WO2023040945A1 - 特异性结合pd-1的蛋白及其医药用途 - Google Patents

特异性结合pd-1的蛋白及其医药用途 Download PDF

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WO2023040945A1
WO2023040945A1 PCT/CN2022/118979 CN2022118979W WO2023040945A1 WO 2023040945 A1 WO2023040945 A1 WO 2023040945A1 CN 2022118979 W CN2022118979 W CN 2022118979W WO 2023040945 A1 WO2023040945 A1 WO 2023040945A1
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pvrig
binding protein
tigit
antigen
binding
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PCT/CN2022/118979
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English (en)
French (fr)
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曹志亮
王宝辉
林�源
林侃
廖成
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江苏恒瑞医药股份有限公司
上海盛迪医药有限公司
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Priority to KR1020247011478A priority Critical patent/KR20240055080A/ko
Priority to CA3231320A priority patent/CA3231320A1/en
Priority to CN202280057192.2A priority patent/CN117940453A/zh
Publication of WO2023040945A1 publication Critical patent/WO2023040945A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present disclosure relates to the field of biomedicine, in particular to a protein specifically binding to PD-1, a protein specifically binding to PD-1/PVRIG/TIGIT, and a method for treating cancer and a pharmaceutical use thereof.
  • Immunotherapy is a hotspot in the field of cancer treatment, and anti-PD-1 antibodies have shown high effectiveness and safety in clinical practice.
  • PD-1 Programmed Cell death-1 belongs to the CD28 receptor family and is an immunosuppressive receptor (Riley et al. 2009, Immunol. Rev. 29:114-25). This family also includes CD28, CTLA-4, ICOS, PD-1 and BTLA.
  • PD-1 is a type I transmembrane protein, which is very similar to CTLA-4 in structure, but PD-1 lacks the MYPPPY sequence that binds to B7-1 and B7-2. PD-1 is mainly expressed in activated B cells, T cells and myeloid cells (Chen et al. 2013, Nat. Rev. Immunol. 13:227-42).
  • PD-1 has two glycoprotein ligands on the cell surface, PD ligand 1 (PD-L1, also known as CD274, B7-H1) and PD ligand 2 (PD-L2, also known as B7-DC). Neither PD-L1 nor PD-L2 binds to other CD28 receptor family members.
  • PD-L1 is widely expressed in lymphocytes (such as CD4 + T cells and CD8 + T cells, macrophages, etc.), as well as peripheral tissues, various tumor cells, and virus-infected cells.
  • PD-L2 is mainly expressed on activated dendritic cells and macrophages (Dong et al. 1999, Nat. Med. 5:1365-9). After PD-1 binds to its ligand PD-L1 or PD-L2, it will down-regulate the function of T cells, including reducing the activation, differentiation and proliferation of T cells, and the secretion of cytokines.
  • PVRIG Polyvirus receptor-related Ig domain containing protein
  • CD112R Polyvirus receptor-related Ig domain containing protein
  • B7/CD28 superfamily like TIGIT T cell immunoglobulin and ITIM domain
  • PVRL2 also known as CD112
  • the ligand of PVRIG binds to PVRIG, it will activate the ITIM domain in the intracellular region of PVRIG, making PVRIG play an immunosuppressive role.
  • PVRIG is mainly expressed on the surface of CD4 + T cells, CD8 + T cells and NK cells.
  • PVRIG and its ligand PVRL2 are highly expressed in a variety of solid tumors, including lung cancer, breast cancer, ovarian cancer, kidney cancer, gastric cancer, endometrial cancer, head and neck cancer, etc.
  • the expression of PVRIG in these cancers is highly correlated with TIGIT and PD-1. Similar to PD-1 and TIGIT, PVRIG-positive T cells were also Eomes-positive and Tbet-negative, suggesting that PVRIG is involved in the exhaustion of T cells. Therefore, PVRIG may represent a new immune checkpoint in addition to PD-1 and TIGIT, and play a role of redundancy.
  • TIGIT is highly expressed on lymphocytes, including cancer-infiltrating lymphocytes (TILs) and Tregs that infiltrate different types of cancer. Binding of TIGIT to its cognate ligand PVR (aka CD155) has been shown to directly inhibit NK cell cytotoxicity through its cytoplasmic ITIM domain. PVR is also widely expressed in cancer, suggesting that the TIGIT-PVR signaling axis may be a major immune escape mechanism in cancer.
  • Inhibitory receptors TIGIT, PVRIG and activating receptor DNAM-1 bind the same ligands CD155 and CD112, but the affinity of inhibitory receptors is higher (Y.Zhu et al. 2016, J Exp Med.213:167-176 ).
  • the down-regulation of DNAM-1 expression in NK cells isolated from tumor patients makes NK cells more susceptible to inhibition by TIGIT or PVRIG, blocking the combination of TIGIT and PVRIG can activate the cell killing function of NK cells (L.Martinet et al. 2015, Cell Reports .11:85-97).
  • TIGIT and PVRIG are currently the most clinically successful immune checkpoint inhibitor monoclonal antibody, the up-regulation of other immune checkpoints on the surface of T cells will limit its efficacy.
  • TIGIT and PVRIG also regulate the activity of NK cells, suggesting that they may synergize with the function of PD-1/PD-L1 in NK cells. It has been reported in the literature that the high expression of TIGIT and PVRIG ligands CD155 and CD112 in lung cancer, ovarian cancer, colorectal cancer, and melanoma is significantly associated with poor prognosis after PD-1/PD-L1 treatment.
  • patients with high CD155 expression have a poor response to anti-PD-1 antibodies, while patients with low CD155 expression have a good response to PD-1 (S. Whelan et al. 2019, Cancer Immunol Res. 7:257-268; A. Lepletier et al. 2020 , Clin Cancer Res. 26:3671-3681).
  • Compugen's COM701 is the world's first anti-PVRIG humanized antibody approved by the FDA for clinical use. It is currently in Phase I clinical stage for the treatment of cancer. Surface Oncology also has an anti-PVRIG antibody, SRF-813, in development.
  • Anti-TIGIT antibodies include Genentech’s tiragolumab, BMS-986207 jointly developed by Ono Pharmaceutical and BMS, Merck’s MK-7684, iTeos Therapeutics’ EOS-884448, and Arcus Biosciences’ AB-154, all of which are in phase II clinical trials.
  • anti-PD-1 antibodies with new structures and related anti-PD-1/PVRIG/TIGIT trispecific antibodies, which are expected to enhance the activation of T cells and NK cells, overcome PD-1/PD-L1 drug resistance, expand Responsive patient populations to improve the efficacy of cancer immunotherapy.
  • anti-PD-1/PVRIG/TIGIT trispecific antibodies can simultaneously bind to multiple immune checkpoints on the same cell surface, potentially having an avidity effect.
  • PVRIG antibody drug or its combination has a better curative effect.
  • the present disclosure provides PD-1 binding protein, PD-1/PVRIG/TIGIT binding protein, PD-1/PVRIG binding protein, PD-1/TIGIT binding protein, encoding nucleic acid, vector, host cell, pharmaceutical composition, etc. Methods and related pharmaceutical uses for treating or preventing cancer.
  • the present disclosure provides a PD-1 binding protein comprising an immunoglobulin single variable domain comprising:
  • CDR1, CDR2 and CDR3 in any one of the amino acid sequences shown in SEQ ID NO: 3, 13-15, 17-35, or
  • the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system.
  • the single variable structure of the immunoglobulin specifically binds to the PD-1 antigen or a fragment thereof.
  • the PD-1 binding protein comprises at least one immunoglobulin single variable structure.
  • a PD-1 binding protein comprising any one of the above-mentioned CDR1, CDR2 and CDR3 or any combination thereof.
  • PD-1 binding protein is provided, according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are respectively
  • the amino acid sequence of CDR1 of the immunoglobulin single variable domain in the aforementioned PD-1 binding protein is shown in SEQ ID NO: 7, and the amino acid sequence of CDR2 is shown in SEQ ID NO: 8, 38-40, As shown in any one of 101, the amino acid sequence of CDR3 is shown in any one of SEQ ID NO: 9, 41, 42.
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain in the aforementioned PD-1 binding protein are shown in SEQ ID NO: 7, 38, and 41, respectively.
  • the immunoglobulin single variable domain in the aforementioned PD-1 binding protein is humanized, affinity matured, engineered to remove T cell epitopes, reduce antibody deamidation and/or reduce antibody isomerization.
  • the immunoglobulin single variable domain is obtained through affinity maturation and has one or more changes in one or more CDRs that result in an affinity for PD-1 compared to the parent There was an increase in immunoglobulin single variable domains.
  • the humanization process uses the heavy chain framework region IGHV3-23*01 or IGHV3-23*04 of the human germline template.
  • the amino acid sequence of the immunoglobulin single variable domain in the aforementioned PD-1 binding protein is shown in any of SEQ ID NO: 3, 13-15, 17-35, or shown in SEQ ID NO : any of 2, 16, or having at least 80% sequence identity with any of the preceding sequences.
  • At least 90% (sequence) identity encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99% or 100% (sequence) identity;
  • at least 80% (sequence) identity encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86 %, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, At least 99% or 100% (sequence) identity.
  • the aforementioned PD-1 binding protein comprises or is an antibody or an antigen-binding fragment thereof that specifically binds to PD-1 or a fragment thereof.
  • the antibody or an antigen-binding fragment thereof is, for example, a camelid antibody, a chimeric antibody, a humanized antibody, a fully human antibody or an antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof is, for example, a recombinant antibody or fragment thereof.
  • the antigen-binding fragment is a linear antibody, single chain antibody, nanobody, peptibody, domain antibody, and multispecific antibody (bispecific antibody, diabody, triabody and tetrabody, tandem di-scFv , tandem tri-scFv).
  • the immunoglobulin single variable domain in the aforementioned PD-1 binding protein is VHH.
  • the present disclosure provides a PD-1 binding protein comprising one or more (e.g., 2, 3, 4, 5, 6) of the aforementioned immunoglobulin single variable domains that can be
  • the variable domains can be the same or different and can form dimeric or multimeric molecules.
  • the aforementioned PD-1 binding protein further comprises a human immunoglobulin Fc region; for example, the Fc region is the Fc region of human IgG1, IgG2 or IgG4.
  • the Fc region may have mutations, exemplary mutations are selected from any one or combination of:
  • Hybrid IgG2/4 Fc domains may also be used, for example an Fc having residues 117-260 from IgG2 and residues 261-447 from IgG4.
  • the Fc region of human IgG4 has 228P, 234A, 235A and/or 447A mutations.
  • the Fc region contained in the aforementioned PD-1 binding protein can enable the binding protein to form a dimer molecule, prolonging the in vivo half-life of the binding protein.
  • the immunoglobulin single variable domain in the aforementioned PD-1 binding protein is connected to the Fc region directly or through a linker.
  • the linker can be a non-functional amino acid sequence with a length of 1-20 or more amino acids and no secondary or higher structure.
  • the linker is a flexible linker, such as G 4 S, GS, GAP, (G 4 S) 2 , (G 4 S) 3 , (G 4 S) 4 , (G 4 S) 5 , ASGS, etc. .
  • the PD-1 binding protein of the present disclosure is an anti-PD-1 antibody or an antigen-binding fragment thereof, or a conjugate or fusion protein comprising the antibody or antigen-binding fragment.
  • the aforementioned PD-1 binding protein has at least one activity selected from the following:
  • the aforementioned PD-1 binding protein of the present disclosure can bind PD-1 with a K D value of ⁇ 1 ⁇ 10 -7 M, such as ⁇ 1 ⁇ 10 -8 M, or ⁇ 1 ⁇ 10 -9 M, or ⁇ 1 ⁇ 10 -10 M.
  • the PD-1 binding protein of the present disclosure can specifically bind human PD-1 and block the interaction between PD-1 and PD-L1, and/or block the interaction between PD-1 and PD-L2 .
  • the aforementioned PD-1 binding proteins of the present disclosure are capable of inhibiting tumor growth by at least about 10%, such as at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%.
  • the foregoing PD-1 binding proteins of the present disclosure encompass variants that have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations; the amino acid mutations can be conservative substitutions, substitutions or modifications, and/or deletions or additions that do not affect the function; the amino acid mutations can be Occurs in the CDR and/or FR regions.
  • amino acid mutations can be conservative substitutions, substitutions or modifications, and/or deletions or additions that do not affect the function
  • the amino acid mutations can be Occurs in the CDR and/or FR regions.
  • an anti-PD-1 antibody or antigen-binding fragment thereof that binds or competes for binding to the same epitope as the immunoglobulin single variable domain in the aforementioned PD-1 binding protein of the present disclosure.
  • an anti-PD-1 antibody or antigen-binding fragment thereof that blocks the interaction of the immunoglobulin single variable domain and PD-1 (such as human PD-1) in the aforementioned PD-1 binding protein of the present disclosure. combination.
  • anti-PD-1 antibodies or antigen-binding fragments thereof are provided, the binding of which to PD-1 (such as human PD-1) is controlled by the immunoglobulin single variable structure in the aforementioned PD-1 binding protein of the present disclosure domain blocking.
  • the aforementioned PD-1 binding protein of the present disclosure reduces the binding of PD-1 to PD-L1 and/or PD-L2 by at least 60%, at least 70%, at least 80%, at least 90%, at least 95% , at least 98% or above.
  • a protein or molecule comprising any one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) of the aforementioned PD-1 binding proteins of the present disclosure
  • the immunoglobulin single variable domains are the same or different.
  • the protein or molecule is a conjugate, which may, for example, comprise any detectable label.
  • the present disclosure provides a PD-1/PVRIG/TIGIT binding protein comprising a first antigen-binding domain that specifically binds PD-1, a second antigen-binding domain that specifically binds PVRIG, and a third antigen-binding domain that specifically binds TIGIT
  • the domains can specifically bind PD-1, PVRIG and TIGIT simultaneously or separately.
  • the aforementioned PD-1/PVRIG/TIGIT binding protein comprises or is an anti-PD-1/PVRIG/TIGIT trispecific antibody.
  • each antigen-binding domain in the aforementioned PD-1/PVRIG/TIGIT binding protein is selected from Fab, Fv, sFv, Fab', F(ab') 2 , linear antibody, single-chain antibody, scFv, sdAb , sdFv, nanobody, peptibody, domain antibody or any combination thereof.
  • the first antigen-binding domain specifically binding to PD-1 comprises or is VHH
  • the second antigen-binding domain specifically binding to PVRIG comprises or is VHH
  • the third antigen-binding domain specifically binding to TIGIT The domain is Fab or F(ab') 2 .
  • the aforementioned PD-1/PVRIG/TIGIT binding protein is humanized, affinity matured, engineered to remove T cell epitopes, reduce antibody deamidation and/or reduce antibody isomerization.
  • the aforementioned PD-1/PVRIG/TIGIT binding protein is a recombinant antibody, a camelid antibody, a chimeric antibody, a humanized antibody, a fully human antibody or an antigen-binding fragment thereof.
  • the first antigen-binding domain that specifically binds to PD-1 comprises (at least one) immunoglobulin single variable domain, and the immunoglobulin Protein single variable domains contain:
  • CDR1, CDR2 and CDR3 in any amino acid sequence shown in SEQ ID NO: 3, 13-15, 17-35,
  • the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system.
  • amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are respectively:
  • the amino acid sequence of CDR1 of the immunoglobulin single variable domain is shown in SEQ ID NO: 7, and the amino acid sequence of CDR2 is shown in any one of SEQ ID NO: 8, 38-40, or 101 , the amino acid sequence of CDR3 is shown in any of SEQ ID NO: 9, 41, 42.
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NO: 7, 38, and 41, respectively.
  • the second antigen-binding domain that specifically binds PVRIG comprises (at least one) a single variable domain of an immunoglobulin, and the single immunoglobulin
  • the variable domain contains:
  • CDR1, CDR2 and CDR3 in any one of the amino acid sequences shown in SEQ ID NO: 45, 62-66, 95-96, or
  • CDR1, CDR2 and CDR3 in any one of the amino acid sequences shown in SEQ ID NO: 44, 57-61.
  • amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are respectively:
  • the third antigen-binding domain that specifically binds to TIGIT comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein :
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NO: 73, 74 and 75 respectively, and the light chain variable region comprises the amino acid sequences shown in SEQ ID NO: 76, 77 and 78 respectively.
  • the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown.
  • the first antigen-binding domain that specifically binds to PD-1 comprises such as:
  • the second antigen-binding domain that specifically binds PVRIG comprises such as:
  • the third antigen-binding domain that specifically binds to TIGIT comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein :
  • the heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO: 79-81 or having at least 80%, at least 90% identity thereto,
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 82 or 83 or having at least 80%, at least 90% identity thereto.
  • the third antigen-binding domain specifically binding to TIGIT comprises a full-length heavy chain (HC) and a full-length light chain (LC).
  • the full-length heavy chain is of the IgG1, IgG2 or IgG4 isotype and the full-length light chain is of the Kappa isotype;
  • the heavy chain sequence is an amino acid sequence shown in SEQ ID NO: 67 or having at least 80% or at least 90% sequence identity therewith, and the light chain sequence is shown in SEQ ID NO: 68 or an amino acid sequence with it Amino acid sequences having at least 80%, at least 90% sequence identity.
  • the first antigen-binding domain that specifically binds PD-1 the second antigen-binding domain that specifically binds PVRIG, and the third antigen-binding domain that specifically binds TIGIT
  • the antigen binding domains are linked directly or via a linker.
  • the linker has an amino acid sequence shown as (G 4 S) x , wherein x is independently selected from the integer of 1-20; for another example, the linker is (G 4 S) 2 , ( Amino acid sequences represented by G 4 S) 3 and (G 4 S) 4 .
  • the antigen-binding domain that specifically binds to PD-1 is located at the N-terminal or C-terminal of the antigen-binding domain that specifically binds to PVRIG.
  • the antigen-binding domain that specifically binds to PD-1 is located at the N-terminal or C-terminal of the antigen-binding domain that specifically binds to TIGIT.
  • the antigen-binding domain that specifically binds PVRIG is located at the N-terminal or C-terminal of the antigen-binding domain that specifically binds TIGIT.
  • the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain are on the same polypeptide chain or not on the same polypeptide chain.
  • the aforementioned PD-1/PVRIG/TIGIT binding protein comprises a first polypeptide chain and a second polypeptide chain, wherein it is shown in a structure selected from the following:
  • the first polypeptide chain [the first antigen-binding domain specifically binding to PD-1]-[linker 1]a-[the second antigen-binding domain specifically binding to PVRIG]-[linker 2 ]b-[VH specifically binding to the third antigen-binding domain of TIGIT]-CH1-Fc
  • the second polypeptide chain [VL specifically binding to the third antigen-binding domain of TIGIT]-C ⁇ ;
  • the first polypeptide chain [the second antigen-binding domain specifically binding to PVRIG]-[Linker 2]b-[VH specifically binding to the third antigen-binding domain of TIGIT]-CH1-Fc
  • the second polypeptide chain [the first antigen-binding domain that specifically binds to PD-1]-[Linker 3]c-[VL that specifically binds to the third antigen-binding domain of TIGIT]-C ⁇ ; or
  • the first polypeptide chain [the second antigen-binding domain that specifically binds PVRIG]-[Linker 2]b-[the VH that specifically binds the third antigen-binding domain of TIGIT]-CH1-Fc- [Linker 4] d-[the first antigen-binding domain that specifically binds PD-1], and the second polypeptide chain: [VL that specifically binds the third antigen-binding domain of TIGIT]-C ⁇ ; or
  • the first polypeptide chain [the second antigen-binding domain specifically binding to PVRIG]-[Linker 2]b-[VH specifically binding to the third antigen-binding domain of TIGIT]-CH1-Fc, And the second polypeptide chain: [VL that specifically binds to the third antigen-binding domain of TIGIT]-C ⁇ -[Linker 5]e-[the first antigen-binding domain that specifically binds to PD-1].
  • the above first and second polypeptide chains are all sequenced from N-terminus to C-terminus.
  • linker 1 linker 2, linker 3, linker 4, and linker 5 can be the same or different; a, b, c, d , e can optionally be 0 or 1 independently.
  • each linker is independently EPKSS or is a (G x S) y linker, wherein x is selected from an integer of 1-5 (eg, 1, 2, 3, 4, 5) and y is selected from 1-6 integers (for example, 1, 2, 3, 4, 5, 6).
  • the linker is a (G x S) y linker, it is, for example, any one of (G 4 S) 2 , (G 4 S) 3 , and (G 4 S) 4 .
  • a PD-1/PVRIG/TIGIT binding protein comprising a first polypeptide chain and a second polypeptide chain, wherein:
  • the first and second polypeptide chains respectively comprise the amino acid sequences shown in SEQ ID NO: 87, 68;
  • the first and second polypeptide chains respectively comprise the amino acid sequences shown in SEQ ID NO: 84, 88;
  • the first and second polypeptide chains respectively comprise the amino acid sequences shown in SEQ ID NO: 89, 68;
  • the first and second polypeptide chains respectively comprise the amino acid sequences shown in SEQ ID NO: 84, 90; or
  • the first and second polypeptide chains respectively comprise the amino acid sequences shown in SEQ ID NO: 91, 68;
  • the first and second polypeptide chains respectively comprise the amino acid sequences shown in SEQ ID NO: 85, 92;
  • the first and second polypeptide chains respectively comprise the amino acid sequences shown in SEQ ID NO: 93, 68;
  • the first and second polypeptide chains respectively comprise the amino acid sequences shown in SEQ ID NO: 85, 94;
  • the first and second polypeptide chains respectively comprise the amino acid sequences shown in SEQ ID NO: 97, 68;
  • the first and second polypeptide chains respectively comprise the amino acid sequences shown in SEQ ID NO: 98, 68;
  • the aforementioned PD-1/PVRIG/TIGIT binding protein of the present disclosure comprises two identical or different first polypeptide chains, and comprises two identical or different second polypeptide chains.
  • the PD-1/PVRIG/TIGIT binding protein comprises two identical first polypeptide chains and two identical second polypeptide chains.
  • the PD-1/PVRIG/TIGIT-binding protein of the present disclosure has all or any of its properties and functions because it comprises the antigen-binding domain of the present disclosure that specifically binds to PD-1.
  • the PD-1/PVRIG/TIGIT binding protein also has at least one of the following characteristics because it comprises a PVRIG/TIGIT binding domain:
  • the aforementioned PD-1/PVRIG/TIGIT binding proteins of the present disclosure are capable of inhibiting tumor growth by at least about 10%, such as at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%.
  • the aforementioned PD-1/PVRIG/TIGIT binding proteins of the present disclosure encompass variants that have one or more of the aforementioned combinations of the first polypeptide chain and the second polypeptide chain (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid mutations; the amino acid mutations may be conservative substitutions, substitutions or modifications, and/or deletions that do not affect function, Add to.
  • the mutation may occur in the antigen-binding domain specifically binding to PD-1, the antigen-binding domain specifically binding to PVRIG, or the antigen-binding domain specifically binding to TIGIT (such as CDR region and/or FR region).
  • a PD-1/PVRIG/TIGIT binding protein which binds or competes with the aforementioned PD-1/PVRIG/TIGIT binding protein of the present disclosure to bind to the same PD-1, PVRIG and/or TIGIT or its epitope; or block the aforementioned PD-1/PVRIG/TIGIT binding protein of the present disclosure from combining with PD-1, PVRIG and/or TIGIT; PD-1/PVRIG/TIGIT binding proteins of the present disclosure block.
  • PD-1/PVRIG binding protein PD-1/TIGIT binding protein
  • the present disclosure provides a PD-1/PVRIG binding protein, which comprises an antigen-binding domain that specifically binds to PD-1 and an antigen-binding domain that specifically binds to PVRIG, and can specifically bind to PD-1 and PVRIG simultaneously or separately.
  • the present disclosure provides a PD-1/TIGIT-binding protein, which comprises an antigen-binding domain that specifically binds to PD-1 and an antigen-binding domain that specifically binds to TIGIT, and can specifically bind to PD-1 and TIGIT simultaneously or separately.
  • the aforementioned PD-1/PVRIG binding protein comprises or is an anti-PD-1/PVRIG bispecific antibody
  • the aforementioned PD-1/TIGIT binding protein comprises or is an anti-PD-1/TIGIT bispecific antibody.
  • each antigen-binding domain in the aforementioned PD-1/PVRIG binding protein and PD-1/TIGIT binding protein is selected from Fab, Fv, sFv, Fab', F(ab') 2 , linear antibody, monoclonal antibody Chain antibody, scFv, sdAb, sdFv, nanobody, peptibody, domain antibody or any combination thereof.
  • the antigen-binding domain specifically binding to PD-1 comprises or is VHH
  • the antigen-binding domain specifically binding to PVRIG comprises or is VHH
  • the antigen-binding domain specifically binding to TIGIT is Fab or F(ab ') 2 .
  • the aforementioned PD-1/PVRIG binding protein and PD-1/TIGIT binding protein are humanized, affinity matured, removed T cell epitopes, reduced antibody deamidation and/or modified to reduce antibody isomerization.
  • the aforementioned PD-1/PVRIG binding protein and PD-1/TIGIT binding protein are recombinant antibodies, camelid antibodies, chimeric antibodies, humanized antibodies, fully human antibodies or antigen-binding fragments thereof.
  • the antigen binding domain that specifically binds to PD-1 comprises at least one immunoglobulin single variable domain
  • the immunoglobulin A globulin single variable domain contains:
  • CDR1, CDR2 and CDR3 in any amino acid sequence shown in SEQ ID NO: 3, 13-15, 17-35,
  • the CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system.
  • amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are:
  • the amino acid sequence of CDR1 of the immunoglobulin single variable domain is shown in SEQ ID NO: 7, and the amino acid sequence of CDR2 is shown in any one of SEQ ID NO: 8, 38-40, or 101 , the amino acid sequence of CDR3 is shown in any of SEQ ID NO: 9, 41, 42.
  • amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are shown in SEQ ID NO: 7, 38, and 41, respectively.
  • the antigen-binding domain that specifically binds PVRIG comprises at least one immunoglobulin single variable domain, and the immunoglobulin single variable domain comprises:
  • CDR1, CDR2 and CDR3 in any one of the amino acid sequences shown in SEQ ID NO: 45, 62-66, 95-96, or
  • CDR1, CDR2 and CDR3 in any one of the amino acid sequences shown in SEQ ID NO: 44, 57-61.
  • amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are respectively:
  • the antigen-binding domain that specifically binds to TIGIT comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NO: 73, 74 and 75 respectively, and the light chain variable region comprises the amino acid sequences shown in SEQ ID NO: 76, 77 and 78 respectively.
  • the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown.
  • the antigen binding domain that specifically binds to PD-1 comprises:
  • amino acid sequence having at least 80%, at least 85%, at least 90% sequence identity to any of said sequences.
  • the antigen binding domain that specifically binds to PVRIG comprises such as:
  • amino acid sequence having at least 80%, at least 85%, at least 90% sequence identity to any of said sequences.
  • the antigen binding domain that specifically binds TIGIT comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
  • the heavy chain variable region comprises an amino acid sequence as shown in any of SEQ ID NO: 79-81 or having at least 80%, at least 90% identity thereto,
  • the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 82 or 83 or having at least 80%, at least 90% identity thereto.
  • the third antigen-binding domain specifically binding to TIGIT comprises a full-length heavy chain (HC) and a full-length light chain (LC).
  • the full-length heavy chain is of the IgGl, IgG2 or IgG4 isotype and the full-length light chain is of the Kappa isotype.
  • the heavy chain sequence is an amino acid sequence shown in SEQ ID NO: 67 or having at least 80% or at least 90% sequence identity with it
  • the light chain sequence is shown in SEQ ID NO: 68 or an amino acid sequence with it Amino acid sequences having at least 80%, at least 90% sequence identity.
  • the linker is independently such as (G 4 S) the amino acid sequence shown by x , wherein, x is independently selected from the integer of 1-20 (for example, 1, 2, 3, 4, 5, 6, 7, 8); for another example, the linker is independently It is the amino acid sequence represented by (G 4 S) 2 and (G 4 S) 3 .
  • the antigen-binding domain that specifically binds to PD-1 is located at the N-terminal or C-terminal of the antigen-binding domain that specifically binds to PVRIG.
  • the antigen-binding domain that specifically binds to PD-1 is located at the N-terminal or C-terminal of the antigen-binding domain that specifically binds to TIGIT.
  • the aforementioned PD-1/PVRIG binding protein and PD-1/TIGIT binding protein further comprise a human immunoglobulin Fc region; for example, the Fc region is the Fc region of human IgG1, IgG2 or IgG4.
  • the Fc region may have mutations, exemplary mutations are S228P, F234A, L235A and/or K447A mutations.
  • the Fc region can enable the binding protein to form a dimer molecule, prolonging the in vivo half-life of the binding protein.
  • the PD-1/PVRIG-binding protein and PD-1/TIGIT-binding protein of the present disclosure have all or any of their properties and functions because they have the antigen-binding domain specifically binding to PD-1 of the present disclosure.
  • the aforementioned PD-1/PVRIG binding protein of the present disclosure is capable of inhibiting tumor growth by at least about 10%, such as at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70% , about 80%, about 90% or more.
  • the PD-1/PVRIG binding proteins, PD-1/TIGIT binding proteins of the present disclosure encompass variants compared to the aforementioned PD-1/PVRIG binding proteins, PD-1/TIGIT binding proteins
  • a PD-1/PVRIG binding protein, PD-1/TIGIT binding protein which binds or competes for binding to the same PD as the aforementioned PD-1/PVRIG binding protein, PD-1/TIGIT binding protein of the present disclosure -1, PVRIG and/or TIGIT or its epitope; or blocking the aforementioned PD-1/PVRIG binding protein of the present disclosure, PD-1/TIGIT binding protein binding to PD-1, PVRIG and/or TIGIT; or Its binding to PD-1, PVRIG and/or TIGIT is blocked by the aforementioned PD-1/PVRIG binding protein and PD-1/TIGIT binding protein of the present disclosure.
  • a PVRIG-binding protein which comprises an antigen-binding domain that specifically binds PVRIG in the aforementioned PD-1/PVRIG-binding protein.
  • the PVRIG antibody can be any one of CPA.7.002, CPA.7.005, CPA.7.021 and CPA.7.050 (see WO2016134333).
  • the aforementioned patents are incorporated herein in their entirety.
  • a TIGIT-binding protein which comprises an antigen-binding domain that specifically binds TIGIT in the aforementioned PD-1/TIGIT-binding protein.
  • TIGIT antibody in WO2019232484, WO2019062832.
  • the TIGIT antibody can be any of CPA.9.083.H4(S241P), CPA.9.086.H4(S241P), CHA.9.547.7.H4(S241P) and CHA.9.547.13.H4(S241P) (See WO2019232484).
  • the aforementioned patents are incorporated herein in their entirety.
  • a PVRIG/TIGIT-binding protein which comprises the antigen-binding domain that specifically binds TIGIT and the antigen-binding domain that specifically binds PVRIG in the aforementioned PD-1/PVRIG/TIGIT-binding protein.
  • the present disclosure provides polynucleotides encoding PD-1 binding proteins, PD-1/PVRIG/TIGIT binding proteins, PD-1/PVRIG binding proteins, PD-1/TIGIT binding proteins, PVRIG binding proteins, TIGIT binding proteins of the present disclosure .
  • a polynucleotide of the present disclosure may be RNA, DNA or cDNA. According to some embodiments of the present disclosure, the polynucleotides of the present disclosure are substantially isolated polynucleotides.
  • a polynucleotide of the present disclosure may also be in the form of, may be present in, and/or may be part of a vector, such as a plasmid, cosmid, YAC, or viral vector.
  • the vector may especially be an expression vector, i.e. a vector providing for in vitro and/or in vivo (i.e. in a suitable host cell, host organism and/or expression system) expression of the PD-1 binding protein.
  • the expression vector typically comprises at least one polynucleotide of the present disclosure operably linked to one or more suitable expression control elements (eg, promoters, enhancers, terminators, etc.). It is common knowledge of those skilled in the art to select said elements and their sequences for expression in a particular host.
  • PD-1 Binding Proteins PD-1/PVRIG/TIGIT Binding Proteins, PD-1/PVRIG Binding Proteins, PD-1/TIGIT Binding Proteins, PVRIG Binding Proteins, TIGIT Binding Proteins of the Disclosure and other elements such as promoters, enhancers, terminators, integrators, selectable markers, leader sequences, reporter genes.
  • polynucleotides of the present disclosure may be prepared or obtained by known means (e.g., by automated DNA synthesis and/or recombinant DNA techniques) based on information on the amino acid sequence of the polypeptides of the present disclosure, and/or may be isolated from suitable natural sources .
  • the present disclosure provides expression or capable of expressing one or more PD-1 binding protein, PD-1/PVRIG/TIGIT binding protein, PD-1/PVRIG binding protein, PD-1/TIGIT binding protein, PVRIG binding protein of the present disclosure , a TIGIT binding protein and/or a recombinant host cell comprising a nucleic acid or vector of the present disclosure.
  • the host cell is a bacterial cell, a fungal cell, or a mammalian cell.
  • Bacterial cells include, for example, Gram-negative bacterial strains (such as Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus spp. (Bacillus) strains, Streptomyces (Streptomyces) strains, Staphylococcus (Staphylococcus) strains and Lactococcus (Lactococcus) strains) cells.
  • Gram-negative bacterial strains such as Escherichia coli strains, Proteus strains, and Pseudomonas strains
  • Gram-positive bacterial strains such as Bacillus spp. (Bacillus) strains, Streptomyces (Streptomyces) strains, Staphylococcus (Staphylococcus) strains and Lactococcus (Lactococcus) strains
  • Fungal cells include, for example, cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or Saccharomyces (such as Saccharomyces cerevisiae), fission yeast Schizosaccharomyces (such as Schizosaccharomyces pombe), Pichia (such as Pichia pastoris and Pichia methanolica) and Hansen A cell of a species of Saccharomyces (Hansenula).
  • Saccharomyces such as Saccharomyces cerevisiae
  • fission yeast Schizosaccharomyces such as Schizosaccharomyces pombe
  • Pichia such as Pichia pastoris and Pichia methanolica
  • Hansen A cell of a species of Saccharomyces Hansenula
  • Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
  • the present disclosure can also be used with amphibian cells, insect cells, plant cells, and any other cell used in the art to express heterologous proteins.
  • the cells of the present disclosure do not develop into finished plants or individual animals.
  • the present disclosure provides methods for preparing the PD-1 binding protein, PD-1/PVRIG/TIGIT binding protein, PD-1/PVRIG binding protein, PD-1/TIGIT binding protein, PVRIG binding protein, TIGIT binding protein of the present disclosure, the The method described generally includes the following steps:
  • the PD-1 binding protein, PD-1/PVRIG/TIGIT binding protein, PD-1/PVRIG binding protein, PD-1/TIGIT binding protein, PVRIG binding protein, TIGIT binding protein of the present disclosure can be expressed in cells as described above produced intracellularly (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies), followed by isolation from the host cell and optionally further purified; or it may be produced extracellularly (e.g., in the medium in which the host cell is cultivated), followed by Isolated from the culture medium and optionally further purified.
  • Expression vectors can stably transfect cells. Mammalian-like expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free medium in bioreactors for antibody production. The culture fluid that secretes the antibody can be purified and collected using conventional techniques. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The obtained product needs to be immediately frozen, such as -70°C, or freeze-dried.
  • the PD-1 binding protein, PD-1/PVRIG/TIGIT binding protein, PD-1/PVRIG binding protein, PD-1/TIGIT binding protein, PVRIG binding protein, TIGIT binding protein of the present disclosure can also be obtained through Other known methods of producing proteins, such as chemical synthesis, including solid-phase or solution-phase synthesis.
  • the present disclosure provides a composition comprising the aforementioned PD-1 binding protein, PD-1/PVRIG/TIGIT binding protein, PD-1/PVRIG binding protein, PD-1/TIGIT binding protein, PVRIG binding protein and/or TIGIT binding protein.
  • a pharmaceutical composition which contains the above-mentioned PD-1 binding protein, PD-1/PVRIG/TIGIT binding protein, PD-1/PVRIG binding protein and/or PD effective amount for cancer treatment, mitigation or prevention - 1/TIGIT binding protein and at least one pharmaceutically acceptable excipient, diluent or carrier.
  • the unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of PD-1 binding protein, PD-1/PVRIG/TIGIT binding protein, PD-1/PVRIG binding protein and/or PD -1/TIGIT binding protein, or the amount of PD-1 binding protein, PD-1/PVRIG/TIGIT binding protein, PD-1/PVRIG binding protein and/or PD-1/TIGIT binding protein in the unit dose of the pharmaceutical composition 0.1-2000 mg, in some embodiments 1-1000 mg.
  • a product or kit comprising at least one container each independently comprising the aforementioned PD-1 binding protein, PD-1/PVRIG/TIGIT binding protein, PD-1/PVRIG binding protein and/or PD- 1/TIGIT binding protein.
  • the kit comprises a container and a label.
  • Containers such as bottles, syringes and test tubes.
  • the container contains a composition effective for treating a condition.
  • the label on or associated with the container indicates that the composition is used to treat the condition of choice.
  • the composition contains the aforementioned PD-1 binding protein, PD-1/PVRIG/TIGIT binding protein, PD-1/PVRIG binding protein and/or PD-1/TIGIT binding protein.
  • a pharmaceutical composition comprising a PD-1 binding protein of the present disclosure and a PVRIG binding protein.
  • Other pharmaceutical compositions comprise the PD-1 binding protein and TIGIT binding protein of the present disclosure.
  • Other pharmaceutical compositions comprise the PD-1 binding protein, TIGIT binding protein and PVRIG binding protein of the present disclosure.
  • the PD-1 binding protein, TIGIT binding protein and/or PVRIG binding protein can be present in the form of an effective amount for treating or alleviating diseases (such as cancer), and the pharmaceutical composition can also include at least one pharmaceutically acceptable excipient, dilution or carrier.
  • composition, product or kit comprising any one or combination of the following:
  • Any PD-1/PVRIG binding protein provided by the present disclosure is any PD-1/PVRIG binding protein provided by the present disclosure.
  • Any PD-1 binding protein provided by the present disclosure and any PVRIG binding protein provided by the present disclosure are any PD-1 binding protein provided by the present disclosure and any PVRIG binding protein provided by the present disclosure;
  • composition when it is a pharmaceutical composition, it also contains at least one pharmaceutically acceptable excipient, diluent or carrier.
  • the present disclosure provides the aforementioned PD-1 binding protein, PD-1/PVRIG/TIGIT binding protein, PD-1/PVRIG binding protein, PD-1/TIGIT binding protein, PVRIG/TIGIT binding protein, PVRIG binding protein, TIGIT binding protein, Polynucleotides, compositions (including pharmaceutical compositions) are used in methods for treating, alleviating, preventing, and diagnosing diseases or conditions.
  • a method for improving, alleviating, treating or preventing a disease comprising administering to a subject an effective amount of improving, alleviating, treating or preventing the aforementioned PD-1 binding protein, PD-1/PVRIG/TIGIT binding protein, PD-1/PVRIG binding protein, PD-1/TIGIT binding protein, PVRIG binding protein, TIGIT binding protein, polynucleotide or composition (including pharmaceutical composition).
  • the PD-1 binding protein, PD-1/PVRIG/TIGIT binding protein, PD-1/PVRIG binding protein, PD-1/TIGIT binding protein or PVRIG/TIGIT binding protein of the present disclosure is provided to improve the preparation, The use of drugs to alleviate, treat or prevent disease.
  • the use of the PD-1 binding protein of the present disclosure in combination with the PVRIG binding protein in the preparation of a drug for improving, alleviating, treating or preventing diseases is provided.
  • the use of the PD-1 binding protein of the present disclosure in combination with the TIGIT binding protein in the preparation of a drug for improving, alleviating, treating or preventing diseases is provided.
  • the combination of the PD-1 binding protein, PVRIG binding protein and TIGIT binding protein of the present disclosure is provided in the preparation of a drug for improving, alleviating, treating or preventing diseases.
  • the use of the PD-1 binding protein of the present disclosure in combination with the PVRIG/TIGIT binding protein in the preparation of a drug for improving, alleviating, treating or preventing diseases is provided.
  • the PD-1 binding protein of the present disclosure is provided for improving, alleviating, treating or preventing diseases, in combination with PVRIG binding protein and/or TIGIT binding protein, or in combination with PVRIG/TIGIT binding protein.
  • the method for improving, alleviating, treating or preventing diseases of the PD-1 binding protein of the present disclosure is provided, further comprising administering PVRIG binding protein and/or TIGIT binding protein, or further comprising administering PVRIG/TIGIT binding protein.
  • a method for improving, alleviating, treating or preventing a disease with the PVRIG-binding protein and/or TIGIT-binding protein of the present disclosure further comprising administering the PD-1-binding protein of the present disclosure.
  • a method for improving, alleviating, treating or preventing a disease with the PVRIG/TIGIT binding protein of the present disclosure further comprising administering the PD-1 binding protein of the present disclosure.
  • the aforementioned disease is a proliferative disease or any other disease or condition characterized by uncontrolled cell growth (such as cancer, cancer and tumor are used interchangeably in this disclosure), such as PD-L1 overexpression Or related diseases (such as cancer) related to the abnormal expression of PVRIG and TIGIT.
  • a proliferative disease or any other disease or condition characterized by uncontrolled cell growth such as cancer, cancer and tumor are used interchangeably in this disclosure
  • PD-L1 overexpression Or related diseases such as cancer
  • the aforementioned cancer is a solid tumor or a hematological tumor.
  • the aforementioned cancer is advanced or metastatic.
  • the aforementioned cancer is selected from the following or combinations thereof: prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, pancreatic cancer, gastric (stomach/gastric ) carcinoma, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial carcinoma, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma) , glioma, renal carcinoma (RCC), lymphoma (NHL or HL), acute myelogenous leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, Testicular germ cell tumors, mesothelioma, esophageal cancer, Merkel Cells cancer, MSI-high cancer, KRAS-mutated tumors, adult T-cell leukemia/lymphoma
  • lung cancer selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cancer, hematologic cancer, or any other disease or condition characterized by uncontrolled cell growth.
  • the aforementioned subject has a condition associated with PVRIG and/or TIGIT.
  • the subject's condition includes cancer that expresses or does not express PVRIG and further includes non-metastatic or non-invasive and invasive or metastatic cancer, wherein expression of PVRIG by immune cells, stromal cells, or diseased cells is inhibited Anti-tumor responses and anti-invasive immune responses.
  • the methods of the present disclosure are suitable, for example, for the treatment of vascularized cancers.
  • a method for treating or preventing infection or sepsis in a subject comprising administering to the subject an effective amount of the aforementioned PD-1 binding protein, PD-1/PVRIG/TIGIT binding protein , PD-1/PVRIG binding protein, PD-1/TIGIT binding protein, PVRIG binding protein, TIGIT binding protein, polynucleotide, composition (including pharmaceutical composition).
  • the infection is a pathogen infection characterized by varying degrees of dysfunction of virus-specific T cell responses, such as HIV, HCV, HBV.
  • the sepsis includes severe sepsis, septic shock, systemic inflammatory response syndrome (SIRS), bacteremia, sepsis, toxemia, and septic syndrome.
  • methods of activating cytotoxic T cells (CTL) in a subject are provided, methods of activating NK cells in a subject are provided, methods of activating ⁇ T cells in a subject are provided, methods of activating Th1 cells in a subject are provided, Cell method, providing a method for activating, reducing or eliminating at least one cell number and/or activity of regulatory T cells (Treg) in a subject, providing increasing IFN- ⁇ production and/or activity in a subject Or a method for the secretion of pro-inflammatory cytokines, a method for inhibiting the interaction between PVRIG and PVRL2 in a subject, and a method for blocking or inhibiting the interaction between PD-1 and PD-L1/PD-L2 in a subject method, all comprising administering to the subject an effective amount of the aforementioned PD-1 binding protein, PD-1/PVRIG/TIGIT binding protein, PD-1/PVRIG binding protein, PD-1/TIGIT binding protein, PVRIG binding protein , T
  • the present disclosure provides PD-1 binding protein, PD-1/PVRIG/TIGIT binding protein, PD-1/PVRIG binding protein, PD-1/TIGIT binding protein, PVRIG/TIGIT binding protein, PVRIG binding protein, TIGIT binding protein, multinuclear Detection application of nucleotide and composition.
  • the present disclosure also provides methods, systems or devices for detecting PD-1, PVRIG, and TIGIT in vivo or in vitro, which include treating samples with the aforementioned binding proteins, polynucleotides, and compositions of the present disclosure.
  • an in vitro detection method, system or device may, for example, include:
  • a change eg, a statistically significant change in complex formation in a sample or subject as compared to a control sample or subject indicates the presence of PD-1, PVRIG, TIGIT in the sample.
  • the in vivo detection method, system or device may comprise:
  • Detection can include determining where or when a complex is formed.
  • the aforementioned binding proteins and polynucleotides are labeled with detectable substances, and the detection of substances capable of binding proteins and polynucleotides (such as PD-1, PVRIG, TIGIT) can be realized by detecting the labels.
  • Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances.
  • Complex formation of binding proteins, polynucleotides and PD-1, PVRIG, TIGIT can be detected by measuring or visualizing substances that bind or do not bind to PD-1, PVRIG, TIGIT.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • tissue immunohistochemistry for detection purposes, binding proteins, polynucleotides of the disclosure can be labeled with fluorophore chromophores.
  • diagnostic reagents comprising the above-mentioned polynucleotides and binding proteins are also provided, as well as related diagnostic applications.
  • kits in some embodiments, includes the aforementioned binding protein, polynucleotide, and instructions for use in diagnosis.
  • the kit may also contain at least one additional reagent, such as a marker or an additional diagnostic agent.
  • the binding proteins can be formulated as pharmaceutical compositions.
  • Figure 1A to Figure 1B are the results of detecting the activity of anti-PD-1 nanobody by PD-1/PD-L1 NFAT reporter gene system.
  • Pembrolizumab was used as a positive control, and hIgG4 was used as a negative control; among them, Figure 1A shows the results of A6_IgG4 and A17_IgG4.
  • Figure 1B shows the results of A17h1_IgG4, A17m09_hIgG4, A17m0901_hIgG4, A17m0902_hIgG4, A17m0903_hIgG4, A17m0905_hIgG4.
  • Figure 2 is the detection of the degree of activation of T cells by A17m09_hIgG4 through the release of cytokines in the DC cell: T cell mixed lymphatic reaction.
  • Pembrolizumab was used as a positive control
  • hIgG4 was used as a negative control.
  • Figure 3A to Figure 3B are the in vivo drug efficacy test results of A17m09_hIgG4 inhibiting the growth of MC38 colon cancer tumors in mice.
  • Figure 3A is the results of tumor volume in mice
  • Figure 3B is the body weight of mice.
  • Pembrolizumab was used as a positive control
  • PBS was used as a negative control. .
  • Figure 4 shows the results of the mixed lymphocyte reaction (MLR) experiment of anti-PVRIG nanobodies 30 and 151, using Tab5 as a positive control and hIgG4 as a negative control.
  • MLR mixed lymphocyte reaction
  • Figure 5 shows the results of the MLR experiment of the anti-PVRIG/TIGIT bispecific antibody 1708-151H8, and simultaneously detected 151H8, 1708, hIgG4, Tab5, and Pembrolizumab as controls.
  • Figures 6A to 6B show the anti-tumor results of the combination of anti-PVRIG/TIGIT bispecific antibodies 1708-151, 1708 and 151 in a mouse subcutaneous xenograft model of human melanoma A375 mixed with human PBMC.
  • Figure 6B is the corresponding mouse body weight graph.
  • Figures 7A to 7B show the anti-tumor results of anti-PVRIG/TIGIT bispecific antibodies 1708-151H7 and 1708-30H2 in a mouse subcutaneous xenograft tumor model of human melanoma A375 mixed with human PBMC.
  • Figure 7B is the corresponding mouse body weight graph.
  • Figure 8 is a schematic diagram of four antibody configurations of the anti-PD-1/PVRIG/TIGIT trispecific antibody.
  • Figures 9A to 9B are the detection results of the binding of the antibodies of the four configurations of A17h1-1708-30H2 and A17h1-1708-151H7 to the TIGIT antigen, respectively.
  • Figures 10A to 10B are the detection results of the four configurations of antibodies binding to PVRIG antigens of A17h1-1708-30H2 and A17h1-1708-151H7 respectively.
  • Figure 11A, Figure 11B, and Figure 11C respectively show the binding ability of PVRIG sequence optimized in the anti-PD-1/PVRIG/TIGIT trispecific antibody to cells overexpressing PVRIG, TIGIT, and PD-1 antigens.
  • Figure 12A to Figure 12B are the blocking of the binding of PD-L1/PD-1 in the PD-1/PD-L1 NFAT reporter gene system by the antibodies of the four configurations of A17h1-1708-30H2 and A17h1-1708-151H7 respectively .
  • Figures 13A to 13B are the MLR detection results of the four configurations of antibodies of A17h1-1708-30H2 and A17h1-1708-151H7 respectively.
  • Figure 14A to Figure 14B are the MLR detection results of A17m0902-1708-30H2-C in two subjects respectively.
  • Figure 15A to Figure 15B are the results of the NK cell killing function experiments of the antibodies of the four configurations of A17h1-1708-30H2 and A17h1-1708-151H7 respectively.
  • FIG. 16A to Figure 16B Anti-PD-1/PVRIG/TIGIT trispecific antibody evaluation of anti-tumor effect in PD-1 mAb-responsive human melanoma A375 mixed with human PBMC subcutaneous xenograft tumor model in mice.
  • FIG 17A to Figure 17B Anti-PD-1/PVRIG/TIGIT trispecific antibody evaluation of anti-tumor effect in human melanoma A375 mixed with human PBMC mouse subcutaneous xenograft tumor model that does not respond to PD-1 monoclonal antibody.
  • Programmed death 1 "Programmed death 1", “programmed cell death 1”, “protein PD-1”, “PD-1”, “PDCD1” and “hPD-1” are used interchangeably and include variants of human PD-1 Individuals, isotypes, species homologues, and analogs that share at least one common epitope with PD-1.
  • the complete PD-1 sequence can be found from GenBank accession number U64863.
  • “Programmed death ligand-1 (PD-L1)” is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), which downregulates T cells when bound to PD-1 Activation and cytokine secretion.
  • PD-L1 as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and interspecies homologs of hPD-L1, and hPD-L1 having at least one common epitope analogues of .
  • the complete hPD-L1 sequence can be found with GenBank accession number Q9NZQ7.
  • PVRIG or “PVRIG protein” or “PVRIG polypeptide” may optionally include any such protein or variant, conjugate or fragment thereof, including but not limited to known or wild-type PVRIG as described herein, As well as any naturally occurring splice variants, amino acid variants or isoforms, and especially soluble extracellular domain (ECD) fragments of PVRIG.
  • ECD extracellular domain
  • TIGIT or "TIGIT protein” or “TIGIT polypeptide” may optionally include any such protein or variant, conjugate or fragment thereof, including but not limited to known or wild-type TIGIT as described herein, and any naturally occurring splice variants, amino acid variants or isoforms.
  • the complete TIGIT sequence can be found at GenBank accession number AAI01289.1.
  • Binding with PD-1 refers to being able to interact with PD-1 or its epitope, and the PD-1 or its epitope may be of human origin.
  • Binding to PVRIG refers to being capable of interacting with PVRIG or its epitope, which may be of human origin.
  • Binding to TIGIT refers to being capable of interacting with TIGIT or its epitope, which may be of human origin.
  • Antigen-binding site refers to a discrete three-dimensional site on an antigen recognized by an antibody or antigen-binding fragment of the present disclosure.
  • a "PD-1 binding protein” encompasses any molecule capable of specifically binding to a protein or epitope thereof of PD-1.
  • a PD-1 binding protein may comprise an antibody against PD-1 as defined in the present disclosure, an antigen-binding fragment thereof or a conjugate thereof.
  • PD-1 binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR grafted molecules.
  • the "PD-1 binding protein” of the present disclosure may comprise at least one immunoglobulin single variable domain (such as VHH) that binds to PD-1.
  • a "PD-1 binding protein” may comprise 2, 3, 4 or more immunoglobulin single variable domains (eg, VHH) that bind PD-1.
  • the immunoglobulin single variable domain of PD-1 in addition to the immunoglobulin single variable domain of PD-1, it may also include a linker and/or a part with effector function, such as a half-life extending part (such as a serum albumin-binding immunoglobulin protein single variable domain), and/or a fusion partner (such as serum albumin) and/or a conjugated polymer (such as PEG) and/or an Fc region.
  • a linker and/or a part with effector function such as a half-life extending part (such as a serum albumin-binding immunoglobulin protein single variable domain), and/or a fusion partner (such as serum albumin) and/or a conjugated polymer (such as PEG) and/or an Fc region.
  • a linker and/or a part with effector function such as a half-life extending part (such as a serum albumin-binding immunoglobulin protein single variable domain), and/or a fusion partner (
  • a "PD-1 binding protein" of the present disclosure also encompasses bi/multispecific antibodies comprising immunoglobulins that bind different antigens (such as a primary antibody that binds a first antigen (such as PD-1) and a second antibody that binds a second antigen (such as PVRIG), optionally including a third specific antibody that binds a third antigen (such as TIGIT), and further optionally, a fourth antibody that binds a fourth antigen.”
  • PD -1 binding protein encompasses "PD-1/PVRIG binding protein", “PD-1/TIGIT binding protein", “PD-1/PVRIG/TIGIT binding protein”.
  • a “PD-1 binding protein” or “anti-PD-1 antibody” of the present disclosure may comprise one or more effector molecules, for example, in a conjugated manner.
  • effector molecules include, for example, antineoplastic agents, drugs, toxins, biologically active proteins (such as enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof (such as DNA, RNA and fragments thereof ), radionuclides, especially radioiodides, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds detectable by NMR or ESR spectroscopic analysis.
  • the effector molecule when it is a polymer, it may generally be a synthetic or naturally occurring polymer, such as optionally substituted linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymers or branched Polysaccharides or unbranched polysaccharides, such as homo- or heteropolysaccharides.
  • Specific optional substituents that may be present on the synthetic polymers described above include one or more hydroxyl, methyl or methoxy groups.
  • Specific examples of synthetic polymers include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, especially optionally substituted poly(ethylene glycol) alcohol) such as methoxypoly(ethylene glycol) or derivatives thereof.
  • polymers include lactose, amylose, dextran, glycogen or derivatives thereof.
  • the polymer is albumin or a fragment thereof, such as human serum albumin or a fragment thereof.
  • the conjugation of the polymer to the PD-1 binding protein or anti-PD-1 antibody can be achieved by conventional methods.
  • Cytokine is a general term for proteins released by a population of cells that act as intercellular mediators on other cells. Examples of such cytokines include lymphokines, monokines, chemokines and traditional polypeptide hormones. Exemplary cytokines include: human IL-2, IFN- ⁇ , IL-6, TNF ⁇ , IL-17 and IL-5.
  • Antibody encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (such as bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions) as long as they exhibit the desired antigen-binding activity.
  • Antibody can refer to immunoglobulin, which is a tetrapeptide chain structure composed of two heavy chains and two light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five classes, or isotypes, of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are mu, delta, and gamma chains, respectively , ⁇ chain and ⁇ chain.
  • the same class of Ig can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either kappa chains or lambda chains by difference in the constant region.
  • Each of the five Ig classes can have either a kappa chain or a lambda chain.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region (C region).
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDR).
  • Each light chain variable region (VL) and heavy chain variable region (VH) are composed of 3 CDR regions and 4 FR regions, and the sequence from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • Antigen-binding fragment encompasses single chain antibodies (i.e. full length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv , single domain antibody (e.g. VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibody, Bis-scFv, diabody, tribody, triabody, tetrabody and epitope binding fragments of any of the above (see e.g. Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3) 209-217).
  • Antigen-binding fragments of the present disclosure also include Fab and Fab' fragments described in WO2005/003169, WO2005/003170 and WO2005/003171.
  • Multivalent antibodies may comprise multiple specificities such as bispecifics or may be monospecific (see e.g. WO 92/22583 and WO 05/113605), an example of the latter being the Tri-Fab described in WO 92/22583 (or TFM).
  • Bispecific antibody encompasses antibodies (including antibodies or antigen-binding fragments thereof such as single chain antibodies) that specifically bind to two different antigens or to two or at least two different epitopes of the same antigen.
  • Bispecific antibodies of various structures have been disclosed in the prior art. According to the integrity of the IgG molecule, it can be divided into IgG-like bispecific antibodies and antibody fragment bispecific antibodies. According to the number of antigen-binding regions, bispecific antibodies can be divided into bivalent, trivalent, tetravalent or more valent. According to whether the structure is symmetrical, it can be divided into symmetrical structure bispecific antibody and asymmetric structure bispecific antibody.
  • bispecific antibodies based on antibody fragments such as Fab fragments lacking Fc fragments, which form bispecific antibodies by combining two or more Fab fragments in one molecule, which have lower immunogenicity, and It has small molecular weight and high tumor tissue permeability.
  • Typical antibody structures of this type include bispecific antibodies such as F(ab)2, scFv-Fab, and (scFv)2-Fab.
  • IgG-like bispecific antibody for example, with Fc fragment
  • this type of antibody has a relatively large molecular weight, and the Fc fragment is helpful for the purification of the antibody in the later stage, and improves its solubility and stability, and the Fc part may also bind to the receptor FcRn , increase antibody serum half-life.
  • bispecific antibodies such as KiH, CrossMAb, Triomab quadroma, Fc ⁇ Adp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, 1Fab-IgG TDB, FynomAb, two-in- one/DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab) 2 -CrossMAb, IgG-(scFv) 2 , Bs4Ab, DVD-Ig, Tetravalent - Bispecific antibodies such as DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab) 4 -CrossMAb (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585–608 (2019 ); Chen S1 e
  • Trispecific antibody refers to the ability to specifically bind three or at least three different epitopes, which may be epitopes of different antigens or different epitopes of the same antigen.
  • deterministic delineation of the CDRs and identification of the residues of the binding site can be accomplished by resolving the structure of the antibody and/or resolving the structure of the antibody-ligand complex. This can be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography.
  • Various analytical methods can be used to identify CDRs, including but not limited to Kabat numbering system, Chothia numbering system, AbM numbering system, IMGT numbering system, contact definition, conformation definition.
  • the Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see eg Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8).
  • the Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the location of certain structural ring regions. (See eg Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83).
  • the AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group for modeling antibody structures (see, e.g., Martin et al., 1989, ProcNatl Acad Sci (USA), 86:9268-9272; "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd).
  • the AbM numbering system uses a combination of knowledge base and ab initio methods to model the tertiary structure of antibodies from basic sequences (see Samudrala et al., 1999, "AbM” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198. Initio Protein Structure Prediction Using a Combined Hierarchical Approach” described).
  • CDR may refer to a CDR defined by any method known in the art, including combinations of methods. Correspondence between various numbering systems is well known to those skilled in the art, and is exemplarily shown in Table 1 below.
  • the CDR amino acid residues of the VL and VH regions of the disclosed antibodies conform to the known Kabat numbering system in number and position.
  • Antibodies of the present disclosure may be polyclonal, monoclonal, xenogeneic, allogeneic, isogenic, or modified forms thereof, with monoclonal antibodies being particularly useful in various embodiments.
  • antibodies of the present disclosure are recombinant antibodies.
  • Recombinant as used herein generally refers to a product such as a cell or nucleic acid, protein or vector, and means that the cell, nucleic acid, protein or vector has been modified by introducing a heterologous nucleic acid or protein or altering a native nucleic acid or protein.
  • a recombinant cell expresses a gene that is not present in the native (non-recombinant) form of the cell or expresses a native gene that would otherwise be aberrantly expressed, underexpressed, or not expressed at all.
  • a “domain” of a polypeptide or protein refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein.
  • a domain is responsible for a single functional property of a protein, and in many cases can be added, removed or transferred to other proteins without loss of the rest of the protein and/or the function of the domain.
  • Immunoglobulin domain refers to the globular region of an antibody chain. Immunoglobulin domains are characterized by their maintenance of the folded characteristics of antibody molecules.
  • Immunoglobulin variable domain means a domain consisting essentially of "framework region 1" or “FR1”, “framework region 2" or “FR2”, “framework region 3" or “FR3”, and “framework region 4" or the four “framework regions” of "FR4", the "complementarity-determining region 1" or “CDR1”, the “complementarity-determining region 2" or “CDR2”, and the three “complementarity-determining regions” or “CDR3” Immunoglobulin domains consisting of "determining regions” or "CDRs".
  • the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains are endowed with antigen-specificity by having an antigen-binding site.
  • Antibody framework (FR) refers to the portion of a variable domain that serves as a scaffold for the antigen-binding loops (CDRs) of the variable domain.
  • Immunoglobulin single variable domain is commonly used to refer to a variable domain that can be used without interaction with other variable domains (e.g. without the need for between the VH and VL domains of a conventional four-chain monoclonal antibody).
  • an immunoglobulin variable domain (which may be a heavy or light chain domain, including a VH, VHH or VL domain) that forms a functional antigen binding site.
  • immunoglobulin single variable domains include Nanobodies (including VHH, humanized VHH and/or camelized VH, such as camelized human VH), IgNAR, domains, as VH domains or derived from VH Domain (single domain) antibodies such as dAbs TM and (single domain) antibodies that are or are derived from VL domains such as dAbs TM .
  • Immunoglobulin single variable domains based on and/or derived from heavy chain variable domains, such as VH or VHH domains are generally preferred.
  • a specific example of an immunoglobulin single variable domain is a "VHH domain” (or simply "VHH") as defined below.
  • VHH domains also known as heavy chain single domain antibodies, VHH, VHH domains, VHH antibody fragments, VHH antibodies, nanobodies, are termed “heavy chain antibodies” (i.e. "antibodies lacking light chains”) ) antigen-binding variable domains of immunoglobulins (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature 363, 446-448 (1993)).
  • the term "VHH domain” is used to distinguish the VHH from the VH and VL present in conventional tetrapeptide chain structured antibodies.
  • the VHH domain specifically binds the epitope without the need for an additional antigen-binding domain (however, in conventional tetrapeptide chain antibodies, the epitope is recognized by the VL domain together with the VH domain).
  • the VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain.
  • the terms "heavy chain single domain antibody”, “VHH domain”, “VHH”, “VHH domain”, “ VHH antibody fragment”, “VHH antibody”, as well as” "Nanobody”("Nanobody” is a trademark of Ablynx NV, Ghent, Belgium) can be used interchangeably.
  • VHH domain includes but is not limited to natural antibodies produced by camelids, and can also be antibodies produced by camelids After humanization, it can also be obtained by phage display technology.
  • the total number of amino acid residues in the VHH domain will usually be in the range of 110 to 120, often between 112 and 115. However, it should be Note that smaller and longer sequences may also be suitable for the purposes described in this disclosure. Methods for obtaining VHHs that bind specific antigens or epitopes have been previously disclosed in: R.
  • the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e. one according to Kabat numbering). or positions may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This means that, in general, numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.
  • Other numbering systems or coding conventions for VHH include Chothia, IMGT, AbM.
  • Humanized antibody also known as CDR-grafted antibody (CDR-grafted antibody) refers to an antibody produced by grafting a non-human CDR sequence into a human antibody variable region framework. It can overcome the strong immune response induced by chimeric antibodies due to carrying a large number of non-human protein components. In order to avoid a decrease in activity while reducing immunogenicity, minimal reverse mutations can be performed on the variable region of the fully human antibody to maintain activity.
  • humanization examples include: replacing the amino acids of the original VHH sequence with one or more amino acid residues present in the VHH domain derived from Camelidae at the corresponding position in the VH domain of an antibody with a conventional tetrapeptide chain structure "Humanization" by one or more amino acid residues in the VHH sequence (also referred to as “sequence optimization” in this disclosure, in addition to humanization, “sequence optimization” can also cover one or more amino acid residues by providing VHH improved properties. other modifications to the sequence by mutation, such as removal of potential post-translational modification sites).
  • a humanized VHH domain may contain one or more fully human framework region sequences. In some embodiments, the human framework region sequences of IGHV3 may be included.
  • humanization includes grafting mouse CDR sequences into human antibody variable region frameworks, ie antibodies produced from different types of human germline antibody framework sequences. It can overcome the strong antibody variable antibody response induced by chimeric antibodies due to carrying a large number of heterologous protein components.
  • Humanization methods such as protein surface amino acid humanization (resurfacing) and antibody humanization universal framework grafting method (CDR grafting to a universal framework), that is, CDR “grafting” to other "scaffolds” (including but not limited to human scaffolds or non-immunoglobulin scaffold). Scaffolds and techniques suitable for such CDR grafting are known in the art.
  • the germline DNA sequences of the human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database, and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition.
  • the humanized antibody of the present disclosure also includes the humanized antibody after affinity maturation of CDR by phage display.
  • minimal reverse mutations or back mutations can be performed on the human antibody variable region framework sequence to maintain activity.
  • an “affinity matured” antibody is one that has one or more alterations in one or more hypervariable regions (HVRs) as compared to the parent antibody, such alterations resulting in improved affinity of the antibody for the antigen.
  • HVRs hypervariable regions
  • an "affinity matured" PD-1 binding protein or PD-1 antibody has one or more changes in one or more CDRs that result in an increased affinity for the antigen compared to its parental antibody.
  • Affinity matured antibodies can be prepared by methods known in the art, for example, from Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc. Nat. Acad.
  • Fully human antibodies include antibodies having variable and constant regions of human germline immunoglobulin sequences. Fully human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). "Fully human antibody” does not include “humanized antibody”.
  • binding protein refers to the binding of a binding protein to an epitope on an antigen.
  • a PD-1 binding protein, PD-1/PVRIG binding protein, PD-1/TIGIT binding protein, PD-1/PVRIG/TIGIT binding protein of the present disclosure will have a preferred 10- as measured in a Biacore or KinExA or Fortibio assay.
  • K D dissociation constant of 7 to 10 ⁇ 10 moles/liter (M), more preferably 10 ⁇ 8 to 10 ⁇ 10 moles/liter, even more preferably 10 ⁇ 9 to 10 ⁇ 10 or lower, and/or Bind the antigen to be bound ( i.e.
  • PD- 1 , PVRIG and/or or TIGIT or its epitopes. Any KD value greater than 10 -4 M is generally considered to indicate non-specific binding.
  • Specific binding of a binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and/or competitive binding assays as described in this disclosure (e.g. radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competitive assay).
  • SPR surface plasmon resonance
  • Scatchard assays e.g. radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competitive assay.
  • sandwich competitive assay e.g. radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competitive assay.
  • Epitope refers to the site on an antigen to which an immunoglobulin or antibody binds. Epitopes can be formed from contiguous amino acids, or non-contiguous amino acids that are juxtaposed by the tertiary folding of the protein. Epitopes formed from adjacent amino acids are generally maintained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents.
  • An epitope typically comprises at least 3-15 amino acids in a unique spatial conformation. Methods for determining the epitope bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays, among others. Methods of determining the spatial conformation of an epitope include techniques in the art and techniques described in this disclosure, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.
  • Binding affinity or “affinity” is used in the present disclosure as a measure of the strength of a non-covalent interaction between two molecules (eg, an antibody or portion thereof and an antigen).
  • the binding affinity between two molecules can be quantified by determining the dissociation constant ( KD ).
  • KD can be determined by measuring the kinetics of complex formation and dissociation using, for example, the Surface Plasmon Resonance (SPR) method (Biacore).
  • SPR Surface Plasmon Resonance
  • the rate constants corresponding to the association and dissociation of the monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively.
  • the value of the dissociation constant can be directly determined by well known methods and can even be calculated for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9:340-362).
  • KD can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432).
  • Other standard assays to assess the binding ability of an antibody to a target antigen are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis, among others exemplified in this disclosure.
  • Binding kinetics and binding affinity of antibodies can also be assessed by standard assays known in the art, such as surface plasmon resonance (SPR), for example by using the Biacore TM system or KinExA. Binding affinities associated with different molecular interactions can be compared by comparing the KD values of the individual antibody/antigen complexes, eg, a comparison of the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the K value of an interaction of interest (e.g., a specific interaction between an antibody and an antigen) with that of an unintended interaction (e.g., a control antibody known not to bind PD-1). ) K D value for evaluation.
  • SPR surface plasmon resonance
  • a “conservative substitution” refers to a substitution for another amino acid residue that has similar properties to the original amino acid residue.
  • lysine, arginine, and histidine have similar properties in that they have basic side chains
  • aspartic acid and glutamic acid have similar properties in that they have acidic side chains.
  • glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains
  • alanine , valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have nonpolar side chains.
  • “Homology”, “identity” or “sequence identity” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position of.
  • the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of compared positions x 100%. For example, two sequences are 60% homologous if there are 6 matches or homology at 10 positions in the two sequences when the sequences are optimally aligned. In general, comparisons are made when two sequences are aligned to yield the greatest percent homology.
  • Nucleic acid or “polynucleotide” are used interchangeably herein to refer to any molecule of DNA or RNA, single or double stranded and, in the case of single stranded, its complement, preferably double stranded DNA. Nucleic acids are "operably linked” when they are placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.
  • a “host cell” includes any cell or cell culture that can be or has been a recipient of a vector for the incorporation of a polynucleotide insert.
  • a host cell includes the progeny of a single host cell, and the progeny are not necessarily identical (in morphology or in genomic DNA complement) to the parent cell due to natural, accidental, or deliberate mutation.
  • Host cells include cells transfected and/or transformed in vivo with polynucleotides of the present disclosure.
  • Cell “cell line,” and “cell culture” are used interchangeably, and all such designations include progeny. It should also be understood that all progeny may not be precisely identical in DNA content due to deliberate or unintentional mutations. The term includes mutant progeny that have the same function or biological activity as the cells screened for in the original transformation.
  • Inhibition or “blocking” are used interchangeably and encompass both partial and complete inhibition/blocking. “Inhibition of growth” (eg, in relation to cells) is intended to include any measurable decrease in cell growth.
  • Preventing the growth or “growth inhibiting” means inhibiting the growth or proliferation of cells.
  • Proliferative disease refers to a disorder associated with some degree of abnormal cell proliferation.
  • the proliferative disorder is cancer.
  • Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. "Cancer”, “cancerous”, “proliferative disorder” and “tumor” are not mutually exclusive when referred to in this disclosure.
  • Preventing cancer means delaying, inhibiting or preventing the onset of cancer in a subject in which the onset of cancer or tumorigenesis has not been proven, but has been determined, for example, by genetic screening or other methods, identified susceptibility to cancer. This also includes treating a subject with a precancerous condition to halt progression of the precancerous condition to a malignancy or to cause regression thereof.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid refers to the interaction of an exogenous drug, therapeutic, diagnostic, or composition with an animal. , humans, subjects, cells, tissues, organs or biological fluids, such as therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of cells includes contacting the reagents with the cells, and contacting the reagents with a fluid, wherein the fluid contacts the cells.
  • administering also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnostic, binding composition or by another cell. When applied to human, veterinary or research subjects, it refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent to a subject, for example, comprising any binding protein of the present disclosure or a pharmaceutical composition thereof as a therapeutic agent, the subject has suffered from, suspected of suffering from, prone to Suffering from one or more proliferative diseases, or symptoms thereof, for which the therapeutic agent is known to have a therapeutic effect.
  • a therapeutic agent is administered in a subject or population to be treated in an amount effective to alleviate one or more symptoms of a disease, either by inducing regression of such symptoms or inhibiting the progression of such symptoms to any clinically measurable extent.
  • the amount of a therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on factors such as the disease state, age, and weight of the subject, and the extent to which the drug produces the desired therapeutic effect in the subject. ability. Whether disease symptoms have been alleviated can be assessed by any of the clinical tests commonly used by a physician or other professional health care practitioner to assess the severity or progression of such symptoms.
  • any statistical test method known in the art such as Student's t-test, chi-square test, according to Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test determined that it should reduce the target disease symptoms in a statistically significant number of subjects.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to permit or facilitate diagnosis.
  • the effective amount for a subject may vary depending on factors such as the condition being treated, the general health of the subject, the method, route and dose of administration, and the severity of side effects.
  • An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • a subject of the present disclosure can be an animal or a human subject.
  • Subject and “patient” in the present disclosure refer to mammals, especially primates, especially humans.
  • human PD-1 with His tag was used as immunization antigen
  • human PD-1 and cynomolgus monkey PD-1 with biotin label were used as screening antigens, all of which were purchased from AcroBiosystems.
  • Example 1-1 Screening and preparation of anti-PD-1 nanobody
  • the human PD-1 protein (NP_005009.2) sequence is as follows, where the underline is the sequence of the extracellular region, and the amino acids start and end at Leu25-Gln 167.
  • Vaccine 2 healthy alpacas.
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • PBMCs in peripheral blood were isolated, RNA in PBMCs was extracted, and total cDNA was obtained by reverse transcription.
  • the cDNA was used as the template for the first round of nested PCR.
  • the second round of nested PCR product fragments were ligated with a yeast library vector (yeast library vector pYDN3), and the ligated products were transformed into yeast competent cells by electric shock.
  • the insertion rate and library diversity were verified by colony PCR. According to the number of library transformants, library insertion rate and library diversity, the sequencing results showed that the library capacity of one alpaca library was 9.28 ⁇ 10 7 , and that of the other alpaca library was 1.54 ⁇ 10 8 .
  • FACS flow cytometry sorting
  • library screening and identification are used for antibody screening.
  • the sorting adopts two rounds of sorting steps: the first round of sorting uses biotin-labeled human PD-1 protein (Avitag-His Tag) magnetic beads for enrichment, and the second round uses biotin-labeled monkey PD-1 protein (His, Avitag-His Tag) ) flow sorter for sorting (flow antibody: Mouse anti-HA tag Dylight 488; streptavidin Dylight 650).
  • Avitag-His Tag biotin-labeled human PD-1 protein
  • His, Avitag-His Tag biotin-labeled monkey PD-1 protein
  • flow sorter flow sorter for sorting
  • flow antibody Mouse anti-HA tag Dylight 488; streptavidin Dylight 650.
  • 18 unique sequences of VHH cross-linked with human and monkey PD-1 antigens were obtained through monoclonal identification, sequencing, and sequence analysis. The sequences of A6 and A17 among them are
  • the above sequences were linked to human IgG4 Fc (including the hinge region, and with S228P, according to the Eu numbering system) fragments to construct VHH-Fc antibodies. Construct the plasmid, transiently transfect HEK293 cells, express, use protein A column to purify, wash the column with PBS, and elute with 0.1M glycine buffer, pH 2.5. Dialyzed into PBS pH 7.4 buffer.
  • the sequences of human IgG4 Fc (including the hinge region) and A6 and A17-based VHH-Fc antibodies are as follows:
  • Example 1-2 Affinity Identification of Anti-PD-1 Nanobody and Antigen PD-1
  • Pembrolizumab (purchased from Baiying Biotechnology) was used as a control, and the results are shown in Table 5. Moreover, the EC 50 value of A17_hIgG4 was 0.3568nM, which was significantly better than other antibodies obtained by simultaneous screening (results not shown).
  • Antibody humanization is performed by CDR-grafting method.
  • CDR-grafting method Through the IMGT or NCBI website, compare the parental anti-PD-1 antibody with the full human germline gene in the IMGT or NCBI/igblast database, and select the human germline gene IGHV 3- which is highly homologous to the PD-1 Nanobody 23 (IGHV3-23*01; IGHV3-23*04) was used as humanized template.
  • Transplant CDR retain 4 key residues (Y37, E44, R45, L47) in FR2 of Nanobodies, and perform back mutations on the key core residues close to the CDR region and the Vernier position residues that interact with the CDR, Humanized antibodies A17h1, A17h2, A17h3, A6h1 were obtained.
  • TCE removal modification was carried out.
  • antibody deamidation and antibody isomerization sites are modified. Get the following sequence:
  • A17m09 was further selected for germline transformation of antibody sequences to improve the degree of humanization, and the following sequences were obtained:
  • A17 of the present disclosure has the following general formula sequence:
  • CDR1 DYSMS (SEQ ID NO: 7)
  • CDR2 IISGSGX 1 X 2 X 3 HYVDSVKG, wherein X 1 is selected from V or G, X 2 is selected from I or S, X 3 is selected from T or A (SEQ ID NO: 36)
  • CDR3 VSDWX 4 X 5 Y, wherein X 4 is selected from D or E, and X 5 is selected from D or E (SEQ ID NO: 37).
  • CDR2 can be:
  • CDR3 can be:
  • VSDWDDY (SEQ ID NO: 9)
  • VSDWEDY (SEQ ID NO: 41)
  • Example 1-4 PD-1/PD-L1 reporter gene system detects the blocking of PD-L1 binding to PD-1 by anti-PD-1 nanobody
  • the biological activity of anti-PD-1 antibodies can be detected at the cell level.
  • the system consists of two genetically engineered cell lines: PD-1-overexpressing effector cell Jurkat (containing NFAT reporter gene luciferase) and PD-L1-overexpressing antigen-presenting cell CHO-K1 cells. When these two cells were co-cultured, the PD-1/PD-L1 interaction inhibited TCR-mediated NFAT activation, which in turn suppressed the expression of the NFAT reporter gene luciferase.
  • TCR activation induces the expression of luciferase through the NFAT pathway, which can be measured by adding Bio-Glo reagents and using a luminometer for fluorescence quantitative detection to determine the degree of activation of effector cells and Antibody biological activity.
  • Dilute CHO-K1 cells overexpressing PD-L1 to 4 ⁇ 10 5 /mL add 100 ⁇ L to each well of a 96-well plate, and culture overnight. After aspirating the medium, 40 ⁇ L of 1.25 ⁇ 10 6 /mL PD-1 Jurkat cells and 40 ⁇ L of anti-PD-1 nanobody solutions of different concentrations (diluted in 1640+2% FBS solution) were quickly added. Incubate at 37°C for 6 hours, return to room temperature, add 40 ⁇ L Bright-glo reagent (Cat: E2620, promega) to each well, shake at 350 rpm for 5 minutes in the dark, keep at room temperature for 5 minutes, and read the fluorescence value with a microplate reader. Calculation of IC50 .
  • Bright-glo reagent Cat: E2620, promega
  • Pembrolizumab and hIgG4 isotype were used as controls.
  • the biological function activity IC50 value of A17_hIgG4 is 1.199nM, which is significantly better than other antibodies screened simultaneously in the present disclosure (for example, A3_hIgG4 is 7.62, A13_hIgG4 is 3.208; A3_hIgG4 and A13_hIgG4 do not show the sequence) .
  • Antibody number IC 50 (nM) A6_hIgG4 1.354 A17_hIgG4 1.199
  • the humanized anti-PD-1 nanobody was functionally verified, see Figure 1B and Table 7, the IC 50 activity of A17h1_hIgG4 was similar to that of the positive antibody Pembrolizumab.
  • the biological activity IC 50 values of A17m0901_hIgG4 and A17m0902_hIgG4 antibodies were 0.5307nM and 0.4352nM, respectively, which were better than pembrolizumab.
  • Antibody number IC 50 (nM) A17h1_hIgG4 1.039 A17m09_hIgG4 1.083 A17m0901_hIgG4 0.5307 A17m0902_hIgG4 0.4352 A17m0903_hIgG4 1.284 A17m0905_hIgG4 0.5588 Pembrolizumab 0.9756
  • Example 1-5 Identification of the binding affinity of the modified anti-PD-1 nanobody to the antigen PD-1
  • CM5 sensor chip was used in the experiment, and HBS-EP+buffer solution (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20) was used as the mobile phase.
  • HBS-EP+buffer solution (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20) was used as the mobile phase.
  • the anti-human IgG (Fc) antibody was prepared into a 30 ⁇ g/mL solution with 10 mM sodium acetate buffer (pH 5.0), and the Immobilization program was selected to automatically carry out amino-coupling immobilization of the anti-human IgG (Fc) antibody channel.
  • Each antibody to be tested was prepared as a ligand with HBS-EP+ buffer solution, and captured with an anti-human IgG (Fc) antibody on the chip channel.
  • Human or cynomolgus PD-1 antigen protein (Sino Biological, 10377-H08H; 90311-C08H) was used as the analyte, prepared with HBS-EP+ buffer solution, and the analyte was serially diluted 2 times, at a flow rate of 30 ⁇ L/min Flow down through the experimental channel and reference channel, combine for 1min, and dissociate for 15min.
  • Regeneration buffer 10mM Glycine pH 1.5 (GE Healthcare, BR-1003-54) was run at a flow rate of 10 ⁇ l/min for 30s, and the association rate Ka and dissociation rate Kd, as well as the dissociation constant (ie, affinity K D ), were calculated. See Table 8 and Table 9 for the results.
  • the results of binding to human PD-1 antigen showed that the humanized antibody A17h1_hIgG4 and the subsequent optimized antibodies A17m09_hIgG4, A17m0901_hIgG4, A17m0902_hIgG4 had similar KD values to pembrolizumab.
  • A17h1_hIgG4, A17m09_hIgG4, A17m0901_hIgG4, A17m0902_hIgG4 all had slower dissociation rates than pembrolizumab.
  • A17h1_hIgG4, A17m09_hIgG4, A17m0901_hIgG4, A17m0902_hIgG4 antibodies cross-bind monkey PD-1, and the antibodies have similar affinity to human and monkey PD-1.
  • Example 1-6.DC T Mixed Lymphocyte Reaction (Mix Lymphocyte Reaction, MLR) to test the in vitro efficacy of the modified anti-PD-1 nanobody
  • DC dendritic cells
  • allogeneic T cells can activate T cells and promote T cells to secrete cytokines. This process is inhibited by PD-1/PD-L1 signaling, and the use of anti-PD-1 antibodies can relieve inhibition and promote T cell activation. Therefore, the in vitro efficacy of anti-PD-1 antibodies can be tested using the DC:T mixed lymphoid reaction assay.
  • Mononuclear cells were isolated from fresh peripheral blood (PBMC) of a healthy person using a sorting kit (cat: 19359, stemcell). Using a DC cell induction kit (cat: 10985, stemcell), after seven days of culture, monocytes were induced to differentiate into mature DC cells. Then allogeneic T cells were sorted from fresh PBMCs of another healthy person using a sorting kit (cat: 17951, stemcell). 5,000 mature DC cells were co-cultured with 50,000 allogeneic T cells, and corresponding concentrations of candidate antibodies were added.
  • PBMC peripheral blood
  • a sorting kit catalog: 19359, stemcell
  • the cell supernatant was aspirated, and the Cisbio-HTRF IFN- ⁇ detection kit was used (Cat: 62HIFNGPEH, Perkinelmer) The concentration of IFN- ⁇ was detected and EC 50 was calculated.
  • A17m09_hIgG4 showed basically the same in vitro efficacy as Pembrolizumab in the MLR experiment. Among them, the EC 50 of A17m09_hIgG4 is 0.2931nM, and the EC 50 of Pembrolizumab is 0.3271nM.
  • Example 1-7 Anti-PD-1 nanobody inhibits tumor growth in mouse colon cancer model
  • the MC38 mouse colon cancer cell line was cultured in DMEM medium (10% FBS), and 5 ⁇ 10 5 MC38 cells were inoculated subcutaneously in humanized PD-1C57BL/6 female mice (Jicui Yaokang). When the volume reached about 80mm 3 , they were randomly divided into groups of 8 mice, given intraperitoneal injection of antibody or control, and the tumor volume was measured twice a week, weighed, and recorded. See Table 10 for experimental grouping and dosing regimen. Considering that the molecular weight of A17m09_hIgG4 is about 75kDa, which is only half of the normal IgG molecular weight, the dosage was adjusted to half of that of pembrolizumab to achieve equimolar dosage.
  • human PVRIG h-PVRIG-his recombinant protein with his tag
  • human PVRIG h-PVRIG-mIgG2a Fc
  • mouse PVRIG m-PVRIG-hIgG1 Fc
  • His-tagged PVRL2 was purchased from AcroBiosystem (PV2-H52E2).
  • Example 2-1 Screening and preparation of anti-PVRIG nanobody
  • Example 1-1 the human PVRIG recombinant protein with his tag (h-PVRIG-his) was used to immunize alpacas.
  • PBMCs peripheral blood of camels on day 56, RNA was extracted, reverse transcribed into cDNA, and a phage library of anti-human PVRIG nanobody was constructed. After 3 rounds of screening, 400 clones were sequenced, and the variable region sequences of 2 strains are shown below, and the CDRs are shown in Table 13.
  • variable region of the above antibody was linked to the Fc region of the heavy chain of human IgG4.
  • the Fc region of the heavy chain includes the hinge region and carries S228P, F234A, L235A, K447A mutations (Eu nomenclature system) to construct a full-length antibody.
  • the anti-PVRIG antibody CPA.7.021 shown in WO2016134333 was screened from an antibody phage library, and its subtype is IgG1, which can bind well to human PVRIG but not to cynomolgus monkey PVRIG.
  • the heavy chain and light chain variable regions of CPA.7.021 were respectively connected with the human IgG4 heavy chain constant region (with S228P, F234A, L235A, K447A mutations) and the human Kappa light chain constant region to construct the positive antibody Tab5.
  • the PVRIG recombinant protein with his tag was directly coated, and after adding the antibody, the activity of the antibody binding to the antigen was detected by adding a secondary antibody (HRP-coupled anti-primary anti-Fc antibody) and HRP substrate TMB.
  • Human, cynomolgus or mouse PVRIG protein was coated on a 96-well plate, 100 ⁇ L per well at a concentration of 1 ⁇ g/mL, and incubated overnight at 4°C. Wash with lotion three times. Add 300 ⁇ L/well blocking solution (PBS+0.05% Tween20+1% BSA) and incubate at room temperature for 1 hour. Wash with lotion three times. Add 100 ⁇ L of anti-PVRIG antibody to be tested diluted with diluent to each well. Incubate at 37°C for 1 hour. Wash with lotion three times. 100 ⁇ L of HRP-labeled anti-human IgG secondary antibody (Sigma, A8667) was added to each well.
  • HEK293 stably transfected cell lines expressing human or cynomolgus PVRIG gene were prepared. Seed 2x105 cells per well in a 96-well plate. Centrifuge at 300g for 5 minutes, remove the supernatant, add 100 ⁇ L of the antibody to be tested, and incubate at 4°C for 1 hour. Remove the supernatant by centrifugation, wash 3 times with 200 ⁇ L washing solution (PBS+2% FBS), add 100 ⁇ L 1:500 diluted anti-human IgG secondary antibody (Invitrogen, A-11013) labeled with Alexa Fluor 488, and incubate at 4 °C for 1 Hour.
  • PBS+2% FBS 200 ⁇ L washing solution
  • Human PVRIG recombinant protein (h-PVRIG-mIgG2a Fc) was coated on a 96-well plate, 100 ⁇ L per well at a concentration of 1 ⁇ g/mL, and incubated overnight at 4°C. Wash with lotion three times. Add 300 ⁇ L/well blocking solution and incubate at room temperature for 1 hour. Wash with lotion three times. Add 50 ⁇ L of the diluted anti-PVRIG antibody to be tested and 50 ⁇ L of his-tagged ligand PVRL2 to each well, and incubate at 37°C for 1 hour. Wash with lotion three times. Add 100 ⁇ L of HRP-labeled anti-his-tag secondary antibody (Genscrpit) diluted 1:2000 times to each well.
  • HRP-labeled anti-his-tag secondary antibody (Genscrpit) diluted 1:2000 times to each well.
  • plvx-OS8 G418 resistance
  • transfect 293F cells G418 screening
  • use flow cytometry to detect the expression of OS8 in the cloned cells and detect the activation of OS8 to Jurkat cells, select the clone with a moderate degree of activation, and obtain 293F - OS8 cell line
  • construct plvx-PVRL2 plasmid use it to infect 293F-OS8 cells, and use flow cytometry to screen out the clone with the highest expression of PVRL2, so as to obtain 293F-OS8-PVRL2 cell line.
  • plvx-NFAT-Luc Hygromycin resistance
  • plvx-NFAT-Luc Hygromycin resistance
  • infect Jurkat E6.1 cells add Hygromycin to screen out resistant clones, use OKT3 to stimulate clones, and screen out clones with medium Luciferase signal
  • Obtain the Jurkat-NFAT-Luc cell line construct the plvx-PVRIG (Puromycin resistance) vector, package it into a lentivirus, infect the Jurkat-NFAT-Luc cells, and select the clone with the highest expression of PVRIG by flow cytometry, thereby obtaining Jurkat - NFAT-Luc-PVRIG cell line.
  • plvx-NFAT-Luc Hygromycin resistance
  • 1E4 Jurkat-NFAT-Luc-PVRIG cells were incubated with the antibody to be tested at 37°C for 20 minutes. Add 1E5 293F-OS8-PVRL2 cells and incubate at 37°C for 5 hours. The supernatant was removed by centrifugation, the cells were lysed by adding Luciferase buffer (Promega, E6130), and the fluorescence value was detected. Calculate the EC 50 value to evaluate the in vitro cell activity of the anti-PVRIG antibody. The experimental results are shown in Table 18.
  • PVRIG is expressed on NK cells, while PVRL2 is expressed in many tumor cells, including K562 cells.
  • Anti-PVRIG antibodies can release the inhibitory effect of tumor cells on NK cell activity by blocking the combination of PVRL2 and PVRIG.
  • control group 1 a sample containing only culture medium
  • control group 2 a sample containing only NK92 cells
  • control group 3 a sample containing only K562 cells
  • the killing activity was calculated according to the following formula:
  • Killing activity (%) ⁇ [(R–BG)–(T–BG)–(E–BG)]/[(TL–BGL)–(T–BG)] ⁇ 100;
  • R is the fluorescence value after adding AAF-Glo
  • BG is the fluorescence value of control group 1 after adding AAF-Glo
  • E is the fluorescence value of control group 2 after adding AAF-Glo
  • T is the fluorescence value of control group 3 after adding AAF-Glo.
  • the fluorescence value of Glo is the fluorescence value of the control group three after adding the lysate
  • BGL is the fluorescence value of the control group after adding the lysate again.
  • PVRIG is expressed on T cells, whereas PVRL2 is expressed on DC cells.
  • Anti-PVRIG antibodies can block the combination of PVRL2 and PVRIG, relieve the inhibition of DC cells on T cells, and activate T cells.
  • PBMC peripheral blood of the first individual, and the cells were cultured in RPMI 1640 medium containing 10% FBS, with 50ng/mL GM-CSF (Peprotech, 300-03-100UG) and 50ng/mL IL- Add the final concentration of 4 (Peprotech, 200-04-100UG), add fresh medium containing cytokines every 2-3 days; after culturing for 6 days, add 1 ⁇ g/mL LPS (Sigma, L2880-25MG) to incubate for 24 hours, collect DC cells obtained by differentiation and maturation.
  • GM-CSF Peprotech, 300-03-100UG
  • IL- Add the final concentration of 4 Peprotech, 200-04-100UG
  • PBMCs were isolated from peripheral blood from a second individual, from which CD3 + T cells were isolated using the EasySep Human CD3+ T Cell Isolation Kit (Stemcell, 17952). Adjust the density of CD3 + T cells and DC cells so that 1 ⁇ 10 5 CD3 + T cells and 2 ⁇ 10 4 DC cells are added to each well. Add the antibody to be tested, incubate at 37°C for 120 hours, take the supernatant, and detect the IFN ⁇ content in the supernatant with an ELISA kit (R&D, DY202).
  • a human germline template IGHV3-7*01 with high homology was obtained. Graft the CDRs into the corresponding human templates. The three-dimensional structure simulation and analysis of the transplanted nanobody was performed again, and back mutations were performed on the buried residues, the residues that directly interact with the CDR region, and the residues that have an important impact on the conformation of the variable region. The chemically unstable amino acid residues in the CDR region were optimized to generate a series of humanized nanobodies.
  • the human germline template for each Nanobody and the heavy chain variable region sequence of the humanized antibody are shown below.
  • antibodies 30H1 to 30H5 comprise CDR1 as shown in GDCMG (SEQ ID NO: 46) and CDR2 as shown in TIDNAGRIKYADSVKG (SEQ ID NO: 47) as shown in GWTFGGQCSPAD (SEQ ID NO: 99) CDR3.
  • the heavy chain variable region of the above humanized antibody was linked with the Fc region of the human IgG4 heavy chain to form a full-length anti-PVRIG antibody.
  • the Fc region of the heavy chain includes a hinge region, and carries S228P/F234A/L235A/K447A, or S228P/K447A mutations.
  • the antibody was expressed and purified according to conventional methods, and the target antibody was obtained after detection.
  • Example 2-5 Activity and function verification of humanized anti-PVRIG antibody
  • Example 2-2 FACS was used to detect the binding of anti-PVRIG antibody to human or cynomolgus monkey PVRIG.
  • the experimental results are shown in Table 21.
  • Example 2-2 the affinity of the humanized anti-PVRIG antibody to human PVRIG protein was detected. As shown in Table 22, all antibodies had high affinity to human PVRIG protein.
  • Example 2-3 the activity of the humanized anti-PVRIG antibody in reporter cells was detected.
  • the experimental results are shown in Table 23.
  • the antibodies listed in the table all have the ability to activate Jurkat cells.
  • Example 2-3 the activation ability of the humanized anti-PVRIG antibody on NK cells was detected.
  • the experimental results are shown in Table 24 and Table 25. The results showed that all the antibodies tested had the obvious ability to activate NK cells and promote the killing of target cells K562 by NK cells.
  • the anti-PVRIG nanobody 151 was connected to the N-terminal of the heavy chain or light chain of anti-TIGIT antibody 1708 through the GGGGSGGGGS (SEQ ID NO: 100) linker. Or C-terminal connected.
  • the anti-TIGIT antibody 1708 adopts the human IgG4 subtype with a mutation of S228P (Eu nomenclature system).
  • the anti-TIGIT antibody 1708 and the bispecific antibody sequences formed with 151 are shown in Table 26 below.
  • the sequence information of the anti-TIGIT antibody is shown in Table 27 and Table 28.
  • TIGIT antibodies have been disclosed in WO2019062832A, which is incorporated by reference in its entirety.
  • the double underline in the following sequence is the linker sequence.
  • the second polypeptide chains of 1708-30H2, 1708-151H7, 1708-151H8 were all identical to the light chain of 1708 (SEQ ID NO: 68).
  • Example 2-7 Activity and function verification of anti-PVRIG/TIGIT bispecific antibody
  • the anti-PVRIG antibody is connected to the N-terminal or C-terminal of the heavy and light chain of the anti-TIGIT antibody, it maintains the binding of PVRIG and TIGIT and blocks the ligand, and all show good expression and purity .
  • Biacore was used to detect the affinity of the bispecific antibody to human PVRIG and human TIGIT.
  • the humanized bispecific antibody was captured on the Protein A biosensing chip (GE lifesciences, 29127557) of Biacore instrument (Biacore X100, GE), and then human PVRIG antigen (AcroBiosystem , PVG-H52H4) or human TIGIT antigen (AcroBiosystem, TIT-H52H3), the association and dissociation curves were obtained. The results are shown in Table 31.
  • the activation ability of the humanized anti-PVRIG/TIGIT bispecific antibody on T cells was detected.
  • the experimental results are shown in Figure 5 and Table 32.
  • the results showed that the humanized anti-PVRIG/TIGIT bispecific antibody 1708-151H8 has the obvious ability to activate T cells and promote the secretion of IFN ⁇ by T cells.
  • the bispecific antibody was more active than anti-PVRIG antibody 151H8 alone and anti-TIGIT antibody 1708 alone.
  • Example 2-8 Anti-tumor effect evaluation of anti-PVRIG/TIGIT bispecific antibody in human melanoma A375 mixed with human PBMC subcutaneous xenograft tumor model in mice
  • variable region of the heavy chain of 1708-IgG1 is the same as that of 1708, except that the subtype of the constant region of the heavy chain is changed to IgG1; the full length of the light chain of 1708-IgG1 and the second polypeptide chain of 1708-151-IgG1 are both identical The same light chain.
  • NCG mice female, 4-8 weeks old, weighing about 18-22 g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. All NCG mice were cultured according to the conditions of the IVC constant temperature and pressure system in the SPF animal room.
  • A375 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS). A375 cells in the exponential growth phase were collected and resuspended in HBSS to a suitable concentration for subcutaneous tumor inoculation in NCG mice. The A375 cells used for co-culture need to be treated with Mitomycin C for 2 hours, and washed three times with PBS. The peripheral blood of normal people was taken, and human PBMCs were separated by density gradient centrifugation and counted. Then PBMCs were resuspended to a concentration of 3 ⁇ 10 6 cells/mL in RPMI1640 medium (containing IL2 and 10% FBS), and co-cultured with A375 cells treated with Mitomycin C.
  • FBS fetal bovine serum
  • mice After 6 days of co-culture, PBMCs were harvested, and freshly digested A375 cells were collected at the same time. Inoculate each mouse: 5 ⁇ 10 5 PBMCs, 4 ⁇ 10 6 A375 cells; inoculation volume: 0.2 mL/mouse (containing 50% Matrigel); inoculate subcutaneously on the right side of female NCG mice.
  • the mice were administered in groups at random according to their body weight. The detailed administration method, dosage and route of administration are shown in Table 33. The day of group administration was Day 0. Since the molecular weights of the anti-PVRIG antibody and the anti-TIGIT antibody are different, the dosage ensures that the anti-PVRIG antibody and the anti-TIGIT antibody have the same initial molar concentration.
  • N number of animals used; i.p.: intraperitoneal injection; Q2D: once every two days; administration volume: adjust the administration volume according to the body weight of tumor-bearing mice (0.1mL/10g))
  • mice were administered in groups at random according to their body weight.
  • the detailed administration methods, doses and routes of administration are shown in Table 35.
  • the day of group administration was Day 0.
  • N number of animals used; i.p.: intraperitoneal injection; Q2D: once every two days; administration volume: adjusted according to the body weight of tumor-bearing mice (0.1mL/10g))
  • mice After the administration started, the body weight and tumor volume of the mice were measured twice a week.
  • the experimental results are shown in Table 36 and Figures 7A-7B, respectively.
  • both the 1708-30H2 and 1708-151H7 bispecific antibody groups could effectively inhibit tumor growth at low doses, and there was a significant difference between them ( See Figure 7A, Figure 7B).
  • Example 3-1 Design and preparation of anti-PD-1/PVRIG/TIGIT trispecific antibody
  • trispecific antibodies are divided into four types: A, B, C, and D, as shown in Figure 8.
  • Type A The anti-PD-1 nanobody is connected to the anti-PVRIG nanobody through a linker (such as GGGGSGGGGS), and then connected to the N-terminal of the heavy chain of the anti-TIGIT antibody through a linker ( Figure 8A).
  • a linker such as GGGGSGGGGS
  • Type B The anti-PVRIG antibody is connected to the N-terminal of the heavy chain of the TIGIT IgG molecule through a linker, and the anti-PD-1 antibody is connected to the N-terminal of the light chain of the anti-TIGIT antibody through a linker ( Figure 8B).
  • Type C The anti-PVRIG antibody is connected to the N-terminal of the heavy chain of the TIGIT IgG molecule through a linker.
  • the anti-PD-1 antibody was linked to the C-terminal of the heavy chain of the anti-TIGIT antibody via a linker (GGGGSGGGGS) ( Figure 8C).
  • Type D The anti-PVRIG antibody is connected to the N-terminal of the heavy chain of the TIGIT IgG molecule through a linker.
  • the anti-PD-1 antibody was linked to the C-terminal of the light chain of the anti-TIGIT antibody through a linker ( Figure 8D).
  • A17m0902-1708-30H2-A representing the clones of anti-PD-1, TIGIT and PVRIG antibodies used are A17m0902, 1708 and 30H2, respectively, and the type of trispecific antibody is type A.
  • the A17h1-related molecules in Table 37 are based on the corresponding A17m0902-related molecules, and the A17m0902 (SEQ ID NO: 33) sequence is replaced with the A17h1 (SEQ ID NO: 13) sequence.
  • the second polypeptide chain of A17m0902-1708-30H2-A is shown in SEQ ID NO:68.
  • the first polypeptide chain of A17m0902-1708-30H2-B is shown in SEQ ID NO:84.
  • the second polypeptide chain of A17m0902-1708-30H2-C is shown in SEQ ID NO:68.
  • the first polypeptide chain of A17m0902-1708-30H2-D is shown in SEQ ID NO:84.
  • the second polypeptide chain of A17m0902-1708-151H7-A is shown in SEQ ID NO:68.
  • the first polypeptide chain of A17m0902-1708-151H7-B is shown in SEQ ID NO:85.
  • the second polypeptide chain of A17m0902-1708-151H7-C is shown in SEQ ID NO:68.
  • the first polypeptide chain of A17m0902-1708-151H7-D is shown in SEQ ID NO:85.
  • the target antibody can be obtained after transfection of cells, expression, purification and detection by conventional methods.
  • Example 3-2 Binding affinity of anti-PD-1/PVRIG/TIGIT trispecific antibody to antigen
  • ELISA was used for detection.
  • Human TIGIT or PVRIG protein (his tag, Acrobiosystems, PVG-H52H4) was dissolved in PBS to 1 ⁇ g/mL, 100 ⁇ L/well of the solution was added to a 96-well plate, overnight at 4°C, and antigen was coated. Wash three times with PBST solution. Add PBST/1%BSA solution and incubate at 37°C for 1h to block. After washing three times with PBST solution, different concentrations of trispecific antibodies (diluted in PBST/1% BSA solution) were added, and incubated at 37° C. for 1.5 h. Wash three times with PBST solution.
  • A17h1-1708-30H2 and A17h1-1708-151H7 trispecific antibodies all had strong binding to TIGIT, and the difference in binding ability was small. Binding abilities are similar. See Figure 9A, Figure 9B and Table 38 for results. Therefore, all trispecific antibody configurations did not affect their binding to TIGIT.
  • Antibody number EC 50 (nM) for binding to human TIGIT A17h1-1708-30H2-A 0.08749 A17h1-1708-30H2-B 0.1071 A17h1-1708-30H2-C 0.09511 A17h1-1708-30H2-D 0.1125 1708-30H2 0.1156 1708-151H7 0.1175 A17h1-1708-151H7-A 0.1859 A17h1-1708-151H7-B 0.1293 A17h1-1708-151H7-C 0.148
  • A17h1-1708-30H2 and A17h1-1708-151H7 trispecific antibodies all had strong binding to PVRIG, with little difference in binding ability, and compared with the binding ability of 1708-30H2 and 1708-151H7 bispecific antibodies to PVRIG. Binding abilities are similar. See Table 39 and Figures 10A, 10B. Therefore, all trispecific antibody configurations did not affect their binding to PVRIG.
  • Antibody number EC 50 (nM) for binding to human PVRIG A17h1-1708-30H2-A 0.05891 A17h1-1708-30H2-B 0.05834 A17h1-1708-30H2-C 0.08216 A17h1-1708-30H2-D 0.08852 1708-30H2 0.08462 1708-151H7 0.1249 A17h1-1708-151H7-A 0.09519 A17h1-1708-151H7-B 0.1543 A17h1-1708-151H7-C 0.1596 A17h1-1708-151H7-D 0.1578
  • Example 3-3 PVRIG sequence optimization in anti-PD-1/PVRIG/TIGIT trispecific antibody
  • the first amino acid of the original PVRIG antibody sequence (151H7) is histidine (single-letter abbreviation H), located in the trispecific antibody
  • histidine at the N-terminal of the molecular sequence may be modified by pyridoxal phosphate and pyridoxal after dephosphorylation during the actual antibody production process. Based on this problem, the first histidine in the original PVRIG sequence needs to be mutated .
  • the trispecific antibody molecule whose first amino acid of the PVRIG antibody (151H7) sequence was changed from H to E was named A17m0902-1708-151H7-01-C; the first amino acid of the PVRIG (151H7) sequence was changed from H to Q
  • the trispecific antibody molecule was named A17m0902-1708-151H7-02-C.
  • A17m0902-1708-151H7-01-C and A17m0902-1708-151H7-02-C are type C: that is, the anti-PVRIG antibody is linked to the N-terminal of the heavy chain of the anti-TIGIT antibody via linker 1.
  • the anti-PD-1 antibody was linked to the C-terminal of the heavy chain of the anti-TIGIT antibody via linker 2 (C in Figure 8).
  • polypeptide chain sequences of A17m0902-1708-151H7-01-C and A17m0902-1708-151H7-02-C are as follows:
  • the second polypeptide chain of A17m0902-1708-151H7-01-C is shown in SEQ ID NO:68.
  • the second polypeptide chain of A17m0902-1708-151H7-02-C is shown in SEQ ID NO:68.
  • Example 3-4 Anti-PD-1/PVRIG/TIGIT trispecific antibody in the PVRIG sequence optimized binding affinity with antigen
  • HEK293F stably transfected cell lines expressing human PVRIG gene, CHO-K1 stably transfected cell lines expressing human TIGIT gene and Jurkat cells expressing human PD-1 were used respectively. Seed 2x105 cells per well in a 96-well plate. Centrifuge at 300g for 5 minutes, remove the supernatant, add 100 ⁇ L of the antibody to be tested, and incubate at 4°C for 1 hour.
  • Antibody E MAX EC50 (nM) A17m0902-1708-151H7-01-C 1547 0.1048 A17m0902-1708-151H7-02-C 1521 0.09636 A17m0902-1708-151H7-C 1516 0.0944 1708-151H7 1357 0.1124
  • Antibody E MAX EC50 (nM) A17m0902-1708-151H7-01-C 9964 0.6008 A17m0902-1708-151H7-02-C 9747 0.586 A17m0902-1708-151H7-C 9105 0.5577
  • the binding activity, EC50 is similar, indicating that the mutation of the first amino acid of the PVRIG antibody does not affect the binding of the trispecific antibody to PD-1. See Table 42 and Figure 11C for results.
  • Antibody E MAX EC50 (nM) A17m0902-1708-151H7-01-C 1956 0.5027 A17m0902-1708-151H7-02-C 1937 0.4806 A17m0902-1708-151H7-C 1974 0.4006
  • the two trispecific antibody molecules A17m0902-1708-151H7-01-C and A17m0902-1708-151H7-02-C after the first amino acid mutation of the PVRIG antibody were compatible with PVRIG and TIGIT expressed on the cell surface ,
  • the affinity of the PD-1 antigen is comparable to that of the parental A17m0902-1708-151H7-C antibody, and these mutations do not affect the binding of the trispecific antibody to PVRIG, TIGIT and PD-1.
  • Example 3-5 PD-1/PD-L1 reporter gene system detects the blocking of PD-1 by anti-PD-1/PVRIG/TIGIT trispecific antibody
  • Examples 1-4 The method in Examples 1-4 is used for detection. As shown in Figure 12A, Figure 12B and Table 43, A17h1, as the four configurations of trispecific antibodies composed of PD-1 ends, can block PD-1, but the reporter genes of B and C trispecific antibodies Activity is relatively better.
  • Antibody number IC50(nM) A17h1-1708-30H2-A 0.8007 A17h1-1708-30H2-B 0.3436 A17h1-1708-30H2-C 0.2643 A17h1-1708-30H2-D 0.825 A17m09-hIgG4 0.7716 A17h1-hIgG4 0.696 pembrolizumab 0.573
  • Example 3-6.MLR detects the activation of T cells by anti-PD-1/PVRIG/TIGIT trispecific antibody
  • A17m0902-1708-30H2-C with a better configuration was selected for further DC:T mixed lymph reaction experiment verification. See Figure 14A- Figure 14B and Table 45 and Table 46, the results show that at an antibody concentration of 100nM, the A17m0902-1708-30H2-C trispecific antibody is far superior to the bispecific antibody 1708-30H2 in activating T cells, Better than anti-PD-1 monoclonal antibody Ab 464, slightly better than anti-PD-1 monoclonal antibody A17m0902_hIgG4 on the activation of T cells.
  • Example 3-7 NK92 killing detection of NK cell activation by anti-PD-1/PVRIG/TIGIT trispecific antibody
  • NK92 cells were used as effector cells and K562 cells were used as target cells for cell killing experiments.
  • different concentrations of candidate antibodies were incubated with NK92 cells at 37°C for 30 minutes, and then the NK92 cells pre-incubated with the antibodies were compared with K562 cells labeled with CTV (Cat: C34557, Thermo) according to the effect-to-target ratio of 10:1.
  • CTV Cat: C34557, Thermo
  • Mix, culture condition is 1640+10% FBS+10ng/mL IL-2, incubate at 37°C for 4 hours.
  • the cells were resuspended in 10 ⁇ g/mL propidium iodide solution for staining and marking of dead cells. After labeling at room temperature for 10 minutes, the proportion of dead cells (PI positive) in CTV-labeled K562 cells was detected by FACS, and the killing activity was calculated.
  • the trispecific antibody retained the biological function of the anti-PVRIG/TIGIT bispecific antibody to promote NK cell killing, and the IC 50 values of each configuration were similar, see Table 47.
  • Example 3-8 Evaluation of anti-tumor effect of anti-PD-1/PVRIG/TIGIT trispecific antibody in human melanoma A375 mixed with human PBMC subcutaneous xenograft tumor model in mice responding to PD-1 monoclonal antibody
  • the in vivo efficacy of the PD-1/PVRIG/TIGIT trispecific antibody was verified on a human melanoma A375 mixed human PBMC in vivo tumor model responsive to PD-1 monoclonal antibody.
  • NCG mice female, 4-8 weeks old, weighing about 18-22 g (the mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.). The mice were raised in the IVC constant temperature and pressure system in the SPF grade animal room.
  • Donor PBMCs that responded to PD-1 monoclonal antibody in the in vivo pharmacodynamic model were screened from frozen PBMCs and counted. Resuspend PBMC to a certain concentration with RPMI 1640 medium containing IL-2 and 10% FBS. PBMCs were added to A375 cells treated with Mitomycin C for 2 hours. After co-cultivating with A375 for 5 days, PBMCs were harvested. A375 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS), and freshly digested A375 cells were harvested.
  • FBS fetal bovine serum
  • mice were inoculated: 5 ⁇ 10 5 PBMCs, 4 ⁇ 10 6 A375 cells; the inoculation volume was 0.2ml/mouse (containing 50% Matrigel); inoculated subcutaneously on the right side of NCG mice. The day of inoculation was Day 0, and mice were randomly divided into groups according to body weight on Day 1.
  • A17m0902_hIgG4 has a molecular weight of 76 kDa
  • 1708-151H7 has a molecular weight of 173.5 kDa
  • A17m0902-1708-151H7-C has a molecular weight of 200 kDa.
  • a dosing regimen of equimolar concentrations was designed. See Table 48 for the detailed dosing regimen, dosage and route of administration.
  • N number of animals used; i.p.: intraperitoneal injection; BIW*8: 2 times a week, a total of 8 times
  • mice After the administration started, the body weight and tumor volume of the mice were measured twice a week.
  • TGI tumor growth inhibition rate
  • the efficacy of the trispecific antibody A17m0902-1708-151H7-C administration group was significantly better than that of the anti-PD-1 monoclonal antibody A17m0902_hIgG4 administration group, and better than that of PVRIG/TIGIT bispecific antibody + PD-1 monoclonal antibody combination Group.
  • the experimental results are shown in Table 49 and Fig. 16A and Fig. 16B respectively.
  • Tumor volume TGI(%) Tumor weight TGI (%) 1 blank control / / 2 1708-151H7 7.05 3.84 3 A17m0902_hIgG4 79.91 77.20 4 A17m0902_hIgG4+1708-151H7 88.25 87.60 5 A17m0902-1708-151H7-C 91.63 91.50
  • N number of animals used; i.p.: intraperitoneal injection; BIW: 2 times a week
  • Example 3-9 Evaluation of anti-tumor effect of anti-PD-1/PVRIG/TIGIT trispecific antibody in human melanoma A375 mixed with human PBMC mouse subcutaneous xenograft tumor model that does not respond to PD-1 monoclonal antibody
  • the in vivo efficacy of the anti-PD-1/PVRIG/TIGIT trispecific antibody was verified on the human melanoma A375 mixed human PBMC in vivo tumor model that did not respond to the anti-PD-1 monoclonal antibody.
  • NCG mice female, 4-8 weeks old, weighing about 18-22 g (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), were raised in an IVC constant temperature and pressure system in an SPF animal room.
  • Donor PBMCs that did not respond to anti-PD-1 mAb in the in vivo pharmacodynamic model were screened from cryopreserved PBMCs.
  • the experimental scheme is the same as that described in Examples 3-8. See Table 50 for the mouse experimental grouping and dosing regimen.
  • N number of animals used; i.p.: intraperitoneal injection; BIW*8: 2 times a week, a total of 8 times
  • mice After the administration started, the body weight and tumor volume of the mice were measured twice a week.
  • the experimental group was compared with the IgG control group, and the tumor growth inhibition rate (TGI %) were 4.53%, 20.75%, 36.70% and 31.95%, respectively.
  • the experiment was terminated, all mice were euthanized, their tumors were stripped off, weighed and photographed. According to the determination of the tumor weight of mice in each group, compared with the IgG control group in the experimental group, the tumor growth inhibition rate (TGI%) of 1708-151H7, A17m0902_hIgG4, A17m0902-1708-151H7-C, 1708-151H7+A17m0902_hIgG4 tumor weight TGI was respectively 0%, 16.8%, 38.2%, and 33.4%. The trend of TGI of tumor weight was consistent with that of TGI of tumor volume. The experimental results are shown in Table 51 and Fig. 17A and Fig. 17B respectively.

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Abstract

涉及特异性结合PD-1的蛋白及其医药用途。具体而言，提供了一种特异性结合PD-1的蛋白、特异性结合PD-1/PVRIG/TIGIT的蛋白及其用于治疗疾病(例如癌症)的用途。

Description

特异性结合PD-1的蛋白及其医药用途
本公开要求2021年09月15日提交的中国专利申请(申请号CN202111078222.1)的优先权。
技术领域
本公开涉及生物医药领域，具体涉及特异性结合PD-1的蛋白、特异性结合PD-1/PVRIG/TIGIT的蛋白及其用于治疗癌症的方法和制药用途。
背景技术
免疫治疗是癌症治疗领域的热点，以抗PD-1抗体为代表的免疫途径治疗药物已经在临床上体现出高有效性和高安全性。
PD-1(Programmed Cell death-1)属于CD28受体家族，是免疫抑制性受体(Riley等人2009，Immunol.Rev.29:114-25)。该家族还包括CD28、CTLA-4、ICOS、PD-1和BTLA。PD-1是I型跨膜蛋白，与CTLA-4结构很相似，但PD-1缺失了与B7-1和B7-2结合的MYPPPY序列。PD-1主要表达在活化的B细胞、T细胞和骨髓细胞(Chen等人2013，Nat.Rev.Immunol.13：227-42)。
PD-1有两种细胞表面的糖蛋白配体，分别是PD配体1(PD-L1，又名CD274，B7-H1)和PD配体2(PD-L2，又名B7-DC)。PD-L1和PD-L2均不与其它CD28受体家族成员结合。PD-L1广泛表达于淋巴细胞(例如CD4 +T细胞和CD8 +T细胞、巨噬细胞等)以及如外周组织、各种肿瘤细胞和病毒感染细胞等。PD-L2主要表达于活化的树突状细胞和巨噬细胞(Dong等人1999，Nat.Med.5:1365-9)。PD-1与其配体PD-L1或PD-L2结合后，会下调T细胞的功能，包括降低T细胞的活化、分化增殖和细胞因子的分泌等。
关于PVRIG(Poliovirus receptor-related Ig domain containing protein)又名CD112R，与TIGIT(T cell immunoglobulin and ITIM domain)、CD96和CD226等同属于B7/CD28超家族，在免疫***中起重要作用。当PVRIG的配体PVRL2(又名CD112)结合PVRIG时，会活化PVRIG胞内区的ITIM结构域，使PVRIG起到免疫抑制的作用。PVRIG主要表达在CD4 +T细胞，CD8 +T细胞和NK细胞的表面。PVRIG和其配体PVRL2在多种实体瘤中高表达，包括肺癌、乳腺癌、卵巢癌、肾癌、胃癌、子宫内膜癌、头颈癌等。PVRIG在这些癌症中的表达与TIGIT和PD-1有高度相关性。与PD-1和TIGIT相似，PVRIG阳性的T细胞也会呈现Eomes阳性和Tbet阴性，表明PVRIG与T细胞的耗竭有关。因此，PVRIG可能代表了除了PD-1和TIGIT之外的有一个新的免疫检查点，并起到冗余(redundancy)的作用。体外细胞实验和小鼠模型中显示，对小鼠PVRIG的敲除或抑制，可以有效抑制癌症的生长，并与PD-1和TIGIT抑制剂发生协同作用。TIGIT在淋巴细胞上高 度表达，包括浸润不同类型癌症的癌症浸润淋巴细胞(TIL)和Treg。已经证明TIGIT与其同源配体PVR(又名CD155)的结合通过其细胞质ITIM结构域直接抑制NK细胞的细胞毒性。PVR也在癌症中广泛表达，表明TIGIT-PVR信号轴可能是癌症的主要免疫逃逸机制。抑制性受体TIGIT、PVRIG与激活型受体DNAM-1结合同样的配体CD155与CD112，但抑制性受体的亲和力更高(Y.Zhu等人2016，J Exp Med.213:167-176)。肿瘤患者体内分离的NK细胞DNAM-1表达下调，导致NK细胞更易受到TIGIT或PVRIG的抑制，阻断TIGIT与PVRIG的结合，能够激活NK细胞的细胞杀伤功能(L.Martinet等人2015，Cell Reports.11:85-97)。
虽然PD-1/PD-L1抗体是目前临床最成功的免疫检查点抑制剂单抗，但T细胞表面其他免疫检查点的表达上调会限制其疗效。TIGIT和PVRIG除了调控T细胞的功能，还调控NK细胞活性，提示与PD-1/PD-L1在NK细胞中的功能可能产生协同。文献报道TIGIT与PVRIG的配体CD155与CD112在肺癌、卵巢癌、结直肠癌、黑色素瘤中的高表达与PD-1/PD-L1治疗不良预后显著相关。如CD155表达高的患者对抗PD-1抗体响应差，而CD155表达低的患者对PD-1响应好(S.Whelan等人2019，Cancer Immunol Res.7:257-268；A.Lepletier等人2020，Clin Cancer Res.26:3671-3681)。
尚未有任何PVRIG抗体、抗PVRIG/TIGIT双特异性抗体药物上市。Compugen公司的COM701是全球首个获FDA批准进入临床的抗PVRIG人源化抗体，目前处于I期临床阶段，用于治疗癌症。Surface Oncology也有抗PVRIG抗体SRF-813在开发。抗TIGIT抗体有Genentech的tiragolumab、Ono Pharmaceutical与BMS合作开发的BMS-986207、默沙东的MK-7684、iTeos Therapeutics的EOS-884448、Arcus Biosciences的AB-154，均在临床II期阶段。
因此，本公开提供新结构的抗PD-1抗体及相关的抗PD-1/PVRIG/TIGIT三特异性抗体，有望增强T细胞和NK细胞激活、克服PD-1/PD-L1耐药、拓展应答患者人群，从而提高癌症免疫治疗的效果。此外，抗PD-1/PVRIG/TIGIT三特异性抗体可同时结合同一细胞表面的多个免疫检查点，潜在具有亲和力效应(avidity effect)，因此有潜力在临床提供比现有PD-1、TIGIT、PVRIG抗体药物或其联用更佳的疗效。
发明内容
本公开提供了PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白，其编码核酸、载体、宿主细胞、药物组合物、其用于治疗或预防癌症的方法和相关制药用途。
PD-1结合蛋白
本公开提供PD-1结合蛋白，其包含免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含：
SEQ ID NO：3、13-15、17-35任一所示氨基酸序列中的CDR1、CDR2和CDR3，或
SEQ ID NO：2、16任一所示氨基酸序列中的CDR1、CDR2和CDR3，
所述CDR1、CDR2和CDR3是根据Kabat、IMGT、Chothia、AbM或Contact编号***定义的。所述免疫球蛋白单一可变结构是特异性结合PD-1抗原或其片段的。
一些实施方案中，所述PD-1结合蛋白包含至少一个免疫球蛋白单一可变结构。
一些实施方案中，提供PD-1结合蛋白，包含上述CDR1、CDR2和CDR3中的任意一个或其任意组合。
一些实施方案中，提供PD-1结合蛋白，根据Kabat编号***，所述免疫球蛋白单一可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别
如SEQ ID NO：7、36、37所示，或
如SEQ ID NO：4、5、6所示。
一些实施方案中，前述PD-1结合蛋白中的免疫球蛋白单一可变结构域的CDR1的氨基酸序列如SEQ ID NO：7所示，CDR2的氨基酸序列如SEQ ID NO：8、38-40、101任一所示，CDR3的氨基酸序列如SEQ ID NO：9、41、42任一所示。
一些实施方案中，前述PD-1结合蛋白中的免疫球蛋白单一可变结构域的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：7、38、41所示。
一些实施方案中，前述PD-1结合蛋白中的免疫球蛋白单一可变结构域为人源化的、亲和力成熟、去除T细胞表位、降低抗体脱酰胺和/或降低抗体异构化改造的。一些实施方案中，所述免疫球蛋白单一可变结构域经过亲和力成熟获得的，其在一个或多个CDR中具有一个或多个变化，所述变化导致对PD-1的亲和力相比于亲本免疫球蛋白单一可变结构域有所增加。
一些具体实施方案中，人源化改造过程使用人种系模板的重链框架区IGHV3-23*01或IGHV3-23*04。
一些实施方案中，前述PD-1结合蛋白中所述免疫球蛋白单一可变结构域的氨基酸序列分别如SEQ ID NO：3、13-15、17-35任一所示，或如SEQ ID NO：2、16任一所示，或与前述序列任一具有至少80％序列同一性。
本公开中，“至少90％(序列)同一性”涵盖至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％(序列)同一性；“至少80％(序列)同一性”涵盖至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％(序列)同一性。
一些实施方案中，前述PD-1结合蛋白中包含或为特异性结合PD-1或其片段 的抗体或其抗原结合片段。所述抗体或其抗原结合片段例如为骆驼抗体、嵌合抗体、人源化抗体、全人抗体或其抗原结合片段。所述抗体或其抗原结合片段例如为重组抗体或其片段。
一些具体实施方案中，所述抗原结合片段为线性抗体、单链抗体、纳米抗体、肽抗体peptibody、结构域抗体和多特异性抗体(双特异性抗体、diabody、triabody和tetrabody、串联二-scFv、串联三-scFv)。
一些实施方案中，前述PD-1结合蛋白中的免疫球蛋白单一可变结构域是VHH。
一些实施方案中，本公开提供PD-1结合蛋白，其包含一个或多个(例如2、3、4、5、6个)前述免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域可以是相同或不同的，可以形成二聚体或多聚体分子。
一些实施方案中，前述PD-1结合蛋白还包含人免疫球蛋白Fc区；例如，所述Fc区是人IgG1、IgG2或IgG4的Fc区。所述Fc区可以具有突变，示例性突变是选自以下任一项或组合：
IgG1上的L234A/L235A；
IgG1上的S267E/L328F；
IgG1上的L234F/L235E/D265A；
IgG1上的L234A/L235A/G237A/P238S/H268A/A330S/P331S；
IgG1上的K214T/E233P/L234V/L235A/G236缺失/A327G/P331A/D365E/L358M；
IgG2上的V234A/G237A；
IgG2上的H268Q/V309L/A330S/P331S；
IgG2上的V234A/G237A/P238S/H268A/V309L/A330S/P331S；
IgG4上的F234A/L235A；
IgG4上的S228P/F234A/L235A；
IgG4上的S228P/F234A/L235A/G237A/P238S；
IgG4上的S228P/F234A/L235A/G236缺失/G237A/P238S；以及
IgG1、IgG2、IgG3或IgG4上的N297A。
还可使用杂合IgG2/4Fc域，例如具有来自IgG2的残基117-260和来自IgG4的残基261-447的Fc。一些具体的实施方案中，所述人IgG4的Fc区具有228P、234A、235A和/或447A突变。
在本文中Fc区所包含的突变的上下文中，“/”表示“和”，例如，“F234A/L235A”表示“F234A和L235A，即，Fc中包含F234A和L235A突变；突变的氨基酸位置是根据EU编号***编号的。
一些实施方案中，前述PD-1结合蛋白中包含的Fc区可以使所述结合蛋白形成二聚体分子，延长所述结合蛋白的体内半衰期。
一些实施方案中，前述PD-1结合蛋白中免疫球蛋白单一可变结构域与Fc区直接或通过连接子连接。所述连接子可以是1-20个或更多个氨基酸长、无二级以上结构的非功能性氨基酸序列。例如，所述连接子是柔性连接子，例如G 4S、GS、GAP、(G 4S) 2、(G 4S) 3、(G 4S) 4、(G 4S) 5、ASGS等。
一些实施方案中，本公开的PD-1结合蛋白为抗PD-1抗体或其抗原结合片段，或包含所述抗体、抗原结合片段的缀合物、融合蛋白。
一些实施方案中，前述PD-1结合蛋白，具有选自以下至少一项的活性：
(a)以≤10 -7的K D值与人PD-1或其表位结合；
(b)抑制PD-1与PD-L1的结合；
(c)抑制PD-1与PD-L2的结合；
(d)诱导CD4 +T细胞分泌IFN-γ；
(e)增强PBMC的活化；
(f)增强T细胞的活化；
(g)抑制肿瘤生长。
一些实施方案中，本公开的前述PD-1结合蛋白结合PD-1的K D值可以≤1×10 -7M，例如≤1×10 -8M，或≤1×10 -9M，或≤1×10 -10M。
一些实施方案中，本公开的PD-1结合蛋白能够特异性结合人PD-1并阻断PD-1和PD-L1的相互作用，和/或阻断PD-1和PD-L2的相互作用。
一些实施方案中，本公开的前述PD-1结合蛋白能够抑制肿瘤生长至少约10％，例如至少约20％、约30％、约40％、约50％、约60％、约70％、约80％。
一些实施方案中，本公开的前述PD-1结合蛋白涵盖变体，所述变体与SEQ ID NO：2-3、13-35任一相比有一个或多个(例如，1、2、3、4、5、6、7、8、9、10个)氨基酸突变；所述氨基酸突变可以是保守的替换、取代或修饰，和/或不影响功能的缺失、添加；所述氨基酸突变可以发生在CDR区和/或FR区。
一些实施方案中，提供抗PD-1抗体或其抗原结合片段，其与前述本公开的PD-1结合蛋白中的免疫球蛋白单一可变结构域结合或竞争结合相同的表位。
一些实施方案中，提供抗PD-1抗体或其抗原结合片段，其阻断前述本公开的PD-1结合蛋白中的免疫球蛋白单一可变结构域与PD-1(例如人PD-1)的结合。
一些实施方案中，提供抗PD-1抗体或其抗原结合片段，其与PD-1(例如人PD-1)的结合被前述本公开的PD-1结合蛋白中的免疫球蛋白单一可变结构域阻断。
一些实施方案中，前述本公开的PD-1结合蛋白使PD-1与PD-L1和/或PD-L2的结合降低至少60％、至少70％、至少80％、至少90％、至少95％、至少98％或以上。
一些实施方案中，提供蛋白或分子，其包含前述本公开任意一个或多个(例如，1、2、3、4、5、6、7、8、9、10个)PD-1结合蛋白中的免疫球蛋白单一可 变结构域，所述免疫球蛋白单一可变结构域是相同的或不同的。例如，所述蛋白或分子为缀合物，所述缀合物例如可包含任意可检测标记。
PD-1/PVRIG/TIGIT结合蛋白
本公开提供PD-1/PVRIG/TIGIT结合蛋白，其包含特异性结合PD-1的第一抗原结合结构域、特异性结合PVRIG的第二抗原结合结构域和特异性结合TIGIT的第三抗原结合结构域，能同时或分别特异性结合PD-1、PVRIG和TIGIT。
一些实施方案中，前述PD-1/PVRIG/TIGIT结合蛋白包含或为抗PD-1/PVRIG/TIGIT三特异性抗体。
一些实施方案中，前述PD-1/PVRIG/TIGIT结合蛋白中的各抗原结合结构域选自Fab、Fv、sFv、Fab’、F(ab’) 2、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体peptibody、结构域抗体或由其任意组合组成。在具体的实施方案中，特异性结合PD-1的第一抗原结合结构域包含或为VHH、特异性结合PVRIG的第二抗原结合结构域包含或为VHH、特异性结合TIGIT的第三抗原结合结构域为Fab或F(ab’) 2。
一些实施方案中，前述PD-1/PVRIG/TIGIT结合蛋白为人源化的、亲和力成熟、去除T细胞表位、降低抗体脱酰胺和/或降低抗体异构化改造的。
一些实施方案中，前述PD-1/PVRIG/TIGIT结合蛋白为重组抗体、骆驼抗体、嵌合抗体、人源化抗体、全人抗体或其抗原结合片段。
一些实施方案中，前述PD-1/PVRIG/TIGIT结合蛋白中，所述特异性结合PD-1的第一抗原结合结构域包含(至少一个)免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含：
SEQ ID NO：3、13-15、17-35任一所示氨基酸序列中的CDR1、CDR2和CDR3，
SEQ ID NO：2、16任一所示氨基酸序列中的CDR1、CDR2和CDR3，
所述CDR1、CDR2和CDR3是根据Kabat、IMGT、Chothia、AbM或Contact编号***定义的。
一些实施方案中，根据Kabat编号***，所述免疫球蛋白单一可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别：
如SEQ ID NO：7、36、37所示，或
如SEQ ID NO：4、5、6所示。
一些具体实施方案中，所述免疫球蛋白单一可变结构域的CDR1的氨基酸序列如SEQ ID NO：7所示，CDR2的氨基酸序列如SEQ ID NO：8、38-40、101任一所示，CDR3的氨基酸序列如SEQ ID NO：9、41、42任一所示。
一些具体实施方案中，所述免疫球蛋白单一可变结构域的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：7、38、41所示。
一些实施方案中，前述PD-1/PVRIG/TIGIT结合蛋白中，所述特异性结合PVRIG的第二抗原结合结构域包含(至少一个)免疫球蛋白单一可变结构域，所述免疫 球蛋白单一可变结构域包含：
SEQ ID NO：45、62-66、95-96任一所示氨基酸序列中的CDR1、CDR2和CDR3，或
SEQ ID NO：44、57-61任一所示氨基酸序列中的CDR1、CDR2和CDR3。
一些具体实施方案中，根据Kabat编号***，所述免疫球蛋白单一可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别：
如SEQ ID NO：49、50、51所示，或
如SEQ ID NO：46、47、48或99所示。
一些实施方案中，前述PD-1/PVRIG/TIGIT结合蛋白中，所述特异性结合TIGIT的第三抗原结合结构域包含重链可变区(VH)和轻链可变区(VL)，其中：
所述重链可变区包含分别如SEQ ID NO：73、74和75所示氨基酸序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：76、77和78所示氨基酸序列的LCDR1、LCDR2和LCDR3。
一些实施方案中，前述PD-1/PVRIG/TIGIT结合蛋白中，所述特异性结合PD-1的第一抗原结合结构域包含如：
SEQ ID NO：3、13-15、17-35任一所示或与之具有至少80％、至少90％序列同一性的氨基酸序列；
SEQ ID NO：2、16任一所示或与之具有至少80％、至少90％序列同一性的氨基酸序列。
一些实施方案中，前述PD-1/PVRIG/TIGIT结合蛋白中，所述特异性结合PVRIG的第二抗原结合结构域包含如：
SEQ ID NO：45、62-66、95-96任一所示或与之具有至少80％、至少90％序列同一性的氨基酸序列；或
SEQ ID NO：44、57-61任一所示或与之具有至少80％、至少90％序列同一性的氨基酸序列。
一些实施方案中，前述PD-1/PVRIG/TIGIT结合蛋白中，所述特异性结合TIGIT的第三抗原结合结构域包含重链可变区(VH)和轻链可变区(VL)，其中：
所述重链可变区包含如SEQ ID NO：79-81中任一所示或与之具有至少80％、至少90％同一性的氨基酸序列，
所述轻链可变区包含如SEQ ID NO：82或83所示或与之具有至少80％、至少90％同一性的氨基酸序列。
一些具体实施方案中，所述特异性结合TIGIT的第三抗原结合结构域包含全长重链(HC)和全长轻链(LC)。
例如，所述全长重链为IgG1、IgG2或IgG4同种型，所述全长轻链为Kappa同种型；
又例如，所述重链序列为SEQ ID NO：67所示或与之具有至少80％、至少90％ 序列同一性的氨基酸序列，所述轻链序列为SEQ ID NO：68所示或与之具有至少80％、至少90％序列同一性的氨基酸序列。
一些实施方案中，前述PD-1/PVRIG/TIGIT结合蛋白中，特异性结合PD-1的第一抗原结合结构域、特异性结合PVRIG的第二抗原结合结构域、特异性结合TIGIT的第三抗原结合结构域直接或通过连接子相连接。例如，所述连接子为具有如(G 4S) x所示的氨基酸序列，其中，x独立地选自1-20的整；又例如，所述连接子为(G 4S) 2、(G 4S) 3、(G 4S) 4所示的氨基酸序列。
一些实施方案中，前述PD-1/PVRIG/TIGIT结合蛋白，特异性结合PD-1的抗原结合结构域位于特异性结合PVRIG的抗原结合结构域的N端或C端。
一些实施方案中，前述PD-1/PVRIG/TIGIT结合蛋白，特异性结合PD-1的抗原结合结构域位于特异性结合TIGIT的抗原结合结构域的N端或C端。
一些实施方案中，前述PD-1/PVRIG/TIGIT结合蛋白，特异性结合PVRIG的抗原结合结构域位于特异性结合TIGIT的抗原结合结构域的N端或C端。
一些实施方案中，第一抗原结合结构域、第二抗原结合结构域、第三抗原结合结构域彼此在一条多肽链上，或不在一条多肽链上。
一些实施方案中，前述PD-1/PVRIG/TIGIT结合蛋白，包含第一多肽链和第二多肽链，其中如选自以下的结构所示：
(I)第一多肽链：[特异性结合PD-1的第一抗原结合结构域]-[连接子1]a-[特异性结合PVRIG的第二抗原结合结构域]-[连接子2]b-[特异性结合TIGIT的第三抗原结合结构域的VH]-CH1-Fc，和第二多肽链：[特异性结合TIGIT的第三抗原结合结构域的VL]-Cκ；或
(II)第一多肽链：[特异性结合PVRIG的第二抗原结合结构域]-[连接子2]b-[特异性结合TIGIT的第三抗原结合结构域的VH]-CH1-Fc，和第二多肽链：[特异性结合PD-1的第一抗原结合结构域]-[连接子3]c-[特异性结合TIGIT的第三抗原结合结构域的VL]-Cκ；或
(III)第一多肽链：[特异性结合PVRIG的第二抗原结合结构域]-[连接子2]b-[特异性结合TIGIT的第三抗原结合结构域的VH]-CH1-Fc-[连接子4]d-[特异性结合PD-1的第一抗原结合结构域]，和第二多肽链：[特异性结合TIGIT的第三抗原结合结构域的VL]-Cκ；或
(IV)第一多肽链：[特异性结合PVRIG的第二抗原结合结构域]-[连接子2]b-[特异性结合TIGIT的第三抗原结合结构域的VH]-CH1-Fc，和第二多肽链：[特异性结合TIGIT的第三抗原结合结构域的VL]-Cκ-[连接子5]e-[特异性结合PD-1的第一抗原结合结构域]。
以上第一、第二多肽链均是从N端到C端顺序。
其中，-表示肽键，连接子为能够实现连接功能的多肽，连接子1、连接子2、连接子3、连接子4、连接子5可以相同，也可以不同；a、b、c、d、e可以任选 独立地为0或1。
例如，各连接子独立地为EPKSS或为(G xS) y连接子，其中，x选自1-5的整数(例如，1、2、3、4、5)，y选自1-6的整数(例如，1、2、3、4、5、6)。
当连接子为(G xS) y连接子时，例如为(G 4S) 2、(G 4S) 3、(G 4S) 4任一所示的连接子。
一些实施方案中，提供PD-1/PVRIG/TIGIT结合蛋白，其包含第一多肽链和第二多肽链，其中：
所述第一、第二多肽链分别包含如SEQ ID NO：87、68所示的氨基酸序列；
所述第一、第二多肽链分别包含如SEQ ID NO：84、88所示的氨基酸序列；
所述第一、第二多肽链分别包含如SEQ ID NO：89、68所示的氨基酸序列；
所述第一、第二多肽链分别包含如SEQ ID NO：84、90所示的氨基酸序列；或
所述第一、第二多肽链分别包含如SEQ ID NO：91、68所示的氨基酸序列；
所述第一、第二多肽链分别包含如SEQ ID NO：85、92所示的氨基酸序列；
所述第一、第二多肽链分别包含如SEQ ID NO：93、68所示的氨基酸序列；
所述第一、第二多肽链分别包含如SEQ ID NO：85、94所示的氨基酸序列；
所述第一、第二多肽链分别包含如SEQ ID NO：97、68所示的氨基酸序列；
所述第一、第二多肽链分别包含如SEQ ID NO：98、68所示的氨基酸序列；
或与前述任一的第一、第二多肽链均具有至少80％或均具有至少90％序列同一性的氨基酸序列组合。
一些实施方案中，前述本公开的PD-1/PVRIG/TIGIT结合蛋白包含两条相同或不同的第一多肽链，包含两条相同或不同的第二多肽链。
一些具体实施方案中，PD-1/PVRIG/TIGIT结合蛋白包含两条相同的第一多肽链和两条相同的第二多肽链。
一些实施方案中，本公开PD-1/PVRIG/TIGIT结合蛋白因为包含本公开的特异性结合PD-1的抗原结合结构域，而具有其所有或任意的性质和功能。
PD-1/PVRIG/TIGIT结合蛋白因为包含PVRIG/TIGIT结合结构域，还有具有下述特征中的至少一个：
(a)以小于1×10 -7M的K D值结合人PVRIG；
(b)阻断PVRIG与其配体(例如，PVRL2)的相互作用；
(c)解除树突状细胞(DC)对T细胞的抑制作用，活化T细胞；
(d)解除肿瘤细胞对NK细胞的抑制作用。
一些实施方案中，本公开的前述PD-1/PVRIG/TIGIT结合蛋白能够抑制肿瘤生长至少约10％，例如至少约20％、约30％、约40％、约50％、约60％、约70％、约80％、约90％。
一些实施方案中，本公开的前述PD-1/PVRIG/TIGIT结合蛋白涵盖变体，所述 变体与前述第一多肽链和第二多肽链的任一组合相比有一个或多个(例如，1、2、3、4、5、6、7、8、9、10个)氨基酸突变；所述氨基酸突变可以是保守的替换、取代或修饰，和/或不影响功能的缺失、添加。所述突变可以发生在特异性结合PD-1的抗原结合结构域、特异性结合PVRIG的抗原结合结构域、特异性结合TIGIT的抗原结合结构域(例如CDR区和/或FR区)。
一些实施方案中，提供了一种PD-1/PVRIG/TIGIT结合蛋白，其与前述本公开的PD-1/PVRIG/TIGIT结合蛋白结合或竞争结合相同的PD-1、PVRIG和/或TIGIT或其表位；或阻断前述本公开的前述PD-1/PVRIG/TIGIT结合蛋白与PD-1、PVRIG和/或TIGIT的结合；或其与PD-1、PVRIG和/或TIGIT的结合被前述本公开的PD-1/PVRIG/TIGIT结合蛋白阻断。
PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白
本公开提供PD-1/PVRIG结合蛋白，其包含特异性结合PD-1的抗原结合结构域、特异性结合PVRIG的抗原结合结构域，能同时或分别特异性结合PD-1、PVRIG。
本公开提供PD-1/TIGIT结合蛋白，其包含特异性结合PD-1的抗原结合结构域、特异性结合TIGIT的抗原结合结构域，能同时或分别特异性结合PD-1、TIGIT。
一些实施方案中，前述PD-1/PVRIG结合蛋白包含或为抗PD-1/PVRIG双特异性抗体，前述PD-1/TIGIT结合蛋白包含或为抗PD-1/TIGIT双特异性抗体。
一些实施方案中，前述PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白中的各抗原结合结构域选自Fab、Fv、sFv、Fab’、F(ab’) 2、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体peptibody、结构域抗体或由其任意组合组成。
一些实施方案中，特异性结合PD-1的抗原结合结构域包含或为VHH、特异性结合PVRIG的抗原结合结构域包含或为VHH、特异性结合TIGIT的抗原结合结构域为Fab或F(ab’) 2。
一些实施方案中，前述PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白为人源化的、亲和力成熟、去除T细胞表位、降低抗体脱酰胺和/或降低抗体异构化改造的。
一些实施方案中，前述PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白为重组抗体、骆驼抗体、嵌合抗体、人源化抗体、全人抗体或其抗原结合片段。
一些实施方案中，PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白中，所述特异性结合PD-1的抗原结合结构域包含至少一个免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含：
SEQ ID NO：3、13-15、17-35任一所示氨基酸序列中的CDR1、CDR2和CDR3，
SEQ ID NO：2、16任一所示氨基酸序列中的CDR1、CDR2和CDR3，
所述CDR1、CDR2和CDR3是根据Kabat、IMGT、Chothia、AbM或Contact编号***定义的。
一些实施方案中，根据Kabat编号***，所述免疫球蛋白单一可变结构域的 CDR1、CDR2和CDR3的氨基酸序列分别：
如SEQ ID NO：7、36、37所示，
如SEQ ID NO：4、5、6所示。
一些具体实施方案中，所述免疫球蛋白单一可变结构域的CDR1的氨基酸序列如SEQ ID NO：7所示，CDR2的氨基酸序列如SEQ ID NO：8、38-40、101任一所示，CDR3的氨基酸序列如SEQ ID NO：9、41、42任一所示。
一些具体实施方案中，所述免疫球蛋白单一可变结构域的CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：7、38、41所示。
一些实施方案中，前述PD-1/PVRIG结合蛋白中，所述特异性结合PVRIG的抗原结合结构域包含至少一个免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含：
SEQ ID NO：45、62-66、95-96任一所示氨基酸序列中的CDR1、CDR2和CDR3，或
SEQ ID NO：44、57-61任一所示氨基酸序列中的CDR1、CDR2和CDR3。
一些具体实施方案中，根据Kabat编号***，所述免疫球蛋白单一可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别：
如SEQ ID NO：49、50、51所示，
如SEQ ID NO：46、47、48或99所示。
一些实施方案中，前述PD-1/TIGIT结合蛋白中，所述特异性结合TIGIT的抗原结合结构域包含重链可变区(VH)和轻链可变区(VL)，其中：
所述重链可变区包含分别如SEQ ID NO：73、74和75所示氨基酸序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：76、77和78所示氨基酸序列的LCDR1、LCDR2和LCDR3。
一些实施方案中，前述PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白中，所述特异性结合PD-1的抗原结合结构域包含如
SEQ ID NO：3、13-15、17-35任一所示氨基酸序列；或
SEQ ID NO：2、16任一所示氨基酸序列；或
与所述序列任一具有至少80％、至少85％、至少90％序列同一性的氨基酸序列。
一些实施方案中，前述PD-1/PVRIG结合蛋白中，所述特异性结合PVRIG的抗原结合结构域包含如：
SEQ ID NO：45、62-66、95-96任一所示的氨基酸序列；或
SEQ ID NO：44、57-61任一所示的氨基酸序列；或
与所述序列任一具有至少80％、至少85％、至少90％序列同一性的氨基酸序列。
一些实施方案中，前述PD-1/TIGIT结合蛋白中，所述特异性结合TIGIT的抗 原结合结构域包含重链可变区(VH)和轻链可变区(VL)，其中：
所述重链可变区包含如SEQ ID NO：79-81中任一所示或与之具有至少80％、至少90％同一性的氨基酸序列，
所述轻链可变区包含如SEQ ID NO：82或83所示或与之具有至少80％、至少90％同一性的氨基酸序列。
一些具体实施方案中，所述特异性结合TIGIT的第三抗原结合结构域包含全长重链(HC)和全长轻链(LC)。
例如，所述全长重链为IgG1、IgG2或IgG4同种型，所述全长轻链为Kappa同种型。
又例如，所述重链序列为SEQ ID NO：67所示或与之具有至少80％、至少90％序列同一性的氨基酸序列，所述轻链序列为SEQ ID NO：68所示或与之具有至少80％、至少90％序列同一性的氨基酸序列。
一些实施方案中，前述PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白中，不同抗原结合结构域直接或通过连接子相连接；例如，所述连接子独立地为具有如(G 4S) x所示的氨基酸序列，其中，x独立地选自1-20(例如，1、2、3、4、5、6、7、8)的整；又例如，所述连接子独立地为(G 4S) 2、(G 4S) 3所示的氨基酸序列。
一些实施方案中，前述PD-1/PVRIG结合蛋白，特异性结合PD-1的抗原结合结构域位于特异性结合PVRIG的抗原结合结构域的N端或C端。
一些实施方案中，前述PD-1/TIGIT结合蛋白，特异性结合PD-1的抗原结合结构域位于特异性结合TIGIT的抗原结合结构域的N端或C端。
一些实施方案中，前述PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白还包含人免疫球蛋白Fc区；例如，所述Fc区是人IgG1、IgG2或IgG4的Fc区。所述Fc区可以具有突变，示例性突变为S228P、F234A、L235A和/或K447A突变。一些实施方案中，Fc区可以使所述结合蛋白形成二聚体分子，延长所述结合蛋白的体内半衰期。
一些实施方案中，本公开PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白因为具有本公开的特异性结合PD-1的抗原结合结构域而具有其所有或任意的性质和功能。
一些实施方案中，本公开的前述PD-1/PVRIG结合蛋白能够抑制肿瘤生长至少约10％，例如至少约20％、约30％、约40％、约50％、约60％、约70％、约80％、约90％或以上。
一些实施方案中，本公开的PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白涵盖变体，所述变体与前述PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白相比有一个或多个(例如，1、2、3、4、5、6、7、8、9、10个)氨基酸突变；所述氨基酸突变可以是保守的替换、取代或修饰，和/或不影响功能的缺失、添加；所述突变 可以发生在特异性结合PD-1的抗原结合结构域、特异性结合PVRIG的抗原结合结构域、特异性结合TIGIT的抗原结合结构域的CDR区和/或FR区。
一些实施方案中，提供PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白，其与前述本公开的PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白结合或竞争结合相同的PD-1、PVRIG和/或TIGIT或其表位；或阻断前述本公开的前述PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白与PD-1、PVRIG和/或TIGIT的结合；或其与PD-1、PVRIG和/或TIGIT的结合被前述本公开的PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白阻断。
一些实施方案中，提供PVRIG结合蛋白，其包含前述PD-1/PVRIG结合蛋白中的特异性结合PVRIG的抗原结合结构域。
本公开中，涉及PVRIG结合蛋白或特异性结合PVRIG的抗原结合结构域，其均可包含或选自WO2016134333、WO2016134335、WO2018033798、WO2018220446、WO2019079777、WO2019232484、WO2020018879、WO2021021837、WO2021097294、WO2021091605、WO2021113831、中的PVRIG抗体。例如，PVRIG抗体可以为CPA.7.002、CPA.7.005、CPA.7.021和CPA.7.050中的任一种(参见WO2016134333)。此处全文引入上述专利。
一些实施方案中，提供TIGIT结合蛋白，其包含前述PD-1/TIGIT结合蛋白中的特异性结合TIGIT的抗原结合结构域。
本公开中，涉及TIGIT结合蛋白或特异性结合TIGIT的抗原结合结构域，其均可包含或选自WO2019062832、WO2009126688、WO2014089113、WO2015009856、WO2015143343、WO2015174439、WO2016028656、WO2016106302、WO2017053748、WO2017030823、US20160176963、US20130251720、WO2019232484、WO2019062832中的TIGIT抗体。例如，TIGIT抗体可以为CPA.9.083.H4(S241P)、CPA.9.086.H4(S241P)、CHA.9.547.7.H4(S241P)和CHA.9.547.13.H4(S241P)中的任一种(参见WO2019232484)。此处全文引入上述专利。
一些实施方案中，提供PVRIG/TIGIT结合蛋白，其包含前述PD-1/PVRIG/TIGIT结合蛋白中的特异性结合TIGIT的抗原结合结构域和特异性结合PVRIG的抗原结合结构域。
多核苷酸和载体
本公开提供编码本公开的PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白、PVRIG结合蛋白、TIGIT结合蛋白的多核苷酸。本公开的多核苷酸可为RNA、DNA或cDNA。根据本公开的一些实施方案，本公开的多核苷酸是基本上分离的多核苷酸。
本公开的多核苷酸也可呈载体形式，可存在于载体中和/或可为载体的一部分，该载体例如质粒、粘端质粒、YAC或病毒载体。载体可尤其为表达载体，即可提 供PD-1结合蛋白体外和/或体内(即在适合宿主细胞、宿主有机体和/或表达***中)表达的载体。该表达载体通常包含至少一种本公开的多核苷酸，其可操作地连接至一个或多个适合的表达调控元件(例如启动子、增强子、终止子等)。针对在特定宿主中的表达，对所述元件及其序列进行选择为本领域技术人员的常识。对本公开的PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白、PVRIG结合蛋白、TIGIT结合蛋白的表达有用或必需的调控元件及其他元件，例如为启动子、增强子、终止子、整合因子、选择标记物、前导序列、报告基因。
本公开的多核苷酸可基于本公开的多肽的氨基酸序列的信息通过已知的方式(例如通过自动DNA合成和/或重组DNA技术)制备或获得，和/或可从适合的天然来源加以分离。
宿主细胞
本公开提供表达或能够表达一种或多种本公开的PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白、PVRIG结合蛋白、TIGIT结合蛋白和/或含有本公开的核酸或载体的重组宿主细胞。一些实施方案中，宿主细胞为细菌细胞、真菌细胞或哺乳动物细胞。
细菌细胞例如包括革兰氏阴性细菌菌株(例如大肠杆菌(Escherichia coli)菌株、变形杆菌属(Proteus)菌株及假单胞菌属(Pseudomonas)菌株)及***菌株(例如芽孢杆菌属(Bacillus)菌株、链霉菌属(Streptomyces)菌株、葡萄球菌属(Staphylococcus)菌株及乳球菌属(Lactococcus)菌株)的细胞。
真菌细胞例如包括木霉属(Trichoderma)、脉孢菌属(Neurospora)及曲菌属(Aspergillus)的物种的细胞；或者包括酵母属(Saccharomyces)(例如酿酒酵母(Saccharomyces cerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如粟酒裂殖酵母(Schizosaccharomyces pombe))、毕赤酵母属(Pichia)(例如巴斯德毕赤酵母(Pichia pastoris)及嗜甲醇毕赤酵母(Pichia methanolica))及汉森酵母属(Hansenula)的物种的细胞。
哺乳动物细胞例如包括例如HEK293细胞、CHO细胞、BHK细胞、HeLa细胞、COS细胞等。
然而，本公开也可使用两栖类细胞、昆虫细胞、植物细胞及本领域中用于表达异源蛋白的任何其他细胞。
本公开的细胞不能发育成完成的植株或动物个体。
生产或制备方法
本公开提供制备本公开的PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白、PVRIG结合蛋白、TIGIT结合蛋白的方法，所述方法通常包含以下步骤：
-在允许表达本公开的结合蛋白的条件下培养本公开的宿主细胞；及
-从培养物回收由所述宿主细胞表达的结合蛋白；及
-任选地，包括进一步纯化和/或修饰本公开的结合蛋白。
本公开的PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白、PVRIG结合蛋白、TIGIT结合蛋白可在如上所述细胞中以细胞内方式(例如在细胞质中、在周质中或在包涵体中)产生，接着从宿主细胞分离且任选进一步纯化；或其可以细胞外方式(例如在培养宿主细胞的培养基中)产生，接着自培养基分离且任选进一步纯化。
用于重组产生多肽的方法及试剂，例如特定适合表达载体、转化或转染方法、选择标记物、诱导蛋白表达的方法、培养条件等在本领域中是已知的。类似地，适用于制造本公开的结合蛋白或抗体的分离及纯化技术为本领域技术人员所公知。生产和纯化抗体的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南(5-8章和15章)。本公开工程化的抗体也可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至表达载体。表达载体可以稳定地转染细胞。哺乳动物类表达***会导致抗体的糖基化，特别是在Fc区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。也可以用常规方法去除可溶的混合物和多聚体，比如分子筛，离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
然而，本公开的PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白、PVRIG结合蛋白、TIGIT结合蛋白也可以通过本领域已知的其它产生蛋白质的方法获得，例如化学合成，包括固相或液相合成。
组合物/药物组合物
本公开提供组合物，包含前述PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白、PVRIG结合蛋白和/或TIGIT结合蛋白。
例如，提供药物组合物，其含有对癌症治疗、缓解或预防有效量的如上所述的PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白和/或PD-1/TIGIT结合蛋白和至少一种可药用的赋形剂、稀释或载体。
在一些具体实施方案中，所述药物组合物单位计量中可含有0.01至99重量％的PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白和/或PD-1/TIGIT结合蛋白，或药物组合物单位剂量中含PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白和/或PD-1/TIGIT结合蛋白的量为0.1-2000mg，在一些具体实施方案中为1-1000mg。
一些实施方案中，提供产品或药盒，其含有至少一个容器，各自独立地包含前述PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白和/或 PD-1/TIGIT结合蛋白。可选地，药盒包含容器和标签。容器例如瓶、注射器和试管。容器容纳有效于治疗病症的组合物。容器上或与容器相连的标签表明所述组合物用于治疗所选病症。组合物中含有前述PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白和/或PD-1/TIGIT结合蛋白。
一些实施方案中，提供药物组合物，包含本公开的PD-1结合蛋白和PVRIG结合蛋白。另一些药物组合物，包含本公开的PD-1结合蛋白和TIGIT结合蛋白。另一些药物组合物，包含本公开的PD-1结合蛋白、TIGIT结合蛋白和PVRIG结合蛋白。所述PD-1结合蛋白、TIGIT结合蛋白和/或PVRIG结合蛋白可以治疗或缓解疾病(例如癌症)有效量的形式存在，药物组合物中还可以包含至少一种可药用的赋形剂、稀释或载体。
示例性地，提供组合物、产品或药盒，其含有以下任一项或其组合：
本公开提供的任意PD-1结合蛋白；
本公开提供的任意PD-1/PVRIG/TIGIT结合蛋白；
本公开提供的任意PD-1/PVRIG结合蛋白；
本公开提供的任意PD-1/TIGIT结合蛋白；
本公开提供的任意PD-1结合蛋白和本公开提供的任意PVRIG/TIGIT结合蛋白；
本公开提供的任意PD-1结合蛋白和本公开提供的任意PVRIG结合蛋白；
本公开提供的任意PD-1结合蛋白和本公开提供的任意TIGIT结合蛋白；或
本公开提供的任意PD-1结合蛋白、本公开提供的任意PVRIG结合蛋白和本公开提供的任意TIGIT结合蛋白。
可选地，当组合物为药物组合物时，还含有至少一种可药用的赋形剂、稀释剂或载体。
治疗疾病的方法和制药用途
本公开提供前述PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白、PVRIG/TIGIT结合蛋白、PVRIG结合蛋白、TIGIT结合蛋白、多核苷酸、组合物(包括药物组合物)用于治疗、缓解、预防、诊断疾病或病症的方法。
一些实施方案中，提供改善、缓解、治疗或预防疾病的方法，包括向受试者施用改善、缓解、治疗或预防有效量的前述PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白、PVRIG结合蛋白、TIGIT结合蛋白、多核苷酸或组合物(包括药物组合物)。
一些实施方案中，提供本公开的PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白或PVRIG/TIGIT结合蛋白在制备改善、缓解、治疗或预防疾病的药物的用途。
一些实施方案中，提供本公开的PD-1结合蛋白与PVRIG结合蛋白联合在制备 改善、缓解、治疗或预防疾病的药物的用途。
一些实施方案中，提供本公开的PD-1结合蛋白与TIGIT结合蛋白联合在制备改善、缓解、治疗或预防疾病的药物的用途。
一些实施方案中，提供本公开的PD-1结合蛋白、PVRIG结合蛋白和TIGIT结合蛋白联合在制备改善、缓解、治疗或预防疾病的药物的用途。
一些实施方案中，提供本公开的PD-1结合蛋白与PVRIG/TIGIT结合蛋白联合在制备改善、缓解、治疗或预防疾病的药物的用途。
一些实施方案中，提供本公开的PD-1结合蛋白用于改善、缓解、治疗或预防疾病，联合PVRIG结合蛋白和/或TIGIT结合蛋白，或联合PVRIG/TIGIT结合蛋白。
一些实施方案中，提供本公开的PD-1结合蛋白改善、缓解、治疗或预防疾病的方法，进一步包括施用PVRIG结合蛋白和/或TIGIT结合蛋白，或进一步包括施用PVRIG/TIGIT结合蛋白。
一些实施方案中，提供本公开的PVRIG结合蛋白和/或TIGIT结合蛋白改善、缓解、治疗或预防疾病的方法，进一步包括施用本公开的PD-1结合蛋白。
一些实施方案中，提供本公开的PVRIG/TIGIT结合蛋白改善、缓解、治疗或预防疾病的方法，进一步包括施用本公开的PD-1结合蛋白。
一些实施方案中，前述疾病为增殖性疾病或者任何特征在于不受控细胞生长的其它疾病或病症(例如癌症，本公开中，癌症和肿瘤可互相替代使用)，例如为PD-L1超量表达或与PVRIG、TIGIT异常表达相关的相关疾病(例如癌症)。
一些实施方案中，前述癌症为实体瘤或血液肿瘤。
一些实施方案中，前述癌症为晚期或转移性的。
一些实施方案中，前述癌症选自以下或其组合：***癌、肝癌(HCC)、结直肠癌、卵巢癌、子宫内膜癌、乳腺癌、三阴性乳腺癌、胰腺癌、胃(stomach/gastric)癌、***、头颈癌、甲状腺癌、睾丸癌、尿路上皮癌、肺癌(小细胞肺癌、非小细胞肺癌)、黑素瘤、非黑素瘤皮肤癌(鳞状和基底细胞癌)、神经胶质瘤、肾癌(RCC)、淋巴瘤(NHL或HL)、急性骨髓性白血病(AML)、T细胞急性淋巴母细胞性白血病(T-ALL)、弥漫性大B细胞淋巴瘤、睾丸生殖细胞肿瘤、间皮瘤、食道癌、默克细胞癌(Merkel Cells cancer)、高MSI癌、KRAS突变肿瘤、成人T细胞白血病/淋巴瘤和骨髓增生异常综合征(MDS)。例如，选自肺癌、***癌、乳腺癌、头颈部癌、食管癌、胃癌、结肠癌、结直肠癌、膀胱癌、***、子宫癌、卵巢癌、肝癌、黑色素瘤、肾癌、鳞状细胞癌、血液***癌症或者任何特征在于不受控细胞生长的其它疾病或病症。
一些实施方案中，前述受试者具有与PVRIG和/或TIGIT相关的病况。一些具体方案中，受试者病况包括表达或不表达PVRIG的癌症并且进一步包括非转移性或非浸润性以及浸润性或转移性癌症，其中免疫细胞、基质细胞或发生病变的细 胞的PVRIG表达抑制抗肿瘤反应和抗浸润性免疫反应。本公开的方法例如适合于治疗血管化癌症。
一些实施方案中，提供治疗或预防受试者感染或脓毒症的方法，包括向所述受试者施用治疗或预防有效量的前述PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白、PVRIG结合蛋白、TIGIT结合蛋白、多核苷酸、组合物(包括药物组合物)。一些具体方案中，所述感染是病原体感染，以病毒特异性T细胞反应的不同程度的功能性障碍为特征，如HIV、HCV、HBV。一些具体方案中，所述脓毒症包括重度脓毒症、脓毒性休克、全身炎症反应综合征(SIRS)、菌血症、败血症、毒血症、脓毒性综合征。
一些实施方案中，提供活化受试者的细胞毒性T细胞(CTL)的方法，提供活化受试者的NK细胞的方法，提供活化受试者的γδT细胞的方法，提供活化受试者的Th1细胞的方法，提供活化、减少或消除受试者体内的调节性T细胞(Treg)中的至少一种的细胞数量和/或活性的方法，提供增加受试者体内的IFN-γ产生和/或促炎性细胞因子分泌的方法，提供抑制受试者体内的PVRIG和PVRL2的相互作用的方法，提供阻断或抑制受试者体内PD-1与PD-L1/PD-L2的相互作用的方法，均包括向所述受试者施用有效量的前述PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白、PVRIG结合蛋白、TIGIT结合蛋白、多核苷酸、组合物(包括药物组合物)。
检测和试剂盒
本公开提供PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白、PVRIG/TIGIT结合蛋白、PVRIG结合蛋白、TIGIT结合蛋白、多核苷酸、组合物的检测用途。本公开还提供用于体内或体外检测PD-1、PVRIG、TIGIT的方法、***或装置，其包括用本公开的前述结合蛋白、多核苷酸、组合物处理样品。
一些实施方案中，体外检测方法、***或装置可能例如包括：
(1)使样品与PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白、PVRIG/TIGIT结合蛋白、PVRIG结合蛋白、TIGIT结合蛋白、多核苷酸、组合物接触；
(2)检测在前述结合蛋白、多核苷酸和样品之间形成的复合物；和/或
(3)使参比样品(例如，对照样品)与结合蛋白、核酸接触；和
(4)通过与参比样品比较，确定复合物形成的程度。如与对照样品或受试者中相比，样品或受试者中复合物形成的变化(例如，统计学上的显著变化)表示样品中存在PD-1、PVRIG、TIGIT。
另一些实施方案中，体内检测方法、***或装置可以包括：
(1)向受试者施用前述结合蛋白、多核苷酸；和
(2)检测在结合前述结合蛋白、多核苷酸和受试者之间复合物的形成。
检测可以包括确定形成复合物的位置或时间。用可检测物质对前述结合蛋白、多核苷酸标记，通过对所述标记检测以实现对能结合的蛋白、多核苷酸的物质(例如PD-1、PVRIG、TIGIT)的检测。合适的可检测物质包括多种酶、辅基、荧光物质、发光物质和放射性物质。可以通过测量与PD-1、PVRIG、TIGIT结合或不结合的物质或使其可视化，检测结合蛋白、多核苷酸与PD-1、PVRIG、TIGIT的复合物形成。可以使用常规检测测定法，例如，酶联免疫吸附测定(ELISA)、放射免疫测定(RIA)或组织免疫组织化学。出于检测目的，本公开的结合蛋白、多核苷酸可以用荧光团发色团标记。一些实施方案中，还提供包含上述多核苷酸、结合蛋白的诊断试剂，以及提供相关诊断用途。
一些实施方案中，还提供试剂盒，所述试剂盒包含前述结合蛋白、多核苷酸，还可以包含诊断使用说明。试剂盒还可以含有至少一种额外的试剂，如标记物或额外的诊断剂。对于体内使用，所述结合蛋白可以配制为药物组合物。
附图说明
图1A至图1B为PD-1/PD-L1 NFAT报告基因***检测抗PD-1纳米抗体的活性结果。使用Pembrolizumab作为阳性对照，hIgG4作为阴性对照；其中，图1A为A6_IgG4和A17_IgG4结果。图1B为A17h1_IgG4、A17m09_hIgG4、A17m0901_hIgG4、A17m0902_hIgG4、A17m0903_hIgG4、A17m0905_hIgG4的结果。
图2为通过DC细胞：T细胞混合淋巴反应中细胞因子的释放检测A17m09_hIgG4对T细胞激活程度，使用Pembrolizumab作为阳性对照，hIgG4作为阴性对照。
图3A至图3B为A17m09_hIgG4抑制小鼠MC38结肠癌肿瘤生长的体内药效试验结果，图3A为小鼠肿瘤体积结果图，图3B为小鼠体重图，使用Pembrolizumab作为阳性对照，PBS作为阴性对照。
图4为抗PVRIG纳米抗体30、151的混合淋巴细胞反应(MLR)实验结果，使用Tab5作为阳性对照，hIgG4作为阴性对照。
图5为抗PVRIG/TIGIT双特异性抗体1708-151H8的MLR实验结果，同时检测151H8、1708、hIgG4、Tab5、Pembrolizumab作为对照。
图6A至图6B为抗PVRIG/TIGIT双特异性抗体1708-151、1708和151的联用在人黑色素瘤A375混合人PBMC的小鼠皮下移植瘤模型中的抗肿瘤结果。图6B为对应的小鼠体重图。
图7A至图7B为抗PVRIG/TIGIT双特异性抗体1708-151H7和1708-30H2在人黑色素瘤A375混合人PBMC的小鼠皮下移植瘤模型中的抗肿瘤结果。图7B为对应的小鼠体重图。
图8为抗PD-1/PVRIG/TIGIT三特异性抗体的4种抗体构型示意图。
图9A至图9B分别为A17h1-1708-30H2、A17h1-1708-151H7各自4种构型的抗体结合TIGIT抗原的检测结果。
图10A至图10B分别为A17h1-1708-30H2、A17h1-1708-151H7各自4种构型的抗体结合PVRIG抗原的检测结果。
图11A、图11B、图11C分别为抗PD-1/PVRIG/TIGIT三特异性抗体中PVRIG序列优化后与过表达PVRIG、TIGIT、PD-1抗原的细胞的结合能力。
图12A至图12B分别为A17h1-1708-30H2、A17h1-1708-151H7各自4种构型的抗体在PD-1/PD-L1 NFAT报告基因***中对PD-L1/PD-1结合的阻断。
图13A至图13B分别为A17h1-1708-30H2、A17h1-1708-151H7各自4种构型的抗体的MLR检测结果。
图14A至图14B分别为两个受试者中A17m0902-1708-30H2-C的MLR检测结果。
图15A至图15B分别为A17h1-1708-30H2、A17h1-1708-151H7各自4种构型的抗体的NK细胞杀伤功能实验结果。
图16A至图16B抗PD-1/PVRIG/TIGIT三特异性抗体在PD-1单抗响应的人黑色素瘤A375混合人PBMC的小鼠皮下移植瘤模型中抗肿瘤作用评估。
图17A至图17B抗PD-1/PVRIG/TIGIT三特异性抗体在PD-1单抗不响应的人黑色素瘤A375混合人PBMC的小鼠皮下移植瘤模型中抗肿瘤作用评估。
具体实施方式
术语定义
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除非在本公开中另有明确定义，本公开使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
“程序性死亡1”、“细胞程序性死亡1”、“蛋白PD-1”、“PD-1”、“PDCD1”和“hPD-1”可互换使用，且包括人PD-1的变体、同种型、物种同源物、以及与PD-1具有至少一个共同表位的类似物。完整的PD-1序列可以从GenBank登录号U64863找到。“程序性死亡配体-1(PD-L1)”是PD-1的两种细胞表面糖蛋白配体之一(另一种为PD-L2)，它在与PD-1结合时下调T细胞活化和细胞因子分泌。如本文中使用的“PD-L1”包括人PD-L1(hPD-L1)，hPD-L1的变体、同种型、和种间同源物，以及与hPD-L1具有至少一个共同表位的类似物。完整的hPD-L1序列可以用GenBank登录号Q9NZQ7查到。
“PVRIG”或“PVRIG蛋白质”或“PVRIG多肽”可以任选地包括任何这类蛋白质或其变异体、结合物或片段，包括(但不限于)如本文所述的已知或野生型PVRIG， 以及任何天然产生的剪接变异体、氨基酸变异体或同工型，并且尤其是PVRIG的可溶性胞外域(ECD)片段。此处ECD的定义如专利WO2016134333所述。完整的人类PVRIG序列可以GenBank登录号AAH73861.1找到。
“TIGIT”或“TIGIT蛋白质”或“TIGIT多肽”可以任选地包括任何这类蛋白质或其变异体、结合物或片段，包括(但不限于)如本文所述的已知或野生型TIGIT，以及任何天然产生的剪接变异体、氨基酸变异体或同工型。完整的TIGIT序列可以GenBank登录号AAI01289.1找到。
“与PD-1结合”，指能与PD-1或其表位相互作用，所述PD-1或其表位可以是人源的。“与PVRIG结合”，指能与PVRIG或其表位相互作用，所述PVRIG或其表位可以是人源的。“与TIGIT结合”，指能与TIGIT或其表位相互作用，所述TIGIT或其表位可以是人源的。“抗原结合位点”指抗原上不连续的，由本公开抗体或抗原结合片段识别的三维空间位点。
“PD-1结合蛋白”涵盖任何能够特异性结合PD-1的蛋白或其表位的任何分子。PD-1结合蛋白可以包括针对PD-1的如本公开定义的抗体、其抗原结合片段或其缀合物。PD-1结合蛋白还涵盖免疫球蛋白超家族抗体(IgSF)或CDR移植分子。本公开的“PD-1结合蛋白”可以包含至少一个结合PD-1的免疫球蛋白单一可变结构域(如VHH)。在一些实施方案中，“PD-1结合蛋白”可以包含2、3、4或更多个结合PD-1的免疫球蛋白单一可变结构域(如VHH)。本公开的PD-1结合蛋白除包含PD-1的免疫球蛋白单一可变结构域外，也可包含连接子和/或具有效应功能的部分，例如半衰期延长部分(如结合血清白蛋白的免疫球蛋白单一可变结构域)、和/或融合配偶体(如血清白蛋白)和/或缀合的聚合物(如PEG)和/或Fc区。在一些实施方案中，本公开的“PD-1结合蛋白”还涵盖双/多特异性抗体，其含有结合不同抗原的免疫球蛋白(如结合第一抗原(如PD-1)的第一抗体和结合第二抗原(如PVRIG)的第二抗体，任选包括结合第三体原(如TIGIT)的第三特异性抗体，进一步可选的，包括结合第四抗原的第四抗体。“PD-1结合蛋白”涵盖“PD-1/PVRIG结合蛋白”、“PD-1/TIGIT结合蛋白”、“PD-1/PVRIG/TIGIT结合蛋白”。
本公开的“PD-1结合蛋白”或“抗PD-1抗体”可以例如缀合的方式包含一个或多个效应分子。所述“效应分子”包括例如抗肿瘤剂、药物、毒素、生物活性蛋白(例如酶)、其它抗体或抗体片段、合成或天然存在的聚合物、核酸及其片段(例如DNA、RNA及其片段)、放射性核素，特别地放射性碘化物、放射性同位素、螯合金属、纳米颗粒和报道基团例如荧光化合物或可通过NMR或ESR光谱分析检测的化合物。当效应分子是聚合物时，其通常可以是合成或天然存在的聚合物，例如任选地取代的直链或支链聚亚烷基、聚亚烯基或聚氧化亚烷基聚合物或分支多糖或未分支多糖，例如同聚或异聚多糖。可存在于上述合成聚合物上的具体的任选取代基包括一个或多个羟基、甲基或甲氧基。合成聚合物的具体实例包括任选地取代的直链或支链聚(乙二醇)、聚(丙二醇)、聚(乙烯醇)或其衍生物， 特别地任选地取代的聚(乙二醇)例如甲氧基聚(乙二醇)或其衍生物。具体的天然存在的聚合物包括乳糖、直链淀粉、葡聚糖、糖原或其衍生物。在一个实施方案中，聚合物是白蛋白或其片段，例如人血清白蛋白或其片段。聚合物与PD-1结合蛋白或抗PD-1抗体的缀合方式可以通过常规方法实现。
“细胞因子”是由一个细胞群体释放的、作为细胞间介质作用于其它细胞的蛋白质的一般术语。这样的细胞因子的例子包括淋巴因子、单核因子、趋化因子和传统的多肽激素。示例性的细胞因子包括：人IL-2、IFN-γ、IL-6、TNFα、IL-17和IL-5。
“抗体”涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体；单特异性抗体，多特异性抗体(例如双特异性抗体)，全长抗体和抗体片段(或抗原结合片段，或抗原结合部分)，只要它们展现出期望的抗原结合活性。抗体可以指免疫球蛋白，是由两条重链和两条轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的框架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1，LCDR2，和LCDR3；重链的3个CDR区指HCDR1，HCDR2和HCDR3。
“抗原结合片段”涵盖单链抗体(即全长重链和轻链)；Fab、修饰的Fab、Fab’、修饰的Fab’、F(ab’)2、Fv、Fab-Fv、Fab-dsFv、单结构域抗体(例如VH或VL或VHH)、scFv、二价或三价或四价抗体、Bis-scFv、diabody、tribody、triabody、tetrabody和上述任意一种的表位结合片段(参见例如Holliger and Hudson,2005,Nature Biotech.23(9)：1126-1136；Adair and Lawson,2005,Drug Design Reviews-Online 2(3)209-217)。
产生和制备这些抗原结合片段的方法在本领域是公知的(参见例如Verma等人,1998,Journal ofImmunological Methods,216,165-181)。Fab-Fv形式首先公开于WO2009/040562，其二硫键稳定化形式Fab-dsFv首先公开于WO2010/035012。本公开的抗原结合片段还包括描述于WO2005/003169、WO2005/003170和WO2005/003171中的Fab和Fab’片段。多价抗体可包含多特异性例如双特异性或 可以是单特异性的(参见例如WO92/22583和WO05/113605)，后者的一个示例是描述于WO 92/22583中的Tri-Fab(或TFM)。
“双特异性抗体”涵盖对两个不同抗原或同一抗原的两个或至少两个不同抗原表位特异性结合的抗体(包括抗体或其抗原结合片段，如单链抗体)。现有技术已公开了各种结构的双特异性抗体。根据IgG分子的完整性，可分为IgG样双特异性抗体和抗体片段型双特异性抗体。根据抗原结合区域的数量，可分为二价、三价、四价或更多价的双特异性抗体。根据结构左右是否对称，可分为对称结构双特异性抗体和不对称结构双特异性抗体。其中，基于抗体片段的双特异性抗体，例如缺乏Fc片段的Fab片段，其通过将2个或多个Fab片段结合在一个分子中形成双特异性抗体，其具有较低的免疫原性，且分子量小，具有较高的肿瘤组织渗透性，该类型的典型的抗体结构如F(ab)2、scFv-Fab、(scFv)2-Fab等双特异性抗体。IgG样双特异性抗体(例如具有Fc片段)，这类抗体相对分子量较大，Fc片段有助于抗体后期的纯化，并提高其溶解性、稳定性，Fc部分还可能会与受体FcRn结合，增加抗体血清半衰期。典型的双特异性抗体结构模型如KiH、CrossMAb、Triomab quadroma、FcΔAdp、ART-Ig、BiMAb、Biclonics、BEAT、DuoBody、Azymetric、XmAb、2：1 TCBs、1Fab-IgG TDB、FynomAb、two-in-one/DAF、scFv-Fab-IgG、DART-Fc、LP-DART、CODV-Fab-TL、HLE-BiTE、F(ab) 2-CrossMAb、IgG-(scFv) 2、Bs4Ab、DVD-Ig、Tetravalent-DART-Fc、(scFv)4-Fc、CODV-Ig、mAb2、F(ab) 4-CrossMAb等双特异性抗体(参见Aran F.Labrijn等，Nature Reviews Drug Discovery volume 18，pages585–608(2019)；Chen S1等，J Immunol Res.2019 Feb 11；2019：4516041)。
“三特异性抗体”指能特异性结合三个或至少三个不同抗原表位，可以是不同抗原的抗原表位或同一抗原的不同抗原表位。
对于CDR的确定或定义，能够通过分辨抗体的结构和/或分辨抗体-配体复合物的结构，来完成对CDR的确定性描绘和对结合位点的残基的鉴定。这可通过本领域技术人员已知的各种技术中的任一种，例如X射线晶体学来实现。多种分析方法可用于鉴定CDR，包括但不限于Kabat编号***、Chothia编号***、AbM编号***、IMGT编号***、接触定义、构象定义。
Kabat编号***是用于编号抗体中残基的标准并且通常用于鉴定CDR区域(参见例如Johnson&Wu，2000，Nucleic Acids Res.，28：214-8)。Chothia编号***与Kabat编号***类似，但Chothia编号***考虑了某些结构环区域的位置。(参见例如Chothia等，1986，J.Mol.Biol.，196：901-17；Chothia等人，1989，Nature，342：877-83)。AbM编号***使用建模抗体结构的由Oxford Molecular Group生产的计算机程序集成套件(参见例如Martin等，1989，ProcNatl Acad Sci(USA)，86：9268-9272；“AbMTM，A Computer Program for ModelingVariable Regions of Antibodies，”Oxford，UK；Oxford Molecular，Ltd)。AbM编号***使用知识数据 库和从头开始方法的组合，从基本序列建模抗体的三级结构(参见Samudrala等，1999，在PROTEINS，Structure，Function and Genetics Suppl.，3：194-198中的“Ab Initio Protein Structure Prediction Using a Combined HierarchicalApproach”描述的那些)。接触定义基于可用复杂晶体结构的分析(参见例如MacCallum等，1996，J.Mol.Biol.，5：732-45)。构象定义中，CDR的位置可鉴定为对抗原结合做出焓贡献的残基(参见例如Makabe等，2008，Journal ofBiological Chemistry，283：1156-1166)。另外其它的CDR边界定义可能不严格遵循上述方法之一，但仍然与Kabat CDR的至少一部分重叠。根据特定残基或残基组不显著影响抗原结合的预测或实验结果，CDR的边界可缩短或延长。如本公开使用的，CDR可指通过本领域已知的任何方法(包括方法的组合)定义的CDR。各种编号***之间的对应关系是本领域技术人员熟知的，示例性地，如下表1中所示。
表1.CDR编号***之间的关系
CDR IMGT Kabat AbM Chothia Contact
HCDR1 27-38 31-35 26-35 26-32 30-35
HCDR2 56-65 50-65 50-58 52-56 47-58
HCDR3 105-117 95-102 95-102 95-102 93-101
LCDR1 27-38 24-34 24-34 24-34 30-36
LCDR2 56-65 50-56 50-56 50-56 46-55
LCDR3 105-117 89-97 89-97 89-97 89-96
本公开的抗体的VL区和VH区的CDR氨基酸残基在数量和位置符合已知的Kabat编号***。
本公开的抗体可以是多克隆的、单克隆的、异种的、同种异体的、同基因的或其经过修饰的形式，其中单克隆抗体尤其适用于多个实施例中。一般来说，本公开的抗体是重组抗体。
如本文所用的“重组”泛指例如细胞或核酸、蛋白质或载体等产品，表示所述细胞、核酸、蛋白质或载体已经通过引入异源核酸或蛋白质或改变天然核酸或蛋白质而加以修饰。例如，重组细胞表达天然(非重组)细胞形式内不存在的基因或表达原本异常表达、低表达或完全不表达的天然基因。
多肽或蛋白的“结构域”是指折叠蛋白结构，其能够独立于蛋白的其余部分维持其三级结构。一般而言，结构域负责蛋白的单个功能性质，且在许多情况下可添加、移除或转移至其它蛋白而不损失蛋白的其余部分和/或结构域的功能。
“免疫球蛋白结构域”是指抗体链的球形区域。免疫球蛋白结构域的特征在于其维持抗体分子的折叠特征。
“免疫球蛋白可变结构域”是指基本上由“框架区1”或“FR1”、“框架区2”或“FR2”、“框架区3”或“FR3”、及“框架区4”或“FR4”的四个“框架区”、“互补决定区1”或“CDR1”、“互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3”的三个“互补决定区”或“CDR”组成的免疫球蛋白结构域。因此，免疫球蛋白可变结构域的一 般结构或序列可如下表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。免疫球蛋白可变结构域因具有抗原结合位点而赋予其对抗原的特异性。
“抗体框架(FR)”，是指可变结构域的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。
“免疫球蛋白单一可变结构域”通常用于指可以在不与其他可变结构域相互作用的情况下(例如在没有如常规四链单克隆抗体的VH和VL结构域之间所需要的VH/VL相互作用的情况下)，形成功能性抗原结合位点的免疫球蛋白可变结构域(其可以是重链或轻链结构域，包括VH、VHH或VL结构域)。“免疫球蛋白单一可变结构域”的实例包括纳米抗体(包括VHH、人源化VHH和/或骆驼化VH，例如骆驼化人VH)、IgNAR、结构域、作为VH结构域或衍生自VH结构域的(单结构域)抗体(诸如dAbs TM)和作为VL结构域或衍生自VL结构域的(单结构域)抗体(诸如dAbs TM)。基于和/或衍生自重链可变结构域(诸如VH或VHH结构域)的免疫球蛋白单一可变结构域通常是优选的。免疫球蛋白单一可变结构域的一个具体实例为如下文定义的“VHH结构域”(或简称为“VHH”)。
“VHH结构域”，亦称为重链单域抗体、VHH、V HH结构域、VHH抗体片段、VHH抗体、纳米抗体，是称为“重链抗体”(即“缺乏轻链的抗体”)的抗原结合免疫球蛋白的可变结构域(Hamers-Casterman C，Atarhouch T，Muyldermans S，Robinson G，Hamers C，Songa EB，Bendahman N，Hamers R.:“Naturally occurring antibodies devoid of light chains”；Nature363，446-448(1993))。使用术语“VHH结构域”以将所述VHH与存在于常规四肽链结构抗体中的VH以及VL进行区分。VHH结构域特异性结合表位而无需其他抗原结合结构域(然而，常规四肽链结构抗体中，表位由VL结构域与VH结构域一起识别)。VHH结构域为由单一免疫球蛋白结构域形成的小型稳定及高效的抗原识别单元。术语“重链单域抗体”、“VHH结构域”、“VHH”、“V HH结构域”、“VHH抗体片段”、“VHH抗体”、
Figure PCTCN2022118979-appb-000001
以及“
Figure PCTCN2022118979-appb-000002
结构域”(“Nanobody”为Ablynx N.V.公司，Ghent，Belgium的商标)可互换使用。“VHH结构域”包括但不限于经骆驼科动物产生的天然抗体，也可以是骆驼科动物产生的抗体后再经人源化的，也可以是经噬菌体体展示技术筛选获得的。VHH结构域中的氨基酸残基的总数将通常在110至120范围内，常常介于112与115之间。然而应注意较小及较长序列也可适于本公开所述的目的。获得结合特定抗原或表位的VHH的方法，先前已公开于以下文献中：R.van der Linden et al.，Journal of Immunological Methods，240(2000)185-195；Li et al.，J Biol Chem.，287(2012)13713-13721；Deffar et al.，African Journal of Biotechnology Vol.8(12)，pp.2645-2652，17June，2009和WO94/04678。
如本领域中对于VH结构域及VHH结构域所公知的，各CDR中的氨基酸残基的总数可能不同，且可能不对应于由Kabat编号指示的氨基酸残基的总数(即根据Kabat编号的一个或多个位置可能在实际序列中未被占据，或实际序列可能含有 多于Kabat编号所允许数目的氨基酸残基)。这意味着一般而言，根据Kabat的编号可能对应或可能不对应于实际序列中氨基酸残基的实际编号。VHH的其它编号***或编码规则包括Chothia、IMGT、AbM。
“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将非人CDR序列移植到人的抗体可变区框架中产生的抗体。可以克服嵌合抗体由于携带大量非人蛋白成分，从而诱导的强烈的免疫应答反应。为避免在免疫原性下降的同时引起活性的下降，可对所述的全人抗体可变区可进行最少反向突变，以保持活性。“人源化”的例子包括：在和常规四肽链结构抗体VH结构域中相应位置处，将源自骆驼科的VHH结构域通过存在的一个或多个氨基酸残基置换原始VHH序列的氨基酸序列中的一个或多个氨基酸残基而“人源化”(本公开中亦称为“序列优化”，除人源化外，“序列优化”也可涵盖通过提供VHH改良性质的一个或多个突变对序列进行的其它修饰，例如移除潜在的翻译后修饰位点)。人源化VHH结构域可含有一个或多个完全人框架区序列。在一些具体实施方案中，可含IGHV3的人框架区序列。“人源化”的又一例子包括将小鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体构架序列中产生的抗体。可以克服嵌合抗体由于携带大量异源蛋白成分，从而诱导的强烈的抗体可变抗体反应。人源化方法例如蛋白表面氨基酸人源化(resurfacing)及抗体人源化通用框架移植法(CDR grafting to a universal framework)，即将CDR“移植”于其它“支架”(包括但不限于人支架或非免疫球蛋白支架)上。适于所述CDR移植的支架及技术在本领域中是已知的。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。本公开的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。此外，为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。
“亲和力成熟的”抗体指与亲本抗体相比，在一个或多个高变区(HVR)中具有一处或多处改变的抗体，此类改变导致该抗体对抗原的亲和力改善。例如，“亲和力成熟”的PD-1结合蛋白或PD-1抗体，在一个或多个CDR中具有一个或多个变化，所述变化导致对抗原的亲和力相比于其亲本抗体有所增加。亲和力成熟的抗体可通过例如由以下所述的本领域中已知的方法来制备：Marks等人，1992，Biotechnology 10：779-783或Barbas等人，1994，Proc.Nat.Acad.Sci，USA 91：3809-3813.；Shier等人，1995，Gene 169：147-155；Yelton等人，1995，Immunol.155：1994-2004；Jackson等人，1995，J.Immunol.154(7)：3310-9；及Hawkins等人，1992，J.MoI.Biol.226(3)：889896；KS Johnson及RE Hawkins，“Affinity maturation of antibodies using phage display”，Oxford University Press 1996。
“全人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本公开 的全人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。“全人抗体”不包括“人源化抗体”。
通常，“特异性结合”、“选择性结合”是指结合蛋白与抗原上的表位结合。本公开的PD-1结合蛋白、PD-1/PVRIG结合蛋白、PD-1/TIGIT结合蛋白、PD-1/PVRIG/TIGIT结合蛋白将以如于Biacore或KinExA或Fortibio测定中测量的优选10 -7至10 -10摩尔/升(M)、更优选10 -8至10 -10摩尔/升、甚至更优选10 -9至10 -10或更低的解离常数(K D)，和/或以至少10 -7M、优选至少10 -8M、更优选至少10 -9M，更优选至少10 -10M的缔合常数(KA)结合所要结合的抗原(即PD-1、PVRIG和/或TIGIT)或其表位。任何大于10 -4M的K D值一般都视为指示非特异性结合。结合蛋白对抗原或表位的特异性结合可以以已知的任何适合方式来测定，包括例如本公开所述的表面等离子体共振术(SPR)测定、Scatchard测定和/或竞争性结合测定(例如放射免疫测定(RIA)、酶免疫测定(EIA)及夹心式竞争性测定)。
“表位”是指抗原上与免疫球蛋白或抗体结合的位点。表位可以由相邻的氨基酸、或通过蛋白质的三级折叠而并列的不相邻的氨基酸形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象包括至少3-15个氨基酸。确定表位与给定的抗体结合的方法在本领域中是熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本公开所述的技术，例如X射线晶体分析法和二维核磁共振等。
“结合亲和力”或“亲和力”在本公开中用作两个分子(例如抗体或其部分与抗原)之间的非共价相互作用的强度量度。两个分子之间的结合亲和力可通过确定解离常数(K D)来量化。可通过使用例如表面等离子共振(SPR)方法(Biacore)测量复合物形成和解离的动力学来确定K D。对应于单价复合物的结合和解离的速率常数分别被称为结合速率常数ka(或kon)和解离速率常数kd(或koff)。K D通过方程K D＝kd/ka与ka和kd有关。解离常数的值可通过众所周知的方法直接确定，并且甚至可通过例如Caceci等人(1984，Byte 9：340-362)中所述的那些方法对于复杂混合物进行计算。例如，可使用双重过滤硝化纤维素滤器结合测定如Wong&Lohman(1993，Proc.Natl.Acad.Sci.USA 90：5428-5432)中公开的那种来确定K D。评估抗体针对靶抗原的结合能力的其它标准测定是本领域已知的，包括例如ELISA、蛋白质印迹、RIA和流式细胞术分析、以及本公开例举的其它测定。抗体的结合动力学和结合亲和力也可通过本领域已知的标准测定，例如表面等离子共振(SPR)，例如通过使用Biacore TM***或KinExA来评价。可通过比较各个抗体/抗原复合物的K D值来比较与不同分子相互作用相关的结合亲和力，例如，不同抗体对于给定抗原的结合亲和力的比较。类似地，相互作用的特异性可通过确定和比较目的相互作用(例如抗体和抗原之间的特异性相互作用)的K D值与非目 的相互作用(例如已知不结合PD-1的对照抗体)的K D值进行评价。
“保守性置换”指置换为具有与原始氨基酸残基相似的特性的另一个氨基酸残基。例如，赖氨酸、精氨酸和组氨酸具有相似的特性，在于它们具有碱性侧链，并且天冬氨酸和谷氨酸具有相似的特性，在于它们具有酸性侧链。此外，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸和色氨酸具有相似的特性，在于它们具有不带电荷极性侧链，并且丙氨酸、缬氨酸、亮氨酸、苏氨酸、异亮氨酸、脯氨酸、苯丙氨酸和甲硫氨酸具有相似的特性，在于它们具有非极性侧链。另外，酪氨酸、苯丙氨酸、色氨酸和组氨酸具有相似的特性，在于它们具有芳族侧链。因此，本领域技术人员将显而易见，甚至当置换如上文所述的显示相似特性的组中的氨基酸残基时，它将不显示特性的特定变化。
“同源性”、“同一性”或“序列同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同核苷酸或氨基酸单体占据时，例如如果两个DNA分子的每一个位置都被相同核苷酸占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100％的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
“核酸”或“多核苷酸”在本文可互换使用，指的是单链或双链的任何DNA分子或RNA分子以及在单链的情况下，它的互补序列的分子，优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“可操作连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子可操作地连接至所述编码序列。
“宿主细胞”包括各个细胞或细胞培养物，其可为或已是用于掺入多核苷酸***片段的载体的接受者。宿主细胞包括单个宿主细胞的子代，并且由于天然、偶然或有意的突变，子代不一定与亲本细胞完全相同(在形态学或基因组DNA互补体中)。宿主细胞包括用本公开的多核苷酸在体内转染和/或转化的细胞。“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括其后代。还应当理解的是，由于有意或非有意的突变，所有后代在DNA含量方面不可能精确相同。该术语包括与最初转化中筛选的细胞具有相同的功能或生物学活性的突变后代。
“抑制”或“阻断”可互换使用，并涵盖部分和完全抑制/阻断这两者。“抑制生长”(例如涉及细胞)旨在包括细胞生长任何可测量的降低。
“阻止...的生长”或“生长抑制”是指抑制细胞的生长或增殖。
“增殖性疾病”指与一定程度的异常细胞增殖有关的病症。在一个实施方案中，增殖性病症指癌症。
“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，及所有癌前(pre-cancerous)和癌性细胞和组织。“癌症”、“癌性”、“增殖性病 症”和“肿瘤”在本公开中提到时并不互相排斥。
“预防癌症”是指在受试者中延迟、抑制或防止癌症发作，所述受试者中癌症发生或肿瘤发生的起始尚未得到证实，但是通过例如遗传筛查或其它方法确定，已鉴定了癌症易感性。该还包括治疗具有癌变前病症的受试者以终止所述癌变前病症向恶性肿瘤的进展或导致其消退。
“给予”、“施用”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触，例如治疗、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，其中所述流体与细胞接触。“给予”、“施用”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例如细胞。当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予受试者内用或外用治疗剂，例如包含本公开的任一种结合蛋白或其药物组合物作为治疗剂，所述受试者已经患有、疑似患有、倾向于患有一种或多种增殖性疾病或其症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗受试者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床能测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如受试者的疾病状态、年龄和体重，以及药物在受试者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或产品)在缓解某个受试者中目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的受试者中应当减轻目标疾病症状。
“有效量”包含足以改善或预防医学病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于受试者的有效量可依据以下因素而变化：如待治疗的病症、受试者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。本公开的受试者可以是动物或人类受试者。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。“和/或”应视为特定揭示两种指定特征或组分中的每一者具有或不具有另一者。因此，诸如本公开中“A和/或B”的词组中所用的术语“和/或”包括“A及B”、“A或B”、“A”(单独)及“B”(单独)。除非上下文另外清楚要求，否则在整个说明书和权利要求书中，应将词语“包含”、“具有”、“包括”等理解为具有包含意义，而不是排他性或穷举性意义；也即，“包括但不仅 限于”的意义。
本公开的“受试者”、“患者”意指哺乳动物，尤其灵长类动物，尤其是人。
实施例
以下结合实施例用于进一步描述本公开，但这些实施例并非限制本公开的范围。
本公开实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，分子克隆，实验室手册，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.抗PD-1纳米抗体的筛选、制备和功能验证
本实施例使用带有His标签的人PD-1作为免疫抗原，使用带有生物素标记的人PD-1和食蟹猴PD-1作为筛选抗原，以上均购自AcroBiosystems公司。
实施例1-1.抗PD-1纳米抗体的筛选和制备
1.免疫抗原、筛选抗原序列和制备
表2.免疫羊驼和筛选用PD-1抗原信息
Figure PCTCN2022118979-appb-000003
人PD-1蛋白(NP_005009.2)序列如下，其中下划线为胞外区域序列，氨基酸起止Leu25-Gln 167。
>人PD-1氨基酸序列
Figure PCTCN2022118979-appb-000004
2.羊驼免疫、纳米抗体酵母展示文库构建和抗体筛选
1)羊驼免疫
对2只健康羊驼(alpaca)进行免疫。第0天初次免疫，将0.25mg人PD-1抗原与弗氏完全佐剂(CFA)1ml混匀后皮下注射。第21、42、63天，将0.125mg人PD-1抗原与弗氏不完全佐剂(IFA)1ml混匀后皮下注射。分别在第28天、49天、70天采血10ml、50ml、50ml。检测血清滴度，符合建库标准的羊驼外周 淋巴细胞用于构建纳米抗体酵母展示文库。
2)酵母库构建
分别采集2只羊驼第三次和第四次免疫后50mL外周血，分离出外周血中的PBMC，提取PBMC中的RNA，反转录，获取总cDNA。将cDNA作为巢氏PCR第一轮反应模板。经过两轮巢式PCR扩增，第二轮巢氏PCR产物片段与酵母文库载体(酵母文库载体pYDN3)连接，将连接产物电击转化进入酵母感受态细胞。采用菌落PCR方法验证***率和文库多样性。依据库转化子数、库***率和文库多样性分析测序结果表明，一只羊驼文库库容为9.28×10 7，另一只羊驼文库库容为1.54×10 8。
3)纳米抗体(VHH)筛选
对此酵母展示***采用流式细胞(FACS)分选，以及文库筛选与鉴定进行抗体的筛选。分选采用两轮分选步骤：第一轮分选采用生物素标记人PD-1蛋白(Avitag-His Tag)磁珠富集，第二轮采用生物素标记猴PD-1蛋白(His,Avitag)流式分选仪进行分选(流式抗体为：Mouse anti-HA tag Dylight 488；streptavidin Dylight 650)。两轮分选结束后经过单克隆鉴定、测序、序列分析获得人、猴PD-1抗原交叉结合的18条VHH独特序列。以下示出其中的A6和A17序列。
>A6可变区
Figure PCTCN2022118979-appb-000005
>A17可变区
Figure PCTCN2022118979-appb-000006
表3.抗PD-1纳米抗体的CDR(Kabat编号规则)
Figure PCTCN2022118979-appb-000007
将上述序列分别连接到人IgG4 Fc(包含铰链区，且带有S228P，根据Eu编号***)片段上，构建成VHH-Fc抗体。构建质粒，瞬转HEK293细胞，表达，使用protein A柱纯化，PBS洗柱，0.1M甘氨酸缓冲液洗脱，pH 2.5。透析至PBS pH7.4缓冲液中。其中人IgG4 Fc(包含铰链区)以及基于A6和A17的VHH-Fc抗体序列如下：
>hIgG4
Figure PCTCN2022118979-appb-000008
>A6_hIgG4
Figure PCTCN2022118979-appb-000009
>A17_hIgG4
Figure PCTCN2022118979-appb-000010
实施例1-2.抗PD-1纳米抗体与抗原PD-1的亲和力鉴定
1.ELISA
使用PBS溶解人PD-1蛋白(his tag)至1μg/mL，100uL/孔加入到96孔板中，4℃过夜，包被抗原。使用PBST(PBS+0.05％tween20)溶液洗三次。加入PBST/1％BSA溶液，37℃孵育1h，进行封闭。PBST溶液洗三次后加入不同浓度的候选PD-1VHH-Fc抗体(PBST/1％BSA溶液稀释)，37℃孵育1.5小时。PBST溶液洗三次。加入100μL HRP偶联的抗人IgG4 Fc的二抗(Thermo)溶液，37℃孵育1小时。PBST溶液洗三次后，加入100μL/孔TMB溶液，室温反应5min后，加入50μL终止液，而后使用酶标仪读取450nM值，计算EC 50，
结果如表4，显示A6_hIgG4和A17_hIgG4均与PD-1抗原有较强的结合力。
表4.ELISA检测抗PD-1纳米抗体的EC 50值
抗体编号 EC 50(nM)
A6_hIgG4 0.08605
A17_hIgG4 0.06099
2.FACS
为检测抗PD-1纳米抗体与细胞膜上表达的PD-1的结合能力，将不同浓度的候选抗PD-1VHH-Fc抗体加入到10 5/孔过表达人PD-1的Jurkat细胞(Cat：J1250，Promega)中，室温孵育20min后，PBS洗两次后，加入100μL抗人IgG Fc的荧 光二抗(Biolegend)，室温孵育20min后，PBS洗两次，250μL PBS重悬，使用FACS检测荧光信号，计算EC 50。
使用Pembrolizumab(购自百英生物)作为对照，结果如表5所示。并且，A17_hIgG4的EC 50值为0.3568nM，显著优于同步筛选获得的其它抗体(结果未出示)。
表5.FACS检测抗PD-1纳米抗体结合细胞表面PD-1的EC 50值
抗体编号 EC 50(nM)
A6_hIgG4 1.09
A17_hIgG4 0.3568
Pembrolizumab 0.2169
实施例1-3.抗PD-1纳米抗体的序列改造
1.抗PD-1纳米抗体的人源化和回复突变
抗体人源化通过CDR-grafting方法进行。通过IMGT或NCBI网站，将亲本抗PD-1抗体与IMGT或NCBI/igblast数据库中的全人源胚系基因进行比对，选择与PD-1纳米抗体高度同源的人胚系基因IGHV 3-23(IGHV3-23*01；IGHV3-23*04)作为人源化模板。移植CDR，保留纳米抗体的FR2中的4个关键残基(Y37、E44、R45、L47)，并对接近CDR区的关键核心残基，以及与CDR相互作用的Vernier位置残基进行回复突变，获得人源化抗体A17h1、A17h2、A17h3、A6h1。
>A17h1可变区
Figure PCTCN2022118979-appb-000011
>A17h2可变区
Figure PCTCN2022118979-appb-000012
>A17h3可变区
Figure PCTCN2022118979-appb-000013
>A6h1可变区
Figure PCTCN2022118979-appb-000014
2.去除TCE(T细胞表位)、降低抗体脱酰胺、降低抗体异构化改造
为降低抗体潜在的免疫原性风险，进行去除TCE改造。为提高抗体的成药性，进行抗体脱酰胺、抗体异构化位点的改造。获得如下序列：
>A17m01可变区
Figure PCTCN2022118979-appb-000015
>A17m02可变区
Figure PCTCN2022118979-appb-000016
>A17m03可变区
Figure PCTCN2022118979-appb-000017
>A17m04可变区
Figure PCTCN2022118979-appb-000018
>A17m05可变区
Figure PCTCN2022118979-appb-000019
>A17m06可变区
Figure PCTCN2022118979-appb-000020
>A17m07可变区
Figure PCTCN2022118979-appb-000021
>A17m08可变区
Figure PCTCN2022118979-appb-000022
>A17m09可变区
Figure PCTCN2022118979-appb-000023
>A17m10可变区
Figure PCTCN2022118979-appb-000024
>A17m11可变区
Figure PCTCN2022118979-appb-000025
Figure PCTCN2022118979-appb-000026
>A17m12可变区
Figure PCTCN2022118979-appb-000027
>A17m13可变区
Figure PCTCN2022118979-appb-000028
>A17m14可变区
Figure PCTCN2022118979-appb-000029
>A17m15可变区
Figure PCTCN2022118979-appb-000030
并且，进一步选择A17m09对抗体序列胚系化改造，以提高人源化程度，获得如下序列：
>A17m0901可变区
Figure PCTCN2022118979-appb-000031
>A17m0902可变区
Figure PCTCN2022118979-appb-000032
>A17m0903可变区
Figure PCTCN2022118979-appb-000033
>A17m0905可变区
Figure PCTCN2022118979-appb-000034
即，本公开的A17具有如下的通式序列：
CDR1：DYSMS(SEQ ID NO：7)
CDR2：IISGSGX 1X 2X 3HYVDSVKG，其中，X 1选自V或G，X 2选自I或S，X 3选自T或A(SEQ ID NO：36)
CDR3：VSDWX 4X 5Y，其中，X 4选自D或E，X 5选自D或E(SEQ ID NO：37)。
具体地，CDR2可以为：
IISGSGVIAHYVDSVKG(SEQ ID NO：8)
IISGSGVITHYVDSVKG(SEQ ID NO：38)
IISGSGGIAHYVDSVKG(SEQ ID NO：39)
IISGSGVSAHYVDSVKG(SEQ ID NO：40)
IISGSGGITHYVDSVKG(SEQ ID NO：101)
具体地，CDR3可以为：
VSDWDDY(SEQ ID NO：9)
VSDWEDY(SEQ ID NO：41)
VSDWDEY(SEQ ID NO：42)。
实施例1-4.PD-1/PD-L1报告基因***检测抗PD-1纳米抗体对PD-L1结合PD-1的阻断
使用PD-1/PD-L1 NFAT基因报告***(Cat：J1250，Promega)，可以检测抗PD-1抗体在细胞层面的生物学活性功能。该***由两个基因工程细胞系组成：过表达PD-1的效应细胞Jurkat(内含NFAT报告基因荧光素酶)和过表达PD-L1的抗原递呈细胞CHO-K1细胞。当这两种细胞共培养时，PD-1/PD-L1相互作用会抑制TCR介导的NFAT活化，进而抑制NFAT报告基因荧光素酶的表达。当PD-1/PD-L1相互作用被破坏时，TCR激活会通过NFAT通路诱导荧光素酶的表达，可以通过添加Bio-Glo试剂和使用光度计进行荧光定量检测，从而测定效应细胞活化程度以及抗体的生物学活性。
将过表达PD-L1的CHO-K1细胞稀释到4×10 5/mL，在96孔板中，每孔加入100μL，培养过夜。吸出培养基后，迅速加入40μL 1.25×10 6/mL的PD-1Jurkat细胞以及不同浓度的40μL抗PD-1纳米抗体溶液(1640+2％FBS溶液稀释)。37℃培养6小时，恢复至室温，每孔加入40μL Bright-glo试剂(Cat：E2620，promega)，350rpm避光震荡5min，避光，室温静置5min后，使用酶标仪读取荧光值，计算IC 50。
使用Pembrolizumab和hIgG4 isotype作为对照。如图1A和表6所示，A17_hIgG4的生物学功能活性IC 50值为1.199nM，显著优于本公开同步筛选的其它抗体(例如A3_hIgG4为7.62，A13_hIgG4为3.208；A3_hIgG4和A13_hIgG4未示出序列)。
表6.PD-1/PD-L1 NFAT报告基因***检测抗PD-1纳米抗体的活性IC 50值
抗体编号 IC 50(nM)
A6_hIgG4 1.354
A17_hIgG4 1.199
Pembrolizumab 0.5126
hIgG4 -
采用如上方法，对人源化的抗PD-1纳米抗体进行功能验证，参见图1B和表7，A17h1_hIgG4的IC 50活性与阳性抗体Pembrolizumab相似。经序列改造的候选分子中，A17m0901_hIgG4、A17m0902_hIgG4抗体的生物学活性IC 50值分别为0.5307nM、0.4352nM，优于Pembrolizumab。
表7.PD-1/PD-L1 NFAT报告基因***检测经改造的抗PD-1纳米抗体的活性IC 50
抗体编号 IC 50(nM)
A17h1_hIgG4 1.039
A17m09_hIgG4 1.083
A17m0901_hIgG4 0.5307
A17m0902_hIgG4 0.4352
A17m0903_hIgG4 1.284
A17m0905_hIgG4 0.5588
Pembrolizumab 0.9756
实施例1-5.经改造的抗PD-1纳米抗体与抗原PD-1的结合亲和力鉴定
使用Biacore 8k(GE Healthcare)仪器，采用表面等离子共振技术(surface plasmon resonance，SPR)检测抗PD-1纳米抗体与PD-1抗原的亲和力。实验选用CM5传感器芯片，流动相采用HBS-EP+缓冲溶液(10mM HEPES，150mM NaCl，3mM EDTA，0.05％surfactant P20)。将抗人IgG(Fc)抗体用10mM醋酸钠缓冲液(pH 5.0)，配制成30μg/mL溶液，选择Immobilization程序自动进行抗人IgG(Fc)抗体通道氨基偶联固定。用HBS-EP+缓冲溶液分别配制各待测抗体作为配体，用芯片通道上的抗人IgG(Fc)抗体进行捕获。将人或食蟹猴PD-1抗原蛋白(Sino Biological，10377-H08H；90311-C08H)作为分析物，用HBS-EP+缓冲溶液进行配制，分析物进行2倍梯度稀释，在30μL/min的流速下流过实验通道和参比通道，结合1min，解离15min。再生缓冲液10mM Glycine pH 1.5(GE Healthcare，BR-1003-54)在10μl/min的流速下运行30s，计算结合速率Ka和解离速率Kd，以及解离常数(即亲和力K D)。结果参见表8和表9。
表8.抗PD-1纳米抗体结合人PD-1抗原的SPR亲和力数据
抗体编号 ka(1/Ms) Kd(1/s) K D(M)
A17h1_hIgG4 4.30E+05 8.68E-04 2.02E-09
A17m09_hIgG4 4.58E+05 1.63E-03 3.56E-09
A17m0901_hIgG4 2.97E+05 1.92E-03 6.44E-09
A17m0902hIgG4 2.91E+05 1.77E-03 6.09E-09
Pembrolizumab 1.25E+06 3.89E-03 3.11E-09
表9.抗PD-1纳米抗体结合食蟹猴PD-1抗原的SPR亲和力数据
抗体编号 ka(1/Ms) Kd(1/s) K D(M)
A17h1_hIgG4 4.44E+05 8.07E-04 1.82E-09
A17m09_hIgG4 4.81E+05 8.43E-04 1.75E-09
A17m0901_hIgG4 3.40E+05 1.13E-03 3.32E-09
A17m0902_hIgG4 3.28E+05 1.08E-03 3.28E-09
Pembrolizumab 1.31E+06 1.15E-03 8.82E-10
结合人PD-1抗原结果显示，人源化抗体A17h1_hIgG4及后续改造优化的抗体A17m09_hIgG4、A17m0901_hIgG4、A17m0902_hIgG4与Pembrolizumab具有相似的K D值。A17h1_hIgG4、A17m09_hIgG4、A17m0901_hIgG4、A17m0902_hIgG4均比Pembrolizumab的解离速率慢。A17h1_hIgG4、A17m09_hIgG4、A17m0901_hIgG4、A17m0902_hIgG4抗体交叉结合猴PD-1，抗体与人和猴PD-1具有相似的亲和力。
实施例1-6.DC：T混合淋巴反应(Mix Lymphocyte Reaction，MLR)检验经改造的抗PD-1纳米抗体的体外药效
成熟的树突细胞(Dendritic cells，DC)与异体T细胞共培养，可以激活T细胞，促进T细胞分泌细胞因子。该过程受PD-1/PD-L1信号抑制，使用抗PD-1抗体可以解除抑制，促进T细胞活化。因此，使用DC：T混合淋巴反应实验，可以检验抗PD-1抗体的体外药效。
使用分选试剂盒(cat：19359，stemcell)从一个健康人的新鲜外周血(PBMC)中分选出单核细胞。使用DC细胞诱导试剂盒(cat：10985，stemcell)，培养七天后，诱导单核细胞分化为成熟DC细胞。而后使用分选试剂盒(cat：17951，stemcell)从另一个健康人的新鲜PBMC中分选出异体T细胞。将5000个成熟DC细胞与50000个异体T细胞共培养，并加入相应浓度的候选抗体，共培养六天后，吸取细胞上清，使用Cisbio-HTRF IFN-γ检测试剂盒(Cat：62HIFNGPEH，Perkinelmer)检测IFN-γ浓度，计算EC 50。
如图2所示，A17m09_hIgG4在MLR实验中，显示出与Pembrolizumab基本一致的体外药效。其中，A17m09_hIgG4的EC 50为0.2931nM，Pembrolizumab的EC 50为0.3271nM。
实施例1-7.抗PD-1纳米抗体抑制小鼠结肠癌模型中肿瘤生长
本实施例在人源化PD-1 C57BL/6小鼠的MC38肿瘤模型上，验证抗PD-1抗体的体内抗肿瘤药效。
使用DMEM培养基(10％FBS)培养MC38小鼠结肠癌细胞系，将5×10 5个MC38细胞接种于人源化PD-1C57BL/6雌鼠(集萃药康)皮下，待小鼠平均肿瘤体积达到80mm 3左右时，随机分组，每组8只，给予腹腔注射抗体或对照，每周两次测量肿瘤体积，称体重，记录数据。实验分组与给药方案参见表10。考虑到A17m09_hIgG4的分子量约75kDa，只有正常IgG分子量的一半，故给药剂量调 整为Pembrolizumab的一半，以实现等摩尔数剂量。
如图3A和图3B所示，在同等摩尔剂量下，A17m09_hIgG4的体内抗肿瘤药效与Pembrolizumab一致。肿瘤体积与肿瘤抑制率参见表11。
表10.小鼠实验分组与给药方案
Figure PCTCN2022118979-appb-000035
表11.小鼠肿瘤体积与肿瘤抑制率
Figure PCTCN2022118979-appb-000036
(注：i.p.：腹腔注射；BIW*7：每周2次，一共7次；与对照组比较，*P<0.05，**P<0.01，***P<0.001即认为具有显著性差异)
实施例2.抗PVRIG/TIGIT双特异性抗体
本实施例中，带his标签的人PVRIG(h-PVRIG-his)重组蛋白、带小鼠IgG2a的Fc标签的人PVRIG(h-PVRIG-mIgG2a Fc)重组蛋白、带人IgG1的Fc标签的小鼠PVRIG(m-PVRIG-hIgG1 Fc)购自Acrobiosystems公司。带his标签的PVRL2购自AcroBiosystem(PV2-H52E2)。
实施例2-1.抗PVRIG纳米抗体的筛选和制备
1.免疫抗原、筛选抗原的序列和制备
表12.重组蛋白质的氨基酸序列
名称 氨基酸序列起止 Genbank登录号
h-PVRIG-his Thr41-Asp171 Q6DKI7-1
h-PVRIG-mIgG2a Fc Thr41-Asp171 Q6DKI7-1
m-PVRIG-hIgG1 Fc Ser35-Asp165 A0A1B0GS01-1
带his标签的食蟹猴PVRIG(cyno-PVRIG-his)重组蛋白序列如下：
Figure PCTCN2022118979-appb-000037
2.羊驼免疫、纳米抗体噬菌体展示文库构建和抗体筛选
参照实施例1-1的方法，使用带his标签的人PVRIG重组蛋白(h-PVRIG-his)免疫羊驼。从第56天的骆驼外周血中分离PBMC，提取RNA，反转录成cDNA，构建抗人PVRIG纳米抗体的噬菌体文库。经过3轮筛选，对400个克隆测序，其中2株的可变区序列如下所示，CDR如表13所示。
>30可变区
Figure PCTCN2022118979-appb-000038
>151可变区
Figure PCTCN2022118979-appb-000039
表13.抗PVRIG纳米抗体的CDR(Kabat编号规则)
Figure PCTCN2022118979-appb-000040
将上述抗体可变区与人IgG4重链Fc区域连接，重链Fc区域包括铰链(hinge)区，并带有S228P，F234A，L235A，K447A突变(Eu命名***)，构建全长抗体。
>hIgG4 Fc(S228P/F234A/L235A/K447A)
Figure PCTCN2022118979-appb-000041
>30全长
Figure PCTCN2022118979-appb-000042
>151全长
Figure PCTCN2022118979-appb-000043
Figure PCTCN2022118979-appb-000044
WO2016134333中所示的抗PVRIG抗体CPA.7.021筛选自抗体噬菌体库，其亚型为IgG1，能与人PVRIG较好结合，对食蟹猴PVRIG则无结合。将CPA.7.021的重链和轻链可变区，分别与人IgG4重链恒定区(带有S228P，F234A，L235A，K447A突变)和人Kappa轻链恒定区连接，构建阳性抗体Tab5。
>Tab5重链全长
Figure PCTCN2022118979-appb-000045
>Tab5轻链全长
Figure PCTCN2022118979-appb-000046
将表达上述氨基酸序列的核酸序列克隆到pcDNA3.1表达载体，按常规方法进行抗体表达和纯化，经检测，获得目的抗体。
实施例2-2.抗PVRIG纳米抗体与抗原PVRIG的亲和力鉴定
1.ELISA
用直接包被带his标签的PVRIG重组蛋白，加入抗体后，通过加入二抗(HRP偶联的抗一抗Fc的抗体)和HRP底物TMB检测抗体与抗原结合的活性。
人、食蟹猴或小鼠PVRIG蛋白包被96孔板，按1μg/mL浓度每孔100μL，4℃孵育过夜。洗液洗三遍。加入300μL/孔封闭液(PBS+0.05％Tween20+1％BSA)室温孵育1小时。洗液洗三遍。每孔加100μL用稀释液稀释好的抗PVRIG待测抗体。37℃孵育1小时。洗液洗三遍。每孔加入100μL HRP标记的抗人IgG二抗(Sigma，A8667)。37℃孵育1小时。洗液洗三遍。每孔加入100μL TMB，避光反应15分钟。加入50mL/孔的0.16M硫酸。Thermo MμLtiSkanFc酶标仪读取OD450，计算抗PVRIG抗体对PVRIG重组蛋白的结合EC 50值。表14中所有抗体均对人或食蟹猴的PVRIG重组蛋白有较强的结合能力，但不结合小鼠PVRIG重组蛋白。
表14.抗PVRIG抗体对不同物种PVRIG重组蛋白的结合实验结果
抗体编号 人PVRIG-his ELISA EC 50(nM) 猴PVRIG-his ELISA EC 50(nM)
30 0.26 0.16
151 2.15 2.43
Tab5 2.86 无结合
hIgG4 无结合 无结合
2.FACS检测
制备获得表达人或食蟹猴PVRIG基因的HEK293稳转细胞株。在96孔板中每孔接种2x10 5个细胞。300g离心5分钟，去上清，加入100μL待测抗体，4℃孵育1小时。离心去除上清，用200μL洗液(PBS+2％FBS)洗涤3次，加入100μL 1：500稀释的用Alexa Fluor 488标记的抗人IgG二抗(Invitrogen，A-11013)，4℃孵育1小时。离心去除上清，用200μL洗液(PBS+2％FBS)洗涤3次。用100μL PBS重悬细胞，用流式细胞仪(BD FACS Calibur或BD FACS Canto_II)检测。所有抗体均对细胞表面表达的人或食蟹猴的PVRIG有较强的结合能力，明显强于阳性抗体Tab5，而Tab5甚至完全不结合食蟹猴PVRIG。
表15.抗PVRIG抗体对不同物种PVRIG的细胞结合实验结果
抗体编号 人PVRIG FACS EC 50(nM) 猴PVRIG FACS EC 50(nM)
30 N.A. 0.02
151 0.01 2.23
Tab5 2.13 无结合
hIgG4 无结合 无结合
(注：N.A.，not available，表示结合太强，在低浓度条件下抗体亦不发生解离，无法拟合得到准确的EC 50)
3.Fortebio检测
将Protein A生物传感器(Fortebio，#18-5010)浸泡在200μL的KB缓冲液(PBS，pH 7.4，0.02％tween-20，0.1％BSA)中60秒，进行湿润处理。然后，用KB缓冲液将抗PVRIG抗体稀释到10μg/mL，将传感器置于200μL该溶液中，待读数为1.2nm时停止。将传感器浸泡于KB缓冲液中100秒，以洗脱多余的抗体。将带有his标签的人PVRIG用KB缓冲液以2倍梯度稀释至64nM-4nM之间。将传感器置于该溶液中结合300秒，再置于KB缓冲液中解离600秒。采用动态1：1结合方式拟合，则抗PVRIG抗体与人PVRIG的亲和力如表16所示。
结果显示，所有检测抗体均具有与人PVRIG的高亲和力。
表16.抗PVRIG抗体与人PVRIG的亲和力
抗体编号 Kon(1/Ms) Koff(1/s) KD(M)
30 2.84E+05 2.05E-04 7.23E-10
151 2.61E+05 5.22E-05 2.00E-10
Tab5 7.37E+05 1.61E-05 2.19E-10
实施例2-3.抗PVRIG纳米抗体的功能和活性验证
1.抗PVRIG纳米抗体阻断PVRIG和PVRL2结合实验
人PVRIG重组蛋白(h-PVRIG-mIgG2a Fc)包被96孔板，按1μg/mL浓度每孔100μL，4℃孵育过夜。洗液洗三遍。加入300μL/孔封闭液室温孵育1小时。洗液洗三遍。每孔加50μL稀释好的抗PVRIG待测抗体和50μL带his标签的配体PVRL2，37℃孵育1小时。洗液洗三遍。每孔加入100μL按1：2000倍稀释的用HRP标记的抗his标签的二抗(Genscrpit)。37℃孵育1小时。洗液洗三遍。每孔加入100μL TMB，避光反应15分钟。加入50μL每孔的0.16M硫酸。Thermo MμLtiSkanFc酶标仪读取450nm OD值，计算抗PVRIG抗体对PVRIG与PVRL2结合阻断的IC 50值。
表17结果显示，所检测抗体均可以强烈抑制人PVRIG与人PVRL2的结合。
表17.抗体对人PVRIG/PVRL2结合的阻断实验
抗体编号 ELISA IC 50(nM)
30 1.11
151 0.37
Tab5 1.16
hIgG4 无阻断
2.抗PVRIG纳米抗体报告基因细胞活性实验
首先，构建plvx-OS8(G418抗性)质粒，转染293F细胞，G418筛选，用流式细胞仪检测克隆细胞OS8的表达同时检测OS8对Jurkat细胞的激活，选择激活程度中等的克隆，得到293F-OS8细胞株；构建plvx-PVRL2质粒，用它感染293F-OS8细胞，用流式细胞仪筛选出PVRL2表达量最高的克隆，从而得到293F-OS8-PVRL2细胞株。
其次，构建plvx-NFAT-Luc(Hygromycin抗性)，包装成慢病毒，感染Jurkat E6.1细胞，加Hygromycin筛选出有抗性的克隆，用OKT3去刺激克隆，筛选出Luciferase信号中等的克隆，得到Jurkat-NFAT-Luc细胞系；构建plvx-PVRIG(Puromycin抗性)载体，包装成慢病毒，感染Jurkat-NFAT-Luc细胞，经流式细胞仪筛选出PVRIG表达量最高的克隆，从而得到Jurkat-NFAT-Luc-PVRIG细胞株。
将1E4个Jurkat-NFAT-Luc-PVRIG细胞与待测抗体在37℃孵育20分钟。加入1E5个293F-OS8-PVRL2细胞，37℃孵育5小时。离心去除上清，加入Luciferase缓冲液(Promega，E6130)裂解细胞，检测荧光值。计算EC 50值评价抗PVRIG抗体的体外细胞活性。实验结果如表18所示。
结果显示，所检测抗体有较强的激活Jurkat细胞中Luciferase的能力，活性是阳性抗体的至少10倍以上，证明这些抗体可以结合PVRIG并阻断PVRL2与PVRIG的结合。
表18.抗PVRIG抗体报告基因细胞活性实验结果
抗体编号 PVRIG报告基因细胞活性实验EC 50(nM)
30 0.06
151 0.04
Tab5 0.74
hIgG4 无结合
3.抗PVRIG纳米抗体的NK细胞杀伤实验
PVRIG在NK细胞上表达，而PVRL2在很多肿瘤细胞(包括K562细胞)中表达。抗PVRIG抗体可以通过阻断PVRL2与PVRIG的结合，解除肿瘤细胞对NK细胞活性的抑制作用。
在96孔板中每孔加入50μL(总计1×10 5个)人恶性非霍奇金淋巴瘤NK92细胞。加入50μL 20nM或100nM待测抗体，37℃孵育30分钟。用洗液洗涤两次，重悬至2×10 5个/mL的密度。加入50μL(总计1×10 4个)的人慢性髓系白血病K562细胞，使得NK92细胞与K562细胞个数的比例为10：1。37℃孵育4小时。使用CytoTox-Glo细胞毒性***(Promega，G9292)对杀伤活性进行测量。首先加入50μL AAF-Glo试剂，室温孵育15分钟，测量被NK92细胞杀死的K562细胞的荧光。再加入50μL裂解液，室温孵育15分钟，裂解细胞，测量所有细胞的荧光。准备三种对照组，分别是只包括培养液的样品(对照组一)，只包括NK92细胞的样品(对照组二)，只包括K562细胞的样品(对照组三)，进行同样的操作。
根据如下公式，计算杀伤活性：
杀伤活性(％)＝{[(R–BG)–(T–BG)–(E–BG)]/[(TL–BGL)–(T–BG)]}×100；
其中，R为加入AAF-Glo后的荧光值，BG为对照组一在加入AAF-Glo的荧光值，E为对照组二在加入AAF-Glo的荧光值，T为对照组三在加入AAF-Glo的荧光值；TL为对照组三在加入裂解液后的荧光值，BGL为对照组一再加入裂解液后的荧光值。
实验结果如表19所示，表明所有检测的抗PVRIG抗体均可以明显的激活NK92细胞、杀伤K562细胞。
表19.抗PVRIG抗体的NK细胞杀伤实验
Figure PCTCN2022118979-appb-000047
4.抗PVRIG纳米抗体的混合淋巴细胞反应(MLR)实验
PVRIG在T细胞上表达，而PVRL2在DC细胞上表达。抗PVRIG抗体可以 通过阻断PVRL2与PVRIG的结合，解除DC细胞对T细胞的抑制，活化T细胞。
从第一个体来源的外周血中分离PBMC，将细胞培养于含10％FBS的RPMI 1640培养基中，以50ng/mL GM-CSF(Peprotech，300-03-100UG)和50ng/mL IL-4(Peprotech，200-04-100UG)的终浓度添加，每2-3天添加含细胞因子的新鲜培养基；培养6天后，加入1μg/mL LPS(Sigma，L2880-25MG)孵育24小时，收集分化成熟得到的DC细胞。从第二个体来源的外周血中分离PBMC，使用EasySep人CD3 +T细胞分离试剂盒(Stemcell，17952)从中分离CD3 +T细胞。调整CD3 +T细胞和DC细胞的密度，使得每孔加入1×10 5个CD3 +T细胞和2×10 4个DC细胞。加入待测抗体，37℃孵育120小时，取上清，用ELISA试剂盒(R&D，DY202)检测上清中的IFNγ含量。
如表20和图4所示，相比对照抗体IgG4，所有检测抗PVRIG抗体均可以明显的激活T细胞分泌IFNγ。并且，在低剂量(如4nM、20nM)时，本公开的抗体30和151较之阳性对照Tab5的效果更优。
表20.抗PVRIG抗体混合淋巴细胞反应IFNγ分泌量
Figure PCTCN2022118979-appb-000048
实施例2-4.抗PVRIG纳米抗体的序列改造
通过对选定的抗PVRIG抗体分子进行三维结构同源建模，将抗PVRIG抗体序列与抗体GermLine数据库比较，获得同源性高的人种系模板IGHV3-7*01。将CDR移植到相应的人源模板中。对移植后的纳米抗体再次进行三维结构模拟并分析，对包埋残基、与CDR区有直接相互作用的残基，以及对可变区的构象有重要影响的残基进行回复突变，并对CDR区化学不稳定氨基酸残基优化，产生一系列人源化纳米抗体。各个纳米抗体的人种系模板以及人源化抗体重链可变区序列如下所示。
>30H1可变区
Figure PCTCN2022118979-appb-000049
>30H2可变区
Figure PCTCN2022118979-appb-000050
>30H3可变区
Figure PCTCN2022118979-appb-000051
>30H4可变区
Figure PCTCN2022118979-appb-000052
>30H5可变区
Figure PCTCN2022118979-appb-000053
经人源化后，抗体30H1至30H5包含如GDCMG(SEQ ID NO：46)所示的CDR1，如TIDNAGRIKYADSVKG(SEQ ID NO：47)所示的CDR2，如GWTFGGQCSPAD(SEQ ID NO：99)所示的CDR3。
>151H2可变区
Figure PCTCN2022118979-appb-000054
>151H4可变区
Figure PCTCN2022118979-appb-000055
>151H7可变区
Figure PCTCN2022118979-appb-000056
>151H8可变区
Figure PCTCN2022118979-appb-000057
>151H9可变区
Figure PCTCN2022118979-appb-000058
将上述人源化抗体重链可变区与人IgG4重链Fc区域连接，构造形成全长抗PVRIG抗体。其中重链Fc区域包括铰链(hinge)区，并带有S228P/F234A/L235A/K447A，或S228P/K447A突变。按常规方法进行抗体的表达和纯化，经检测，得到目的抗体。
实施例2-5.人源化抗PVRIG抗体的活性和功能验证
1.人源化抗PVRIG抗体与表达PVRIG的细胞结合实验
依照实施例2-2的方法，FACS检测抗PVRIG抗体与人或食蟹猴PVRIG的结合。实验结果如表21所示。
表21.抗PVRIG纳米抗体对不同物种PVRIG的FACS结合实验结果
抗体编号 人PVRIG FACS EC 50(nM) 猴PVRIG FACS EC 50(nM)
30H1 0.024 0.374
30H2 0.003 0.005
30H3 0.004 0.003
151H4 0.240 0.035
151H7 0.002 0.467
151H8 0.006 N.T.
151H9 0.004 3.942
Tab5 0.160 无结合
hIgG4 无结合 无结合
(注：N.T.，not tested，未测试)
2.人源化抗PVRIG抗体与PVRIG的亲和力测定
依照实施例2-2的Fortebio检测方法，检测人源化抗PVRIG抗体与人PVRIG蛋白的亲和力。如表22所示，所有抗体均具有与人PVRIG蛋白的高亲和力。
表22.人源化抗PVRIG抗体与人PVRIG的亲和力
抗体编号 Kon(1/Ms) Koff(1/s) K D(M)
20H5 1.93E+05 1.35E-05 6.98E-11
30H2 1.69E+05 3.25E-04 1.92E-09
30H3 1.48E+05 3.58E-04 2.41E-09
151H7 1.57E+05 1.88E-04 1.20E-09
3.人源化抗PVRIG抗体报告基因细胞活性实验
依照实施例2-3的方法，检测人源化抗PVRIG抗体在报告基因细胞中的活性。实验结果如表23所示。表中列出的抗体均具有激活Jurkat细胞的能力。
表23.人源化抗PVRIG抗体报告基因细胞活性实验
抗体编号 PVRIG报告基因细胞活性实验EC 50(nM)
30H2 0.176
30H3 0.078
151H7 0.038
151H8 0.058
Tab5 1.380
hIgG4 无激活
4.人源化抗PVRIG抗体的活化NK细胞杀伤能力实验
依照实施例2-3的方法，检测人源化抗PVRIG抗体对NK细胞的活化能力。实验结果如表24和表25所示。结果显示，所测抗体都有明显的活化NK细胞的能力，促进NK细胞对于靶细胞K562的杀伤。
表24.人源化抗PVRIG抗体的NK细胞杀伤实验
Figure PCTCN2022118979-appb-000059
表25.人源化抗PVRIG抗体的NK细胞杀伤实验
Figure PCTCN2022118979-appb-000060
实施例2-6.抗PVRIG/TIGIT双特异性抗体的制备
为探索不同构造的抗PVRIG/TIGIT双特异性抗体对抗体功能的影响，将抗PVRIG纳米抗体151通过GGGGSGGGGS(SEQ ID NO：100)连接子与抗TIGIT抗体1708的重链或轻链的N端或C端相连。形成4个抗PVRIG/TIGIT双特异性抗体，命名为1708-151-1，1708-151-2，1708-151-3，1708-151-4，分别对应151被连接在1708的重链N端，重链C端，轻链N端和轻链C端。抗TIGIT抗体1708采用人IgG4亚型，并带有S228P(Eu命名***)的突变。抗TIGIT抗体1708和其与151形成的双特异性抗体序列如下表26所示。抗TIGIT抗体序列信息如表27和表28所示。TIGIT抗体已经在WO2019062832A中公开，通过引用全文并入。
表26.抗PVRIG/TIGIT双特异性抗体的第一、第二多肽链序列
Figure PCTCN2022118979-appb-000061
Figure PCTCN2022118979-appb-000062
Figure PCTCN2022118979-appb-000063
表27.抗TIGIT抗体重链及轻链CDR区序列(Kabat编号规则)
Figure PCTCN2022118979-appb-000064
表28.抗TIGIT抗体重链VH及轻链VL序列
Figure PCTCN2022118979-appb-000065
将不同的人源化抗PVRIG抗体可变区(30H2，151H7，151H8)连接到抗TIGIT抗体1708的重链N端，即采用1708-151-1类似的双特异性抗体构造，构建双特异性抗体。以下序列中双下划线为连接子序列。
>1708-30H2第一多肽链
Figure PCTCN2022118979-appb-000066
Figure PCTCN2022118979-appb-000067
>1708-151H7第一多肽链
Figure PCTCN2022118979-appb-000068
>1708-151H8第一多肽链
Figure PCTCN2022118979-appb-000069
1708-30H2、1708-151H7、1708-151H8的第二多肽链均与1708的轻链相同(SEQ ID NO：68)。
按常规方法进行抗体的瞬时转染、表达和纯化，经鉴定，得到本公开的全长抗PVRIG/TIGIT双特异性抗体，其均显示了良好的表达量和纯度。
实施例2-7.抗PVRIG/TIGIT双特异性抗体的活性和功能验证
1.双特异性抗体与人PVRIG的结合及对配体PVRL2的阻断
依照实施例2-2和2-3的方法，进行实验。结果表明，不同构型的双特异性抗体1708-151-1、1708-151-2、1708-151-3、1708-151-4，与人PVRIG重组蛋白和过表达人PVRIG细胞的结合，以及对PVRL2结合PVRIG的阻断，无统计学显著差异。
2.双特异性抗体与人TIGIT的结合及对配体PVR的阻断
依照实施例2-2和2-3的方法(相应的受体和配体换为人TIGIT和人PVR)，进行实验，结果如表29所示。结果表明，不同构型的双特异性抗体1708-151-1、1708-151-2、1708-151-3、1708-151-4和抗TIGIT抗体，与人TIGIT重组蛋白和过表达人TIGIT细胞的结合，以及对TIGIT结合其配体PVR的阻断，无统计学显著差异。
可见，抗PVRIG抗体无论是连接到抗TIGIT抗体的重、轻链的N端或C端，都保持了对PVRIG和TIGIT的结合和配体的阻断，并且都显示了良好的表达量、纯度。
3.人源化抗PVRIG/TIGIT双特异性抗体与PVRIG和TIGIT的结合以及对相应配体的阻断
依照实施例2-2和2-3的方法，检测人源化抗PVRIG/TIGIT双特异性抗体对人和食蟹猴PVRIG的结合，对人PVRIG的配体阻断。结果如表29所示。结果表明，各双特异性抗体均可以结合人PVRIG，阻断PVRIG结合PVRL2。
表29.人源化双特异性抗体对PVRIG的结合和配体阻断
Figure PCTCN2022118979-appb-000070
与实施例2-2和2-3类似地，检测人源化抗PVRIG/TIGIT双特异性抗体对人和食蟹猴TIGIT的结合，对人TIGIT与配体结合的阻断，其中将PVRIG蛋白替换为TIGIT，并将PVRL2替换为PVR。结果如表30所示。结果表明，各双特异性抗体均可以结合人的TIGIT，阻断TIGIT结合PVR。
表30.人源化双特异性抗体对TIGIT的结合和配体阻断
Figure PCTCN2022118979-appb-000071
利用Biacore检测双特异性抗体与人PVRIG、与人TIGIT的亲和力。将人源化双特异性抗体捕获于Biacore仪器(Biacore X100，GE)的Protein A生物传感芯片(GE lifesciences，29127557)上，然后于芯片表面流经一系列浓度梯度下的人 PVRIG抗原(AcroBiosystem，PVG-H52H4)或人TIGIT抗原(AcroBiosystem，TIT-H52H3)，获得结合和解离曲线。结果见表31。
表31.人源化双特异性抗体与人PVRIG、人TIGIT的亲和力
Figure PCTCN2022118979-appb-000072
4.抗PVRIG/TIGIT双特异性抗体的混合淋巴细胞反应(MLR)实验
依照实施例2-3第4部分的方法，检测人源化抗PVRIG/TIGIT双特异性抗体对T细胞的活化能力。实验结果如图5和表32所示。结果显示，人源化抗PVRIG/TIGIT双特异性抗体1708-151H8具有明显的活化T细胞的能力，促进T细胞分泌IFNγ。重要的是，双特异性抗体的活性强于单用抗PVRIG抗体151H8，单用抗TIGIT抗体1708。
表32.人源化双特异性抗体混合淋巴细胞反应IFNγ分泌量
Figure PCTCN2022118979-appb-000073
实施例2-8.抗PVRIG/TIGIT双特异性抗体体在人黑色素瘤A375混合人PBMC的小鼠皮下移植瘤模型中的抗肿瘤作用评估
为进一步探究双特异性抗体亚型在动物药效中的作用，进行动物药效试验。其中，1708-IgG1的重链可变区与1708相同，只是重链恒定区亚型换为IgG1；1708-IgG1的轻链全长和1708-151-IgG1的第二多肽链均与与1708的轻链相同。
NCG小鼠，雌性，4-8周，体重约18-22g，购自江苏集萃药康生物科技有限公司。所有的NCG小鼠按照SPF级动物房IVC恒温恒压***条件培养。
A375细胞培养在含10％胎牛血清(FBS)的DMEM培养液中。收集指数生长期的A375细胞，HBSS重悬至适合浓度用于NCG小鼠皮下肿瘤接种。共培养所用的A375细胞需经过Mitomycin C处理2h后，PBS洗三次。取正常人外周血，用密度梯度离心法分离人PBMC，计数。然后用RPMI1640培养基(含IL2和10％FBS)将PBMC重悬至3×10 6个/mL的浓度，与Mitomycin C处理后的A375细胞共培养。共培养6天后，收取PBMC，同时收取新鲜消化下来的A375细胞。每只老鼠接种：PBMC 5×10 5个，A375细胞4×10 6个；接种体积：0.2mL/只(含50％ Matrigel)；接种于雌性NCG小鼠右侧皮下。根据小鼠体重随机进行分组给药，详细的给药方法、给药剂量和给药途径见表33，分组给药当天为第0天。由于抗PVRIG抗体和抗TIGIT抗体的分子量不同，该给药剂量保证了抗PVRIG抗体与抗TIGIT抗体拥有同样的起始摩尔浓度。
表33.给药方案
Figure PCTCN2022118979-appb-000074
(注：N：使用动物数量；i.p.：腹腔注射；Q2D：两天一次；给药体积：根据荷瘤鼠体重调整给药体积(0.1mL/10g))
给药开始后，小鼠每周2次测量肿瘤体积及体重。实验结果分别见表34和图6A、图6B。
表34.抗PVRIG/TIGIT双特异性抗体在小鼠人源A375肿瘤模型中的抑瘤效果
Figure PCTCN2022118979-appb-000075
(注：与对照组(hIgG1)组比较，*P<0.05，**P<0.01，***P<0.001即认为具有显著性差异)
实验结束时(给药后第26天)，与对照组相比，抗PVRIG抗体151-IgG4单药组没有明显差异。抗TIGIT抗体1708-IgG1单药组，抗PVRIG抗体151-IgG4与抗TIGIT抗体1708-IgG1联用组，肿瘤体积下降。而1708-151-IgG4双特异性抗体组甚至可以完全抑制肿瘤的生长，与其它组间具有显著性差异(见图6A)。
根据小鼠体重随机进行分组给药，详细的给药方法、给药剂量和给药途径见表35，分组给药当天为第0天。
表35.给药方案
Figure PCTCN2022118979-appb-000076
(注：N：使用动物数量；i.p.：腹腔注射；Q2D：两天一次；给药体积：根据荷瘤鼠体重调整 给药体积(0.1mL/10g))
给药开始后，小鼠每周2次测量体重及肿瘤体积。实验结果分别见表36和图7A-图7B。
表36.抗PVRIG/TIGIT双特异性抗体在小鼠人源A375肿瘤模型中的抑瘤效果
Figure PCTCN2022118979-appb-000077
(注：与对照组(hIgG1)组比较，*P<0.05，**P<0.01，***P<0.001即认为具有显著性差异)
实验结束时(给药后第28天)，与对照组相比，1708-30H2与1708-151H7双特异性抗体组均可以在低剂量下有效抑制肿瘤生长，与对照组间具有显著性差异(见图7A、图7B)。
实施例3.抗PD-1/PVRIG/TIGIT三特异性抗体
实施例3-1.抗PD-1/PVRIG/TIGIT三特异性抗体的设计和制备
四种类型的三特异性抗体分为A，B，C，D四种类型，示意如图8。
A型：抗PD-1纳米抗体通过连接子(如GGGGSGGGGS)连接抗PVRIG纳米抗体，再经连接子连接在抗TIGIT抗体的重链N端(图8的A)。
B型：抗PVRIG抗体经连接子连接在TIGIT IgG分子的重链N端，而抗PD-1抗体经连接子连接在抗TIGIT抗体的轻链N端(图8的B)。
C型：抗PVRIG抗体经连接子连接在TIGIT IgG分子的重链N端。抗PD-1抗体经连接子(GGGGSGGGGS)连接在抗TIGIT抗体的重链C端(图8的C)。
D型：抗-PVRIG抗体经连接子连接在TIGIT IgG分子的重链N端。抗PD-1抗体经连接子连接在抗TIGIT抗体轻链C端(图8的D)。
本公开中三特异性抗体命名如表37。例如A17m0902-1708-30H2-A，代表使用的抗PD-1、TIGIT和PVRIG抗体的克隆分别为A17m0902、1708和30H2，三特异性抗体类型为A型。
表37.抗PD-1/PVRIG/TIGIT三特异性抗体命名
Figure PCTCN2022118979-appb-000078
Figure PCTCN2022118979-appb-000079
表37中A17h1相关分子，均为在对应的A17m0902相关分子的基础上，将A17m0902(SEQ ID NO：33)序列替换为A17h1(SEQ ID NO：13)序列。
序列如下(下划线为连接子，斜体为Fc或轻链Cκ)：
>A17m0902-1708-30H2-A第一多肽链
Figure PCTCN2022118979-appb-000080
A17m0902-1708-30H2-A第二多肽链如SEQ ID NO：68所示。
A17m0902-1708-30H2-B第一多肽链如SEQ ID NO：84所示。
>A17m0902-1708-30H2-B第二多肽链
Figure PCTCN2022118979-appb-000081
>A17m0902-1708-30H2-C第一多肽链
Figure PCTCN2022118979-appb-000082
A17m0902-1708-30H2-C第二多肽链如SEQ ID NO：68所示。
A17m0902-1708-30H2-D第一多肽链如SEQ ID NO：84所示。
>A17m0902-1708-30H2-D第二多肽链
Figure PCTCN2022118979-appb-000083
>A17m0902-1708-151H7-A第一多肽链
Figure PCTCN2022118979-appb-000084
A17m0902-1708-151H7-A第二多肽链如SEQ ID NO：68所示。
A17m0902-1708-151H7-B第一多肽链如SEQ ID NO：85所示。
>A17m0902-1708-151H7-B第二多肽链
Figure PCTCN2022118979-appb-000085
>A17m0902-1708-151H7-C第一多肽链
Figure PCTCN2022118979-appb-000086
Figure PCTCN2022118979-appb-000087
A17m0902-1708-151H7-C第二多肽链如SEQ ID NO：68所示。
A17m0902-1708-151H7-D第一多肽链如SEQ ID NO：85所示。
>A17m0902-1708-151H7-D第二多肽链
Figure PCTCN2022118979-appb-000088
经常规方法转染细胞，表达，纯化，检测后能够获得目的抗体。
实施例3-2.抗PD-1/PVRIG/TIGIT三特异性抗体与抗原的结合亲和力
采用ELISA进行检测。PBS溶解人TIGIT或PVRIG蛋白(his tag，Acrobiosystems，PVG-H52H4)至1μg/mL，将100μL/孔该溶液加入到96孔板中，4℃过夜，包被抗原。用PBST溶液洗三次。加入PBST/1％BSA溶液，37℃孵育1h，进行封闭。PBST溶液洗三次后加入不同浓度的三特异性抗体(PBST/1％BSA溶液稀释)，37℃孵育1.5h。PBST溶液洗三次。加入100μL HRP耦联的抗人IgG4Fc的二抗(Thermo)溶液，37℃孵育1h。PBST溶液洗三次后，加入100μL/孔TMB溶液，室温反应5min后，加入50μL终止液，使用酶标仪读取OD450值，计算EC 50。
1.与TIGIT的结合
四种构型的A17h1-1708-30H2、A17h1-1708-151H7三特异性抗体与TIGIT均有较强结合，结合能力差异较小，且与1708-30H2、1708-151H7双特异性抗体对TIGIT的结合能力相似。结果参见图9A、图9B和表38。因此，所有三特异性抗体构型不影响其结合TIGIT的结合。
表38.三特异性抗体结合TIGIT抗原的EC 50值
抗体编号 结合人TIGIT的EC 50(nM)
A17h1-1708-30H2-A 0.08749
A17h1-1708-30H2-B 0.1071
A17h1-1708-30H2-C 0.09511
A17h1-1708-30H2-D 0.1125
1708-30H2 0.1156
1708-151H7 0.1175
A17h1-1708-151H7-A 0.1859
A17h1-1708-151H7-B 0.1293
A17h1-1708-151H7-C 0.148
A17h1-1708-151H7-D 0.1624
2.与PVRIG的结合
四种构型的A17h1-1708-30H2、A17h1-1708-151H7三特异性抗体与PVRIG均有较强结合，结合能力差异较小，且与1708-30H2、1708-151H7双特异性抗体对PVRIG的结合能力相似。参见表39和图10A、图10B。因此，所有三特异性抗体构型不影响其结合PVRIG的结合。
表39.三特异性抗体结合PVRIG抗原的EC 50值
抗体编号 结合人PVRIG的EC 50(nM)
A17h1-1708-30H2-A 0.05891
A17h1-1708-30H2-B 0.05834
A17h1-1708-30H2-C 0.08216
A17h1-1708-30H2-D 0.08852
1708-30H2 0.08462
1708-151H7 0.1249
A17h1-1708-151H7-A 0.09519
A17h1-1708-151H7-B 0.1543
A17h1-1708-151H7-C 0.1596
A17h1-1708-151H7-D 0.1578
实施例3-3：抗PD-1/PVRIG/TIGIT三特异性抗体中PVRIG序列优化
PD-1/PVRIG/TIGIT三特异性抗体A17m0902-1708-151H7-C设计中，原PVRIG抗体序列(151H7)的第一位氨基酸为组氨酸(单字母缩写为H)，位于三特异性抗体分子序列N端的组氨酸在实际抗体生产过程中可能会发生磷酸吡哆醛的修饰和脱磷酸后的吡哆醛修饰，基于此问题的考虑需要对原PVRIG序列第一位组氨酸进行突变。由于谷氨酸(单字母缩写为E)和谷氨酰胺(单字母缩写为Q)为纳米抗体序列中第一位出现频率最高的两个氨基酸，因此将H突变为E或者Q。PVRIG抗体151H7的突变序列如下：
>151H7 H1E
Figure PCTCN2022118979-appb-000089
>151H7 H1Q
Figure PCTCN2022118979-appb-000090
将PVRIG抗体(151H7)序列第一位氨基酸由H变为E的三特异性抗体分子命名为A17m0902-1708-151H7-01-C；将PVRIG(151H7)序列第一位氨基酸由H变为Q的三特异性抗体分子命名为A17m0902-1708-151H7-02-C。
A17m0902-1708-151H7-01-C和A17m0902-1708-151H7-02-C分子类型为C型： 即抗PVRIG抗体经连接子1连接在抗TIGIT抗体的重链N端。抗PD-1抗体经连接子2连接在抗TIGIT抗体的重链C端(图8的C)。
A17m0902-1708-151H7-01-C和A17m0902-1708-151H7-02-C多肽链序列如下：
>A17m0902-1708-151H7-01-C第一多肽链
Figure PCTCN2022118979-appb-000091
A17m0902-1708-151H7-01-C第二多肽链如SEQ ID NO：68所示。
>A17m0902-1708-151H7-02-C第一多肽链
Figure PCTCN2022118979-appb-000092
A17m0902-1708-151H7-02-C第二多肽链如SEQ ID NO：68所示。
实施例3-4.抗PD-1/PVRIG/TIGIT三特异性抗体中PVRIG序列优化后与抗原的结合亲和力
分别使用表达人PVRIG基因的HEK293F稳转细胞株，表达人TIGIT基因的CHO-K1稳转细胞株和表达人PD-1的Jurkat细胞。在96孔板中每孔接种2x10 5个细胞。300g离心5分钟，去上清，加入100μL待测抗体，4℃孵育1小时。离心去除上清，用200μL洗液(PBS+2％FBS)洗涤3次，加入100μL 1：500稀释的用Alexa Fluor 488标记的抗人IgG二抗(Invitrogen，A-11013)，4℃孵育1小时。 离心去除上清，用200μL洗液(PBS+2％FBS)洗涤3次。用100μL PBS重悬细胞，用流式细胞仪(BD FACS Calibur或BD FACS Canto_II)检测。
1.与PVRIG的结合
PVRIG抗体第一位氨基酸突变后的两个三特异性抗体分子A17m0902-1708-151H7-01-C和A17m0902-1708-151H7-02-C与突变前的三特异性抗体分子A17m0902-1708-151H7-C和突变前的双特异性抗体分子1708-151H7对细胞表面表达的人PVRIG有较一致的结合活性，EC50值相近，表明PVRIG抗体第一位氨基酸在经过突变后不影响三特异性抗体对于PVRIG的结合。结果参见表40和图11A。
表40.抗体对表达人PVRIG的细胞结合实验E MAX和EC50结果
抗体 E MAX EC 50(nM)
A17m0902-1708-151H7-01-C 1547 0.1048
A17m0902-1708-151H7-02-C 1521 0.09636
A17m0902-1708-151H7-C 1516 0.0944
1708-151H7 1357 0.1124
2.与TIGIT的结合
两个三特异性抗体分子A17m0902-1708-151H7-01-C和A17m0902-1708-151H7-02-C与突变前的三特异性抗体分子A17m0902-1708-151H7-C抗体对细胞表面表达的人TIGIT有较一致的结合活性，EC50相近，表明PVRIG抗体第一位氨基酸在经过突变后不影响三特异性抗体对于TIGIT的结合。结果参见表41和图11B。
表41.三特异性抗体对表达人TIGIT细胞结合实验E MAX和EC50结果
抗体 E MAX EC 50(nM)
A17m0902-1708-151H7-01-C 9964 0.6008
A17m0902-1708-151H7-02-C 9747 0.586
A17m0902-1708-151H7-C 9105 0.5577
3.与PD-1的结合
两个三特异性抗体分子A17m0902-1708-151H7-01-C和A17m0902-1708-151H7-02-C与突变前的A17m0902-1708-151H7-C抗体对细胞表面表达的人PD-1有相近的结合活性，EC50相近，表明PVRIG抗体第一位氨基酸在经过突变后不影响三特异性抗体对于PD-1的结合。结果参见表42和图11C。
表42.三特异性抗体对表达人PD-1的细胞结合实验EMAX和EC50结果
抗体 E MAX EC 50(nM)
A17m0902-1708-151H7-01-C 1956 0.5027
A17m0902-1708-151H7-02-C 1937 0.4806
A17m0902-1708-151H7-C 1974 0.4006
总结上述结合活性实验结果，PVRIG抗体第一位氨基酸突变后的两个三特异性抗体分子A17m0902-1708-151H7-01-C和A17m0902-1708-151H7-02-C与细胞表面表达的PVRIG、TIGIT、PD-1抗原的亲和力与母本A17m0902-1708-151H7-C抗体相当，这些突变不影响三特异性抗体对于PVRIG，TIGIT和PD-1的结合。
实施例3-5.PD-1/PD-L1报告基因***检测抗PD-1/PVRIG/TIGIT三特异性抗体对PD-1的阻断
采用实施例1-4中的方法进行检测。如图12A、图12B和表43，A17h1作为PD-1端组成的四种构型的三特异性抗体均能实现对PD-1阻断，但B型和C型三特异性抗体的报告基因活性相对更好。
表43.三特异性抗体NFAT报告基因***的IC 50值
抗体编号 IC50(nM)
A17h1-1708-30H2-A 0.8007
A17h1-1708-30H2-B 0.3436
A17h1-1708-30H2-C 0.2643
A17h1-1708-30H2-D 0.825
A17m09-hIgG4 0.7716
A17h1-hIgG4 0.696
pembrolizumab 0.573
实施例3-6.MLR检测抗PD-1/PVRIG/TIGIT三特异性抗体对T细胞的活化
按实施例1-6的方法，将来自人受试者的细胞进行DC：T混合淋巴反应实验，结果参见图13A、图13B和表44。结果显示，4种构型的三特异性抗体均能有效促进T细胞活化，解除PD-1/PD-L1的抑制，促进T细胞分泌细胞因子，均具有较强的体外药效活性。
表44.四种构型三特异性抗体的MLR结果
Figure PCTCN2022118979-appb-000093
选择效果较优构型的A17m0902-1708-30H2-C进一步进行DC:T混合淋巴反应实验验证。参见图14A-图14B和表45、表46，结果显示，在100nM的抗体浓 度下，A17m0902-1708-30H2-C三特异性抗体远优于双特异性抗体1708-30H2对T细胞的激活，优于抗PD-1单抗Ab 464，略优于抗PD-1单抗A17m0902_hIgG4对T细胞的激活。
表45.受试者1的MLR检测结果
Figure PCTCN2022118979-appb-000094
表46.受试者2的MLR检测结果
Figure PCTCN2022118979-appb-000095
实施例3-7.NK92杀伤检测抗PD-1/PVRIG/TIGIT三特异性抗体对NK细胞的活化
使用NK细胞杀伤实验，可以检验抗PD-1/PVRIG/TIGIT三特异性抗体的体外药效。使用NK92细胞作为效应细胞，K562细胞作为靶细胞，进行细胞杀伤实验。先将不同浓度的候选抗体与NK92细胞在37℃共孵育30分钟，再将经过抗体预孵育的NK92细胞按照10：1的效靶比与经过CTV(Cat：C34557，Thermo)标记的K562细胞进行混合，培养条件为1640+10％FBS+10ng/mL IL-2，37℃共孵育4小时。离心去上清后，使用10μg/mL碘化丙啶溶液重悬细胞，进行死细胞染色标记。室温标记10分钟后，FACS检测CTV标记的K562细胞中，死细胞(PI阳性)的比例，计算杀伤活性。
如图15A、图15B所示，三特异性抗体保留了抗PVRIG/TIGIT双特异性抗体促进NK细胞杀伤的生物学功能，并且各构型的IC 50值相似，参见表47。
表47.4种构型三特异性抗体的NK细胞杀伤功能
抗体名称 EC 50(nM)
A17h1-1708-151H7-A 0.116
A17h1-1708-151H7-B 0.182
A17h1-1708-151H7-C 0.07
A17h1-1708-151H7-D 0.121
1708-151H7 0.066
A17h1-1708-30H2-A 0.484
A17h1-1708-30H2-B 0.291
A17h1-1708-30H2-C 0.044
A17h1-1708-30H2-D 0.192
1708-30H2 0.325
实施例3-8.抗PD-1/PVRIG/TIGIT三特异性抗体在对PD-1单抗响应的人黑色素瘤A375混合人PBMC的小鼠皮下移植瘤模型中的抗肿瘤作用评估
本实施例在对PD-1单抗响应的人黑色素瘤A375混合人PBMC体内肿瘤模型上，验证PD-1/PVRIG/TIGIT三特异性抗体的体内药效。
NCG小鼠，雌性，4-8周，体重约18-22g(小鼠购自江苏集萃药康生物科技有限公司)。小鼠放置在SPF级动物房IVC恒温恒压***条件下饲养。
从冻存PBMC中筛选获得在体内药效模型中响应PD-1单抗的供体PBMC，计数。用含IL-2和10％FBS的RPMI 1640培养液重悬PBMC到一定的浓度。将PBMC加入经Mitomycin C处理2h后的A375细胞中。与A375共培养5天后，收取PBMC。A375细胞培养在含10％胎牛血清(FBS)的DMEM培养液中，收取新鲜消化下来的A375细胞。每只鼠接种：PBMC 5×10 5个，A375细胞4×10 6个；接种体积为0.2ml/只(含50％Matrigel)；接种于NCG小鼠右侧皮下。接种当天为第0天，第1天时根据小鼠体重随机进行分组给药。
考虑到A17m0902_hIgG4分子量为76kDa；1708-151H7分子量为173.5kDa；A17m0902-1708-151H7-C分子量为200kDa。设计等摩尔浓度的给药方案。详细的给药方案、给药剂量和给药途径见表48。
表48.小鼠实验分组和给药方案
Figure PCTCN2022118979-appb-000096
(注：N：使用动物数量；i.p.：腹腔注射；BIW*8：每周2次，一共8次)
给药开始后，小鼠每周2次测量体重及肿瘤体积。
给药第21天后，与对照组相比，1708-151H7(11.5mg/kg)给药组、A17m0902_hIgG4(5mg/kg)给药组、A17m0902-1708-151H7-C(13.3mg/kg)给药组和1708-151H7(11.5mg/kg)+A17m0902_hIgG4(5mg/kg)给药组的肿瘤生长抑制率(TGI)分别为7.05％、79.91％、91.63％和88.25％。三特异性抗体A17m0902-1708-151H7-C给药组的药效明显优于抗PD-1单抗A17m0902_hIgG4给药组，以及优于PVRIG/TIGIT双特异性抗体+PD-1单抗联用的组。
给药后第21天后结束实验，对所有小鼠实行安乐死，剥离其肿瘤并称重拍照，计算瘤重TGI，与瘤体积TGI趋势一致，结果也显示三特异性抗体A17m0902-1708-151H7–C给药组的药效明显优于PD-1单抗A17m0902_hIgG4给药组，以及优于PVRIG/TIGIT双特异性抗体+PD-1单抗联用的组。实验结果分别见表49和图16A、图16B。
表49.抗PD-1/PVRIG/TIGIT三特异性抗体在PD-1单抗响应的小鼠人源A375
肿瘤模型中的抑瘤效果
组别 药物 瘤体积TGI(％) 瘤重TGI(％)
1 空白对照 / /
2 1708-151H7 7.05 3.84
3 A17m0902_hIgG4 79.91 77.20
4 A17m0902_hIgG4+1708-151H7 88.25 87.60
5 A17m0902-1708-151H7-C 91.63 91.50
(注：N：使用动物数量；i.p.：腹腔注射；BIW：每周2次)
实施例3-9.抗PD-1/PVRIG/TIGIT三特异性抗体在PD-1单抗不响应的人黑色素瘤A375混合人PBMC的小鼠皮下移植瘤模型中的抗肿瘤作用评估
本实施例在抗PD-1单抗不响应的人黑色素瘤A375混合人PBMC体内肿瘤模型上，验证抗PD-1/PVRIG/TIGIT三特异性抗体的体内药效。
NCG小鼠，雌性，4-8周，体重约18-22g(购自江苏集萃药康生物科技有限公司)，放置在SPF级动物房IVC恒温恒压***条件下饲养。
从冻存PBMC中筛选获得在体内药效模型中不响应抗PD-1单抗的供体PBMC。实验方案同实施例3-8描述的方法。小鼠实验分组和给药方案见表50。
表50.小鼠实验分组和给药方案
Figure PCTCN2022118979-appb-000097
(注：N：使用动物数量；i.p.：腹腔注射；BIW*8：每周2次，一共8次)
给药开始后，小鼠每周2次测量体重及肿瘤体积。
给药第17天后，根据测定各组小鼠肿瘤平均体积，实验组与IgG对照组相比，1708-151H7、A17m0902_hIgG4、A17m0902-1708-151H7-C、1708-151H7+A17m0902_hIgG4肿瘤生长抑制率(TGI％)分别为4.53％、20.75％、36.70％和31.95％。
给药后第17天后结束实验，对所有小鼠实行安乐死，剥离其肿瘤并称重拍照。 根据测定各组小鼠肿瘤重量，实验组与IgG对照组相比，1708-151H7、A17m0902_hIgG4、A17m0902-1708-151H7-C、1708-151H7+A17m0902_hIgG4肿瘤生长抑制率(TGI％)瘤重TGI分别为0％、16.8％、38.2％和33.4％。瘤重TGI与瘤体积TGI趋势一致。实验结果分别见表51和图17A、图17B。
与IgG对照组相比，A17m0902-1708-151H7-C给药组和1708-151H7+A17m0902_hIgG4给药组的肿瘤体积与重量均显著性减小(图17A，图17B，P<0.05)，表现出显著的抑瘤效果。本次实验过程中，所有小鼠均未出现明显的体重下降和行为异常，表明小鼠在该实验条件下，对受试药物具有较好的耐受性。
Figure PCTCN2022118979-appb-000098

Claims (25)

  1. PD-1结合蛋白，其包含免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含：
    SEQ ID NO：33、3、13-15、17-32、34-35任一所示氨基酸序列中的CDR1、CDR2和CDR3，或SEQ ID NO：2、16任一所示氨基酸序列中的CDR1、CDR2和CDR3，所述CDR1、CDR2和CDR3是根据Kabat、IMGT、Chothia、AbM或Contact编号***定义的；
    优选地，所述免疫球蛋白单一可变结构域的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO：7、36、37所示，或如SEQ ID NO：4、5、6所示；
    更优选地，所述CDR1的氨基酸序列如SEQ ID NO：7所示，CDR2的氨基酸序列如SEQ ID NO：8、38-40、101任一所示，CDR3的氨基酸序列如SEQ ID NO：41、42、9任一所示；
    最优选地，所述CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：7、38、41所示。
  2. 如权利要求1所述的PD-1结合蛋白，其中所述免疫球蛋白单一可变结构域经过选自以下任一项的改造或其组合：人源化、亲和力成熟、去除T细胞表位、降低抗体脱酰胺、降低抗体异构化；
    优选地，所述人源化改造使用的人种系模板的重链框架区为IGHV3-23*01或IGHV3-23*04。
  3. 如权利要求1或2所述的PD-1结合蛋白，其中，所述免疫球蛋白单一可变结构域的氨基酸序列：
    如SEQ ID NO：33、3、13-15、17-32、34-35任一所示或与之具有至少90％序列同一性；或
    如SEQ ID NO：2、16任一所示或与之具有至少90％序列同一性。
  4. 如权利要求1至3中任一项所述的PD-1结合蛋白，其还包含人免疫球蛋白Fc区；
    优选地，所述Fc区是人IgG1的Fc区或IgG4的Fc区；
    更优选地，所述人IgG4的Fc区具有228P、234A、235A和/或447A突变。
  5. 如权利要求1至4任一项所述的PD-1结合蛋白，其为抗PD-1抗体或其抗原结合片段；
    优选地，所述抗体或其抗原结合片段为骆驼抗体、嵌合抗体、人源化抗体、全人抗体或其抗原结合片段。
  6. PD-1/PVRIG/TIGIT结合蛋白，其包含：
    特异性结合PD-1的第一抗原结合结构域、
    特异性结合PVRIG的第二抗原结合结构域、和
    特异性结合TIGIT的第三抗原结合结构域，
    所述第一抗原结合结构域包含或是权利要求1所限定的免疫球蛋白单一可变结构域；
    优选地，所述PD-1/PVRIG/TIGIT结合蛋白为抗PD-1/PVRIG/TIGIT三特异性抗体。
  7. 如权利要求6所述的PD-1/PVRIG/TIGIT结合蛋白，其中：
    所述第二抗原结合结构域包含免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含：
    SEQ ID NO：95-96、45、62-66任一所示氨基酸序列中的CDR1、CDR2和CDR3，或SEQ ID NO：44、57-61任一所示氨基酸序列中的CDR1、CDR2和CDR3，所述CDR1、CDR2和CDR3是根据Kabat、IMGT、Chothia、AbM或Contact编号***定义的；
    优选地，所述CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO：49、50、51所示，或如SEQ ID NO：46、47、48或99所示。
  8. 如权利要求6或7所述的PD-1/PVRIG/TIGIT结合蛋白，其中：
    所述第三抗原结合结构域包含重链可变区(VH)和轻链可变区(VL)，其中：
    所述VH包含分别如SEQ ID NO：73、74和75所示氨基酸序列的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO：76、77和78所示氨基酸序列的LCDR1、LCDR2和LCDR3。
  9. 如权利要求6至8任一项所述的PD-1/PVRIG/TIGIT结合蛋白，其中：
    所述第一抗原结合结构域包含：
    如SEQ ID NO：33、3、13-15、17-32、34-35任一所示或与之具有至少90％序列同一性的氨基酸序列；或如SEQ ID NO：2、16任一所示或与之具有至少90％序列同一性氨基酸序列；
    优选为SEQ ID NO：33或与之具有至少90％序列同一性的氨基酸序列。
  10. 如权利要求6至9任一项所述的PD-1/PVRIG/TIGIT结合蛋白，其中：
    所述第二抗原结合结构域包含：
    如SEQ ID NO：95-96、45、62-66任一所示或与之具有至少90％序列同一性的氨基酸序列；或如SEQ ID NO：44、57-61任一所示或与之具有至少90％序列同一 性的氨基酸序列；
    优选为SEQ ID NO：95-96、64任一所示或与之具有至少90％序列同一性的氨基酸序列。
  11. 如权利要求6至10任一项所述的PD-1/PVRIG/TIGIT结合蛋白，其中：
    所述第三抗原结合结构域包含VH和VL，其中：
    所述VH包含如SEQ ID NO：79-81中任一所示或与之具有至少90％序列同一性的氨基酸序列，
    所述VL包含如SEQ ID NO：83或82所示或与之具有至少90％序列同一性的氨基酸序列；
    优选为所述VH包含SEQ ID NO：79或与之具有至少90％序列同一性的氨基酸序列，所述VL包含SEQ ID NO：83或与之具有至少90％序列同一性的氨基酸序列。
  12. 如权利要求11所述的PD-1/PVRIG/TIGIT结合蛋白，所述第三抗原结合结构域包含重链(HC)和轻链(LC)；
    优选地，所述HC序列为SEQ ID NO：67所示或与之具有至少90％序列同一性的氨基酸序列，所述LC序列为SEQ ID NO：68所示或与之具有至少90％序列同一性的氨基酸序列。
  13. 如权利要求6至12任一项所述的PD-1/PVRIG/TIGIT结合蛋白，其包含：
    第一多肽链、和
    第二多肽链，
    其中，按从N端到C端顺序：
    (I)第一多肽链是以下结构所示：[第一抗原结合结构域]-[连接子1]a-[第二抗原结合结构域]-[连接子2]b-[第三抗原结合结构域的VH]-CH1-Fc，和
    第二多肽链是以下结构所示：[第三抗原结合结构域的VL]-Cκ；或
    (II)第一多肽链是以下结构所示：[第二抗原结合结构域]-[连接子2]b-[第三抗原结合结构域的VH]-CH1-Fc，和
    第二多肽链是以下结构所示：[第一抗原结合结构域]-[连接子3]c-[第三抗原结合结构域的VL]-Cκ；或
    (III)第一多肽链是以下结构所示：[第二抗原结合结构域]-[连接子2]b-[第三抗原结合结构域的VH]-CH1-Fc-[连接子4]d-[第一抗原结合结构域]，和
    第二多肽链是以下结构所示：[第三抗原结合结构域的VL]-Cκ；或
    (IV)第一多肽链是以下结构所示：[第二抗原结合结构域]-[连接子2]b-[第三抗原结合结构域的VH]-CH1-Fc，和
    第二多肽链是以下结构所示：[第三抗原结合结构域的VL]-Cκ-[连接子5]e-[第一抗原结合结构域]；
    其中，-表示肽键，
    所述连接子1、连接子2、连接子3、连接子4、连接子5可以相同，也可以不同；a、b、c、d、e任选独立地为0或1；
    优选地，所述连接子1、连接子2、连接子3、连接子4、连接子5独立地为(G xS) y，其中，x选自1-5的整数，y选自1-6的整数；更优选地，独立地为(G 4S) 2、(G 4S) 3、(G 4S) 4任一所示的连接子。
  14. 如权利要求6至13中任一项所述的PD-1/PVRIG/TIGIT结合蛋白，其包含第一多肽链和第二多肽链，其选自以下的任一组：
    所述第一、第二多肽链分别包含如SEQ ID NO：97、68所示的氨基酸序列；
    所述第一、第二多肽链分别包含如SEQ ID NO：98、68所示的氨基酸序列；
    所述第一、第二多肽链分别包含如SEQ ID NO：87、68所示的氨基酸序列；
    所述第一、第二多肽链分别包含如SEQ ID NO：84、88所示的氨基酸序列；
    所述第一、第二多肽链分别包含如SEQ ID NO：89、68所示的氨基酸序列；
    所述第一、第二多肽链分别包含如SEQ ID NO：84、90所示的氨基酸序列；
    所述第一、第二多肽链分别包含如SEQ ID NO：91、68所示的氨基酸序列；
    所述第一、第二多肽链分别包含如SEQ ID NO：85、92所示的氨基酸序列；
    所述第一、第二多肽链分别包含如SEQ ID NO：93、68所示的氨基酸序列；
    所述第一、第二多肽链分别包含如SEQ ID NO：85、94所示的氨基酸序列；
    或，与所述第一、第二多肽链的分别具有至少90％序列同一性的氨基酸序列组合；
    优选地，所述PD-1/PVRIG/TIGIT结合蛋白具有两条相同的第一多肽链和两条相同的第二多肽链。
  15. PD-1/PVRIG结合蛋白，其包含：
    -权利要求1至5任一项所述的PD-1结合蛋白、和
    -特异性结合PVRIG的抗原结合结构域；
    优选地，所述特异性结合PVRIG的抗原结合结构域为权利要求7或10所限定的第二抗原结合结构域；
    优选地，所述PD-1/PVRIG结合蛋白为抗PD-1/PVRIG双特异性抗体。
  16. PD-1/TIGIT结合蛋白，其包含：
    -权利要求1至5任一项所述的PD-1结合蛋白、和
    -特异性结合TIGIT的抗原结合结构域；
    优选地，所述特异性结合TIGIT的抗原结合结构域为权利要求8或11所限定的第三抗原结合结构域；
    优选地，所述PD-1/TIGIT结合蛋白为抗PD-1/TIGIT双特异性抗体。
  17. 多核苷酸，其编码：
    权利要求1至5任一项所述的PD-1结合蛋白、或
    权利要求6至14任一项所述的PD-1/PVRIG/TIGIT结合蛋白、或
    权利要求15所述的PD-1/PVRIG结合蛋白、或
    权利要求16所述的PD-1/TIGIT结合蛋白；
    优选地，所述多核苷酸为载体。
  18. 宿主细胞，其含有权利要求17所述的多核苷酸。
  19. 制备PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白或PD-1/TIGIT结合蛋白的方法，包括：
    1)培养权利要求18所述的宿主细胞，以及
    2)从所述宿主细胞中分离所表达的PD-1结合蛋白、PD-1/PVRIG/TIGIT结合蛋白、PD-1/PVRIG结合蛋白或PD-1/TIGIT结合蛋白。
  20. 结合蛋白在制备治疗癌症的药物中的用途，其中所述结合蛋白选自以下任一项或其组合：
    权利要求1至5任一项所述的PD-1结合蛋白；
    权利要求6至14任一项所述的PD-1/PVRIG/TIGIT结合蛋白；
    权利要求15所述PD-1/PVRIG结合蛋白；或
    权利要求16所述的PD-1/TIGIT结合蛋白；
    优选地，所述癌症选自肺癌、***癌、乳腺癌、头颈部癌、食管癌、胃癌、结肠癌、结直肠癌、膀胱癌、***、子宫癌、卵巢癌、肝癌、黑色素瘤、肾癌、鳞状细胞癌、血液***癌症。
  21. 药物组合物，其含有：
    -至少一种可药用的赋形剂、稀释剂或载体；以及
    -选自以下任一项或其组合：
    权利要求1至5任一项所述的PD-1结合蛋白；
    权利要求6至14任一项所述的PD-1/PVRIG/TIGIT结合蛋白；
    权利要求15所述PD-1/PVRIG结合蛋白；
    权利要求16所述的PD-1/TIGIT结合蛋白。
  22. 治疗癌症的方法，包括步骤：
    向受试者施用治疗有效量的权利要求1至5任一项所述的PD-1结合蛋白、权利要求6至14任一项所述的PD-1/PVRIG/TIGIT结合蛋白、权利要求15所述PD-1/PVRIG结合蛋白、权利要求16所述的PD-1/TIGIT结合蛋白或权利要求21的药物组合物；
    优选地，所述癌症选自肺癌、***癌、乳腺癌、头颈部癌、食管癌、胃癌、结肠癌、直肠癌、膀胱癌、***、子宫癌、卵巢癌、肝癌、黑色素瘤、肾癌、鳞状细胞癌或血液***癌症。
  23. 活化NK细胞、γδT细胞和/或Th1细胞的方法，包含步骤：
    向有需要的受试者施用有效量的权利要求1至5任一项所述的PD-1结合蛋白、权利要求6至14任一项所述的PD-1/PVRIG/TIGIT结合蛋白、权利要求15所述PD-1/PVRIG结合蛋白、权利要求16所述的PD-1/TIGIT结合蛋白或权利要求21的药物组合物。
  24. 增加受试者体内的IFN-γ产生和/或促炎性细胞因子分泌的方法，包括步骤：向有需要的受试者施用有效量的权利要求1至5任一项所述的PD-1结合蛋白、权利要求6至14任一项所述的PD-1/PVRIG/TIGIT结合蛋白、权利要求15所述PD-1/PVRIG结合蛋白、权利要求16所述的PD-1/TIGIT结合蛋白或权利要求21的药物组合物。
  25. 抑制受试者体内的PD-1活性和/或促进T细胞增殖的方法，包括步骤：
    向有需要的受试者施用有效量的权利要求1至5任一项所述的PD-1结合蛋白、权利要求6至14任一项所述的PD-1/PVRIG/TIGIT结合蛋白、权利要求15所述PD-1/PVRIG结合蛋白、权利要求16所述的PD-1/TIGIT结合蛋白或权利要求21的药物组合物；
    优选地，所述受试者的PD-L1和/或PD-L2的表达是上调的；
    更优选地，所述受试者患有癌症；
    最优选地，所述癌症选自肺癌、***癌、乳腺癌、头颈部癌、食管癌、胃癌、结肠癌、直肠癌、膀胱癌、***、子宫癌、卵巢癌、肝癌、黑色素瘤、肾癌、鳞状细胞癌、血液***癌症。
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