁇ l per well was inoculated into a CD155-coated 96-well cell culture plate, and 40 ⁇ l of 150ng/ml SEB (manufacturer: Toxin technology, product number BT202) was added; The serially diluted chimeric antibody c7D3 or the same amount of isotype IgG1 blank control antibody or
  • the cell culture plate was taken out, and the cell culture supernatant was collected by centrifugation at 2000 rpm for 10 min.
  • the concentrations of IL-2 (manufacturer: BD, product number: 550611) and IFN- ⁇ (manufacturer: Abcam, product number: ab46025) were detected by ELISA.
  • Figure 9 and Figure 10. The results showed that, compared with the IgG1 control antibody, the chimeric antibody c7D3 could promote the secretion of cytokines IL-2 and IFN- ⁇ from PBMC, indicating that c7D3 could effectively promote T cell activation, and the combination of c7D3 and anti-PD-L1 antibody had a significantly better effect. In the effect of the two drugs alone.

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Abstract

La présente invention relève du domaine de la biologie, et concerne un anticorps anti-TIGIT ou un fragment de liaison à l'antigène de celui-ci, une composition de celui-ci et une utilisation associée. L'anticorps anti-TIGIT et le fragment de liaison à l'antigène de celui-ci selon la présente invention peuvent bloquer de manière efficace la liaison de TIGIT et de ses ligands CD155 et CD112, ce qui favorise efficacement l'activation de lymphocytes T et présente d'excellentes perspectives d'application antitumorale.
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