WO2021180205A1 - Pvrig结合蛋白及其医药用途 - Google Patents

Pvrig结合蛋白及其医药用途 Download PDF

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WO2021180205A1
WO2021180205A1 PCT/CN2021/080470 CN2021080470W WO2021180205A1 WO 2021180205 A1 WO2021180205 A1 WO 2021180205A1 CN 2021080470 W CN2021080470 W CN 2021080470W WO 2021180205 A1 WO2021180205 A1 WO 2021180205A1
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pvrig
seq
antibody
tigit
antigen
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PCT/CN2021/080470
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French (fr)
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林�源
林侃
金薪盛
张曼
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 KR1020227034534A priority Critical patent/KR20220154140A/ko
Priority to US17/905,694 priority patent/US20240043530A1/en
Priority to EP21768888.6A priority patent/EP4119162A4/en
Priority to CA3170025A priority patent/CA3170025A1/en
Priority to JP2022551318A priority patent/JP2023516936A/ja
Priority to CN202180010585.3A priority patent/CN115003333A/zh
Publication of WO2021180205A1 publication Critical patent/WO2021180205A1/zh

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    • A—HUMAN NECESSITIES
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
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    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates to PVRIG binding proteins, such as anti-PVRIG antibodies and bispecific antibodies formed with anti-TIGIT antibodies, and their use as drugs for the treatment of cancer.
  • Tumor immunotherapy is a hot spot in the field of tumor therapy, and tumor immunotherapy with T cells is at its core. Tumor immunotherapy is to make full use of and mobilize the killer T cells in tumor patients to kill tumors. It may be the most effective and safest way to treat tumors. Tumor immunotherapy currently has good prospects for the treatment of several different types of cancer, including disseminated metastatic tumors.
  • T cells in the human body adopts a system of two signal pathways.
  • a series of co-stimulatory molecules are also required to provide the second signal.
  • This dual-signal pathway system plays a vital role in the balance of the immune system in the body. It strictly regulates the body's different immune responses to self and non-self antigens. If the second signal provided by the costimulatory molecule is absent, it will lead to a non-response of T cells or a sustained specific immune response, which will lead to tolerance. Therefore, the second signaling pathway plays a very critical regulatory role in the entire process of the body's immune response.
  • PVRIG also known as CD112R
  • CD112R is a protein expressed on the cell surface. It belongs to the B7/CD28 superfamily equivalent to TIGIT, CD96 and CD226, and plays an important role in the immune system. It contains an extracellular region, a transmembrane region, and an intracellular region. When its ligand PVRL2 (also called CD112) binds to PVRIG, it will activate the ITIM domain of PVRIG's intracellular region, making PVRIG play an immunosuppressive effect.
  • PVRL2 also called CD112
  • 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 many 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 will also be Eomes-positive and Tbet-negative, indicating that PVRIG is related to T cell depletion. Therefore, PVRIG may represent a new immune checkpoint in addition to PD-1 and TIGIT, and play a role of redundancy. In vitro cell experiments and mouse models have shown that knocking out or inhibiting mouse PVRIG can effectively inhibit tumor growth and coordinate with PD-1 and TIGIT inhibitors.
  • TIGIT tumor infiltrating lymphocytes
  • TIL tumor infiltrating lymphocytes
  • Tregs Tregs that infiltrate different types of tumors.
  • PVR cognate ligand PVR
  • Compugen's COM701 is the world's first humanized hybridoma antibody against PVRIG that has been approved by the FDA to enter the clinic. It is currently in phase I clinical phase for the treatment of cancer. Surface Oncology also has anti-PVRIG antibody SRF-813 under development.
  • Anti-TIGIT antibodies include Genentech’s tiragolumab, Ono Pharmaceutical’s BMS-986207 co-developed with BMS, Merck’s MK-7684, iTeos Therapeutics’ EOS-884448, and Arcus Biosciences’ AB-154, all of which are in clinical phase II.
  • the prior art still lacks anti-PVRIG antibodies, anti-PVRIG/TIGIT bispecific antibodies with high affinity, high selectivity, and high biological activity that can inhibit cancer or tumor growth in vivo.
  • the present disclosure aims to provide such antibodies by preventing Cut off the inhibitory pathway of PVRIG and/or TIGIT to activate immunity to treat cancer.
  • the present disclosure provides PVRIG binding proteins, anti-PVRIG antibodies (such as VHH) and their bispecific antibodies with anti-TIGIT antibodies, and their encoding nucleic acids, vectors, host cells, pharmaceutical compositions, methods for treating cancer, and pharmaceutical applications.
  • anti-PVRIG antibodies such as VHH
  • bispecific antibodies with anti-TIGIT antibodies and their encoding nucleic acids, vectors, host cells, pharmaceutical compositions, methods for treating cancer, and pharmaceutical applications.
  • the present disclosure provides PVRIG binding proteins or anti-PVRIG antibodies.
  • the PVRIG binding protein comprises at least one immunoglobulin single variable domain, and the at least one immunoglobulin single variable domain comprises three complementarity determining regions CDR1, CDR2, and CDR3, wherein:
  • CDR1 is selected from SEQ ID NO: 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64
  • CDR2 is selected from SEQ ID NO: 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65
  • CDR3 is selected from SEQ ID NO: 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 150, 151 Any shown amino acid sequence or amino acid sequence with 3, 2, 1 or more amino acid differences.
  • SEQ ID NO: 7-21, 150, 151 are based on the Kabat coding rule
  • SEQ ID NO: 22-36 is based on the Chothia coding rule
  • SEQ ID NO: 37-51 is based on the IMGT coding rule
  • SEQ ID NO: 52 -66 is based on AbM coding rules.
  • the PVRIG binding protein comprises at least one immunoglobulin single variable domain
  • the at least one immunoglobulin single variable domain comprises CDR1 in any of the sequences shown in SEQ ID NO: 2, 75-79 , CDR2, CDR3; or CDR1, CDR2, CDR3 in any of the sequences shown in SEQ ID NO: 3, 80-84; or CDR1, CDR2 in any of the sequences shown in SEQ ID NO: 4, 86-90 , CDR3; or include CDR1, CDR2, and CDR3 in any of the sequences shown in SEQ ID NO: 5, 91-95; or include CDR1, CDR2, and CDR3 in any of the sequences shown in SEQ ID NO: 6, 96-100
  • the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system. In some specific embodiments, the CDR is determined according to the Kabat numbering system.
  • the immunoglobulin single variable domain of the PVRIG binding protein contains three complementarity determining regions CDR1, CDR2, and CDR3, of which:
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 7, 8, and 9 respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 7, 8, 150, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 10, 11, and 12, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 10, 11, and 151, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 13, 14, and 15, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 16, 17, and 18, respectively; or
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 19, 20, and 21, respectively.
  • the immunoglobulin single variable domain of the PVRIG binding protein contains three complementarity determining regions CDR1, CDR2, and CDR3, where:
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 22, 23, and 24, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 25, 26, and 27, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 28, 29, and 30, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 31, 32, 33, respectively; or
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 34, 35, and 36, respectively.
  • the immunoglobulin single variable domain of the PVRIG binding protein contains three complementarity determining regions CDR1, CDR2, and CDR3, where:
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 37, 38, and 39, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 40, 41, and 42 respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 43, 44, and 45, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 46, 47, and 48, respectively; or
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 49, 50, and 51, respectively.
  • the immunoglobulin single variable domain of the PVRIG binding protein contains three complementarity determining regions CDR1, CDR2, and CDR3, where:
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 52, 53, 54 respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 55, 56, 57, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 58, 59, 60, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 61, 62, 63, respectively; or
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 64, 65, and 66, respectively.
  • a PVRIG binding protein comprising an immunoglobulin single variable domain comprising CDR1, CDR2, and CDR3, wherein the immunoglobulin The amino acid sequences of CDR1, CDR2 and CDR3 of the single variable domain of the protein, respectively
  • the PVRIG binding protein of the present disclosure is an antibody or an antigen-binding fragment thereof, preferably a VHH antibody, more preferably a humanized and/or affinity matured VHH antibody.
  • the amino acid sequence of the immunoglobulin single variable domain of the PVRIG binding protein of the present disclosure is shown in any one of SEQ ID NO: 2-6, or has at least 80%, at least 90%, At least 95%, at least 98%, or at least 99% sequence identity.
  • the PVRIG binding protein is a variant protein, which has 3, 2, 1 or more amino acid differences in CDR1 of the above-mentioned PVRIG binding protein, and/or 3, 2, 1 or more amino acids in CDR2 Differences, and/or 3, 2, 1 or more amino acid differences in CDR3.
  • an anti-PVRIG antibody which comprises CDR1, CDR2, and CDR3 in the above PVRIG binding protein. It can be humanized and/or affinity matured.
  • the amino acid sequence of the anti-PVRIG antibody is as shown in SEQ ID NO: 2-6, 75-84, 86-100, or has at least 80%, at least 90%, at least 95%, or at least 98%, at least 99% sequence identity.
  • the anti-PVRIG single domain antibody is linked to the Fc region of human IgG1, IgG2, IgG3, IgG4, for example, it is linked to the Fc region of IgG4 with S228P, F234A, L235A, and/or K447A mutations (e.g., SEQ ID NO: 101 or 153).
  • the immunoglobulin single variable domain in the PVRIG binding protein of the present disclosure is a single domain antibody (VHH), and in some specific embodiments, the VHH is humanized and/or affinity matured VHH.
  • VHH single domain antibody
  • the PVRIG binding protein of the present disclosure comprises an antibody.
  • the PVRIG binding protein of the present disclosure is an antibody (e.g., VHH).
  • the PVRIG binding protein of the present disclosure is a camelid antibody, a humanized antibody, or a fully human antibody.
  • the PVRIG binding protein of the present disclosure or the immunoglobulin single variable domain therein is a camel antibody, wherein the amino acid sequence of VHH is shown in any one of SEQ ID NO: 2-6, or has at least 70% therewith. %, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity.
  • the PVRIG binding protein of the present disclosure or the immunoglobulin single variable domain therein is a humanized antibody
  • the framework region of the antibody is the heavy chain framework region of a human germline template, such as IGHV3-7, specifically, IGHV3 -7*01, IGHV3-30*02.
  • the amino acid sequence of the anti-PVRIG humanized antibody of the present disclosure is shown in any one of SEQ ID NO: 75-84, 86-100, or has at least 70%, at least 80%, or at least 90% therewith. , At least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity.
  • the PVRIG binding protein of the present disclosure comprises or is a humanized antibody that comprises the heavy chain framework region of a human germline template.
  • the heavy chain framework region of the human germline template is IGHV3-7*01 or IGHV3-30*02.
  • the amino acid sequence of the immunoglobulin single variable domain of the humanized antibody is as shown in SEQ ID NO: 75-84 and 86-100, or has at least 90%, At least 95%, at least 98%, or at least 99% sequence identity.
  • the PVRIG binding protein of the present disclosure further comprises a human immunoglobulin Fc region, such as the Fc region of human IgG1, IgG2, IgG3, and IgG4.
  • the human immunoglobulin Fc region is the Fc region of human IgG4.
  • the human immunoglobulin Fc region is the Fc region of human IgG1.
  • the Fc region may have mutations, such as S228P, F234A, L235A, and/or K447A amino acid mutations (for example, SEQ ID NO: 101 or 153).
  • the immunoglobulin single variable domain capable of specifically binding to PVRIG is connected to the immunoglobulin Fc region directly or through a linker.
  • the linker may be a non-functional amino acid sequence with a length of 1-20, 1-30, 1-40, 1-50 or more amino acids and no secondary or higher structure.
  • the joint may be a flexible joint, such as GS, GAP, ASGS, G 4 S, (G 4 S) 2 , (G 4 S) 3 , (G 4 S) 4 , (G 4 S) 5 , (G 4 S) 6 , YGNGT, (YGNGT) 2 , (YGNGT) 3 , (YGNGT) 4 , (YGNGT )5 , (YGNGT )6, etc.
  • the Fc region of the PVRIG binding protein of the present disclosure allows the PVRIG binding protein to form a dimer molecule comprising two or four PVRIG binding domains.
  • PVRIG binding proteins are also called bivalent or tetravalent PVRIG binding proteins.
  • the dimer is, for example, a homodimer.
  • the K D value of the combined human PVRIG is less than 1 ⁇ 10 -7 M;
  • the K D value of the PVRIG binding protein or anti-PVRIG antibody binding to PVRIG of the present disclosure may be less than 1 ⁇ 10 -7 M, less than 1 ⁇ 10 -8 M, less than 1 ⁇ 10 -9 M, and less than 1 ⁇ 10 -10 M.
  • the PVRIG binding protein or anti-PVRIG antibody of the present disclosure can inhibit tumor growth by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least About 80%.
  • the PVRIG binding protein or anti-PVRIG antibody of the present disclosure may be monomeric, and/or PEGylated, and/or glycosylated, and/or albumin conjugated or fused, and/or Fc fused, and/or Or hydroxyethylated, and/or O-glycosylated.
  • the present disclosure provides bispecific antibodies against PVRIG.
  • bispecific antibodies comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to PVRIG.
  • the first antigen-binding domain of the bispecific antibody of the present disclosure specifically binds to PVRIG
  • the first antigen-binding domain comprises at least one immunoglobulin single variable domain (such as VHH), and At least one immunoglobulin single variable domain (such as VHH) contains three complementarity determining regions CDR1, CDR2 and CDR3, of which:
  • CDR1 is selected from SEQ ID NO: 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64
  • CDR2 is selected from SEQ ID NO: 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65
  • CDR3 is selected from SEQ ID NO: 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 150, 151 Any shown amino acid sequence or amino acid sequence with 3, 2, 1 or more amino acid differences.
  • SEQ ID NO: 7-21 is based on Kabat coding rules
  • SEQ ID NO: 22-36 is based on Chothia coding rules
  • SEQ ID NO: 37-51 is based on IMGT coding rules
  • SEQ ID NO: 52-66 is based on AbM coding rules.
  • the first antigen-binding domain that specifically binds to PVRIG in the bispecific antibody comprises at least one immunoglobulin single variable domain, and the at least one immunoglobulin single variable domain comprises SEQ ID NO: 2.
  • the first antigen-binding domain (such as VHH) that specifically binds to PVRIG includes three complementarity determining regions CDR1, CDR2, and CDR3, of which:
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 7, 8, and 9 respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 7, 8, 150, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 10, 11, and 12, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 10, 11, and 151, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 13, 14, and 15, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 16, 17, and 18, respectively; or
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 19, 20, and 21, respectively.
  • the first antigen-binding domain (such as VHH) that specifically binds to PVRIG includes three complementarity determining regions CDR1, CDR2, and CDR3, of which:
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 22, 23, and 24, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 25, 26, and 27, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 28, 29, and 30, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 31, 32, 33, respectively; or
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 34, 35, and 36, respectively.
  • the first antigen-binding domain (such as VHH) that specifically binds to PVRIG includes three complementarity determining regions CDR1, CDR2, and CDR3, of which:
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 37, 38, and 39, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 40, 41, and 42 respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 43, 44, and 45, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 46, 47, and 48, respectively; or
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 49, 50, and 51, respectively.
  • the first antigen-binding domain (such as VHH) that specifically binds to PVRIG includes three complementarity determining regions CDR1, CDR2, and CDR3, of which:
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 52, 53, 54 respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 55, 56, 57, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 58, 59, 60, respectively;
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 61, 62, 63, respectively; or
  • amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NOs: 64, 65, and 66, respectively.
  • the first antigen-binding domain (such as VHH) of the bispecific antibody of the present disclosure comprises the amino acid sequence shown in any one of SEQ ID NO: 2-6, 75-84, 86-100, or has A sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical.
  • the first antigen binding domain is the first antibody, which is VHH;
  • the second antigen binding domain is a second antibody, which includes a heavy chain (HC) and a light chain (LC);
  • the VHH as the first antibody is located at the N-terminus and/or C-terminus of the heavy chain or light chain of the second antibody.
  • the bispecific antibody of the present disclosure includes one second antibody and two VHH first antibodies; the second antibody includes two HCs and two LCs, and one HC of the second antibody is VH It forms an antigen-binding site with the VL of one LC, and the VH of the other HC forms an antigen-binding site with the VL of another LC.
  • one of the first VHH antibodies of the bispecific antibodies of the present disclosure is located at the N-terminus of the heavy chain or light chain of the second antibody, and the other VHH first antibody is located at the heavy chain or the N-terminus of the second antibody.
  • the C-terminus of the light chain is located at the N-terminus of the heavy chain or the light chain of the second antibody.
  • the first antibody of each VHH in the bispecific antibody of the present disclosure is located at the N-terminus of the two heavy chains or the two light chains of the second antibody; or, the first antibody of each VHH is respectively Located at the C-terminus of the two heavy chains or two light chains of the second antibody.
  • the first antibody of each VHH in the bispecific antibodies of the present disclosure is located at the N-terminus of the two heavy chains of the first antibody; or, the first antibody of each VHH is located at the first antibody.
  • the first antibody of the present disclosure may be linked with 1, 2, 3, 4, 5, 6, 7, 8 VHH second antibodies, and the VHH second antibodies may be the same or different , They can all be connected to the N-terminus of the heavy chain of the first antibody, or both are connected to the C-terminus of the heavy chain of the first antibody, or both are connected to the N-terminus of the light chain of the first antibody, or both are connected to the light chain of the first antibody.
  • Chain C-terminus or any combination of heavy chain N-terminus, heavy chain C-terminus, light chain N-terminus, and light chain C-terminus.
  • the first antibody of the VHH in the bispecific antibody of the present disclosure is connected to the N-terminus or C-terminus of each heavy chain of the second antibody directly or through a linker.
  • the linker is selected from the group consisting of: (G m S n ) x or (GGNGT) x or (YGNGT) x amino acid sequences, wherein m and n are each independently selected from an integer of 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7 or 8), x is independently selected from an integer of 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).
  • the linker is an amino acid sequence represented by G 4 S, (G 4 S) 2 , (G 4 S) 3 , (G 4 S) 4 , (G 4 S) 5 , and (G 4 S) 6.
  • the heavy chain of the second antibody of the bispecific antibody of the present disclosure includes a heavy chain variable region (VH) and a heavy chain constant region (CH), and the light chain includes a light chain variable region (VL) and a light chain.
  • the second antibody may be a full-length antibody.
  • the heavy chain of the second antibody of the bispecific antibody of the present disclosure is of the IgG isotype, such as IgG1, IgG2, IgG3, or IgG4, such as the IgG1 isotype; and/or, the second antibody The light chain is of the Kappa isotype.
  • the two HCs of the second antibody of the bispecific antibody of the present disclosure comprise the same CDR and/or the two LCs comprise the same CDR. In some embodiments, the two HCs of the second antibody comprise the same VH and/or the two LCs comprise the same VL. In some specific embodiments, the two HCs of the second antibody have the same amino acid sequence and/or the two LCs have the same amino acid sequence.
  • the first antibodies of the two VHHs of the bispecific antibodies of the present disclosure have the same or different amino acid sequences.
  • the first antibodies of the two VHHs have the same amino acid sequence.
  • the bispecific antibody of the present disclosure comprises two first polypeptide chains and two second polypeptide chains, wherein for each polypeptide chain: a) the first polypeptide chains each independently comprise the first polypeptide chain of VHH The heavy chain (HC) of the first antibody and the second antibody; and b) the second polypeptide chain each independently includes the light chain (LC) of the second antibody; wherein the VHH is connected to the N-terminus of the HC of the first antibody through a linker. / Or C terminal is connected.
  • the first polypeptide chains each independently comprise the heavy chain (HC) of the second antibody; and ii) the second polypeptide chains each independently comprise the light chain (LC) of the first antibody of VHH and the second antibody ;
  • the VHH is connected to the N-terminus and/or C-terminus of the LC of the second antibody directly or through a linker.
  • the bispecific antibodies of the present disclosure comprise two identical first polypeptide chains and two identical second polypeptide chains.
  • the second antigen binding domain of the bispecific antibody of the present disclosure is any anti-TIGIT antibody.
  • the TIGIT antibodies in WO2009126688, WO2014089113, WO2015009856, WO2015143343, WO2015174439, WO2016028656, WO2016106302, WO2017053748, WO2017030823, US20160176963, US20130251720, WO2019232484, WO2019062832 are introduced in full here.
  • 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 second antigen binding domain of the bispecific antibody of the present disclosure is a second antibody.
  • the anti-TIGIT antibody in WO2019062832 is introduced in full here as a secondary antibody.
  • the second antibody is introduced in full here as a secondary antibody.
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 115, 116, and 117, and the light chain variable region includes LCDR1, LCDR2, and HCDR2, as shown in SEQ ID NO: 118, 119, and 120, respectively.
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 121, 122, and 123, respectively; the light chain variable region includes LCDR1, LCDR2, and HCDR2, as shown in SEQ ID NO: 124, 125, and 126, respectively. LCDR3; or
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 127, 128, and 129, respectively; the light chain variable region includes LCDR1, LCDR2, and HCDR2, as shown in SEQ ID NO: 130, 131, and 132, respectively. LCDR3; or
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 133, 134, and 135, respectively; the light chain variable region includes LCDR1, LCDR2, and HCDR2, as shown in SEQ ID NO: 136, 137, and 138, respectively. LCDR3; or
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 139, 140, and 141, respectively; the light chain variable region includes LCDR1, LCDR2, and HCDR2, as shown in SEQ ID NO: 142, 143, and 144, respectively. LCDR3.
  • the first antigen-binding domain or the first antibody (such as VHH) in the bispecific antibody of the present disclosure comprises CDR1, CDR2, CDR3 as shown in SEQ ID NO: 7, 8, 9, or The CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 7, 8, 150;
  • the second antigen-binding domain or the heavy chain variable region of the second antibody includes those shown in SEQ ID NOs: 121, 122, 123, respectively HCDR1, HCDR2, HCDR3, the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 124, 125, and 126, respectively.
  • the first antigen-binding domain or the first antibody (such as VHH) in the bispecific antibody of the present disclosure includes CDR1, CDR2, CDR3 as shown in SEQ ID NO: 10, 11, 12, or includes The CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 10, 11, and 151;
  • the second antigen-binding domain or the heavy chain variable region of the second antibody includes those shown in SEQ ID NOs: 121, 122, and 123, respectively HCDR1, HCDR2, HCDR3, the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 124, 125, and 126, respectively.
  • the first antibody of the VHH in the bispecific antibody of the present disclosure comprises an amino acid sequence as shown in one of SEQ ID NO: 6, 79, 81, 92, 98, 99 or has at least 70% , At least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity;
  • the second antibody comprises any one of SEQ ID NO: 145-147 VH, SEQ ID NO: VL shown in any one of SEQ ID NO: 148-149, or HC shown in SEQ ID NO: 102 and LC shown in SEQ ID NO: 103, or the same as the VH, HC or VL, LC.
  • the bispecific antibodies of the present disclosure comprise:
  • the first polypeptide chain is shown in SEQ ID NO: 104, and the second polypeptide chain is shown in SEQ ID NO: 103;
  • the first polypeptide chain is shown in SEQ ID NO: 105, and the second polypeptide chain is shown in SEQ ID NO: 103;
  • the first polypeptide chain is shown in SEQ ID NO: 102, and the second polypeptide chain is shown in SEQ ID NO: 106;
  • the first polypeptide chain is shown in SEQ ID NO: 102, and the second polypeptide chain is shown in SEQ ID NO: 107;
  • the Fc region of the PVRIG binding protein, anti-PVRIG antibody, and anti-TIGIT bispecific antibody of the present disclosure has mutations, including one or more amino acid mutations selected from the following:
  • antibodies that compete with the PVRIG binding protein, PVRIG ⁇ TIGIT binding protein, anti-PVRIG single domain antibody, anti-PVRIG and TIGIT bispecific antibodies of the present disclosure to bind to the same epitope are provided.
  • the present disclosure provides a PVRIG/TIGIT binding protein, which comprises a first antigen binding domain that specifically binds to PVRIG and a second antigen binding domain that specifically binds to TIGIT,
  • the first antigen-binding domain that specifically binds to PVRIG comprises an immunoglobulin single variable domain, and the immunoglobulin single variable domain comprises:
  • the CDR1, CDR2, CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system.
  • the amino acid sequences of CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are respectively
  • the first antigen-binding domain of the PVRIG/TIGIT binding protein comprises SEQ ID NO: 2-6, 75-84 and 86-100 or has at least 90%, An amino acid sequence with at least 95%, at least 98%, or at least 99% sequence identity.
  • the second antigen binding domain of the PVRIG/TIGIT binding protein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
  • the heavy chain variable region includes the HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 115, 116, and 117, respectively, and the light chain variable region includes the HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 118, 119, and 120, respectively.
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 121, 122, and 123, respectively, and the light chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 124, 125, and 126, respectively.
  • the heavy chain variable region includes the HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 127, 128, and 129, respectively, and the light chain variable region includes the HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 130, 131, and 132, respectively.
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 133, 134, and 135, respectively, and the light chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 136, 137, and 138, respectively.
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 139, 140, and 141, respectively, and the light chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 142, 143, and 144, respectively.
  • the second antigen binding domain of the PVRIG/TIGIT binding protein comprises a full-length heavy chain (HC) and a full-length light chain (LC);
  • the full-length heavy chain is of IgG1 or IgG4 isotype, and the full-length light chain is of Kappa isotype;
  • the heavy chain sequence is shown in SEQ ID NO: 102 or has at least 90% sequence identity with it
  • the light chain sequence is shown in SEQ ID NO: 103 or has at least 90% sequence identity with it sex.
  • the VHH that specifically binds to the first antigen-binding domain of PVRIG is located in the heavy chain variable region or the entire heavy chain that specifically binds to the second antigen-binding domain of TIGIT.
  • the VHH that specifically binds to the first antigen-binding domain of PVRIG is located at the C-terminus of the heavy chain variable region or the full-length heavy chain that specifically binds to the second antigen-binding domain of TIGIT;
  • the VHH that specifically binds to the first antigen-binding domain of PVRIG is located at the N-terminus of the light chain variable region or the full-length light chain that specifically binds to the second antigen-binding domain of TIGIT; and/or
  • the VHH that specifically binds to the first antigen-binding domain of PVRIG is located at the C-terminus of the light chain variable region or the full-length light chain that specifically binds to the second antigen-binding domain of TIGIT.
  • the VHH that specifically binds to the first antigen-binding domain of PVRIG and the second antigen-binding domain that specifically binds to TIGIT are connected directly or through a linker;
  • the linker has an amino acid sequence as shown in (G 4 S) x , wherein x is independently selected from an integer of 1-20;
  • the linker is an amino acid sequence shown by (G 4 S) 2 and (G 4 S) 3.
  • the PVRIG/TIGIT binding protein provided by the present disclosure comprises a first polypeptide chain and a second polypeptide chain, wherein:
  • the first polypeptide chain includes the amino acid sequence shown in any one of SEQ ID NO: 108-112 and 114, and the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 103; or
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 104 or 105
  • the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 103; or
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 102
  • the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 106 or 107.
  • the present disclosure provides polynucleotides that encode the aforementioned PVRIG binding protein, PVRIG/TIGIT binding protein, anti-PVRIG antibody (such as VHH) or anti-PVRIG and TIGIT bispecific antibody.
  • the polynucleotide may be DNA or RNA.
  • a polynucleotide composition comprising:
  • the first nucleic acid encodes the VH or HC including the bispecific antibody of the present disclosure against PVRIG and TIGIT; and the second nucleic acid encodes the VL or LC including the bispecific antibody of the present disclosure against PVRIG and TIGIT.
  • the present disclosure provides an expression vector or expression vector composition containing the polynucleotide or polynucleotide composition as described above.
  • the expression vector may be a eukaryotic expression vector, a prokaryotic expression vector, or a viral vector.
  • an expression vector composition comprising:
  • a first expression vector comprising the first nucleic acid in the above polynucleotide composition
  • the second expression vector includes the second nucleic acid in the above polynucleotide composition.
  • the present disclosure provides a host cell transformed with or containing the expression vector or expression vector composition as described above, which may be a eukaryotic cell or a prokaryotic cell.
  • the host cell is a bacterium, yeast, or mammalian cell. In some specific embodiments, the host cell is Escherichia coli, Pichia pastoris, Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells.
  • the present disclosure provides a preparation method, comprising: expressing PVRIG binding protein, anti-PVRIG antibody (such as VHH), anti-PVRIG and TIGIT bispecific antibody in the host cell as described above, and extracting from the host cell Separate and recover PVRIG binding protein, anti-PVRIG antibodies (such as VHH), anti-PVRIG and TIGIT bispecific antibodies from cells.
  • anti-PVRIG antibody such as VHH
  • anti-PVRIG and TIGIT bispecific antibody from cells.
  • the present disclosure provides a method for preparing PVRIG binding protein, PVRIG/TIGIT binding protein, or anti-PVRIG antibody or antigen binding fragment thereof, including:
  • PVRIG binding protein Isolated and expressed PVRIG binding protein, PVRIG/TIGIT binding protein or anti-PVRIG antibody or antigen binding fragment thereof from the host cell.
  • the present disclosure provides a composition (such as a pharmaceutical composition), which contains a therapeutically effective amount of the above-mentioned PVRIG binding protein, anti-PVRIG antibody (such as VHH), anti-PVRIG and TIGIT bispecific antibody, or PVRIG /TIGIT binding protein, and pharmaceutically acceptable excipients, dilutions or carriers.
  • a composition such as a pharmaceutical composition
  • the composition (such as a pharmaceutical composition) contains the PVRIG binding protein or anti-PVRIG antibody (such as VHH) of the present disclosure, and an anti-TIGIT antibody.
  • the TIGIT antibody may be any anti-TIGIT antibody as described above, for example, the anti-TIGIT antibody in Table 23 and Table 24 of the present disclosure.
  • the combined species may contain pharmaceutically acceptable excipients, diluents or carriers.
  • the anti-TIGIT antibody contains HCDR1, HCDR2, HCDR3 shown in SEQ ID NOs: 121, 122, and 123, and the light chain variable region contains the HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 124, 125, 126, respectively.
  • the pharmaceutical composition unit can contain 0.01 to 99% by weight of PVRIG binding protein, anti-PVRIG antibody (such as VHH), anti-PVRIG and TIGIT bispecific antibody, or pharmaceutical composition unit
  • the amount of bispecific antibody containing PVRIG binding protein, anti-PVRIG antibody (such as VHH), anti-PVRIG and TIGIT in the dose is 0.1-2000mg, or 1-1000mg.
  • anti-PVRIG antibodies such as VHH
  • bispecific antibodies against PVRIG and TIGIT PVRIG/TIGIT binding proteins
  • encoding polynucleotides PVRIG binding proteins of the present disclosure
  • anti-PVRIG antibodies Such as VHH
  • any one of anti-PVRIG and TIGIT bispecific antibodies, coding polynucleotides, or any combination thereof, used in methods for diagnosing, treating, and preventing diseases and preparing drugs and pharmaceutical compositions such as , Used to treat or prevent proliferative disorders (such as cancer or tumors) or delay the progress of related disorders).
  • a method for treating and alleviating a condition of a subject comprising administering to the subject the PVRIG binding protein of the present disclosure, an anti-PVRIG antibody (such as VHH), an anti-PVRIG and TIGIT bispecific antibody, so Said disease is cancer.
  • an anti-PVRIG antibody such as VHH
  • an anti-PVRIG and TIGIT bispecific antibody so Said disease is cancer.
  • a method for activating cytotoxic T cells (CTL) in a subject comprising administering to the subject the PVRIG binding protein of the present disclosure, an anti-PVRIG antibody (such as VHH), an anti-PVRIG and TIGIT dual A specific antibody, wherein the subgroup of the CTL of the subject is activated.
  • an anti-PVRIG antibody such as VHH
  • a method for activating NK cells in a subject comprising administering to the subject a PVRIG binding protein of the present disclosure, an anti-PVRIG antibody (such as VHH), an anti-PVRIG and TIGIT bispecific antibody, wherein The subgroup of NK cells of the subject is activated.
  • an anti-PVRIG antibody such as VHH
  • a method for activating ⁇ T cells in a subject comprising administering to the subject the PVRIG binding protein of the present disclosure, an anti-PVRIG antibody (such as VHH), an anti-PVRIG and TIGIT bispecific antibody, wherein The subgroup of ⁇ T cells of the subject is activated.
  • an anti-PVRIG antibody such as VHH
  • an anti-PVRIG and TIGIT bispecific antibody wherein The subgroup of ⁇ T cells of the subject is activated.
  • a method for activating Th1 cells in a subject comprising administering to the subject a PVRIG binding protein of the present disclosure, an anti-PVRIG antibody (such as VHH), an anti-PVRIG and TIGIT bispecific antibody, wherein The subset of Th1 cells of the subject is activated.
  • a PVRIG binding protein of the present disclosure an anti-PVRIG antibody (such as VHH), an anti-PVRIG and TIGIT bispecific antibody, wherein The subset of Th1 cells of the subject is activated.
  • a method for activating, reducing or eliminating the cell number and/or activity of at least one of regulatory T cells (Treg) in a subject comprising administering the PVRIG of the present disclosure to the subject Binding protein, anti-PVRIG antibody (such as VHH), anti-PVRIG and TIGIT bispecific antibody.
  • Reg regulatory T cells
  • a method for increasing interferon- ⁇ production and/or pro-inflammatory cytokine secretion in a subject comprising administering to the subject the PVRIG binding protein, anti-PVRIG antibody (such as VHH ), bispecific antibodies against PVRIG and TIGIT.
  • anti-PVRIG antibody such as VHH
  • a method for inhibiting the interaction of PVRIG and PVLR2 in a subject comprising administering to the subject the PVRIG binding protein of the present disclosure, an anti-PVRIG antibody (such as VHH), an anti-PVRIG and TIGIT dual Specific antibodies.
  • an anti-PVRIG antibody such as VHH
  • an anti-PVRIG and TIGIT dual Specific antibodies comprising administering to the subject the PVRIG binding protein of the present disclosure, an anti-PVRIG antibody (such as VHH), an anti-PVRIG and TIGIT dual Specific antibodies.
  • a method of treating a subject comprising administering to the subject or subject the PVRIG binding protein, anti-PVRIG antibody (such as VHH), anti-PVRIG and TIGIT bispecific antibody of the present disclosure.
  • the condition of the subject described above is a proliferative disorder (e.g., cancer or tumor) or suffers from a proliferative disorder (e.g., cancer or tumor).
  • the cancer or tumor is selected from the following or a combination thereof: prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, pancreatic cancer, stomach (stomach/gastric) cancer , Cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), nerve Glioma, renal cell carcinoma (RCC), lymphoma (NHL or HL), acute myelogenous leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular reproduction Cell tumors
  • the cancer or tumor is selected from the following cancers or a combination thereof: triple negative breast cancer, gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), Merck cell carcinoma, high MSI cancer, KRAS mutation tumor , Adult T-cell leukemia/lymphoma and myelodysplastic syndrome (MDS).
  • the cancer or tumor is selected from the following cancers or a combination thereof: triple negative breast cancer, gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), Merck cell carcinoma, and high MSI cancer.
  • the aforementioned subject has a condition associated with PVRIG and/or TIGIT.
  • the subject’s condition includes cancers that express or do not express PVRIG and further include non-metastatic or non-invasive and invasive or metastatic cancers, in which immune cells, stromal cells, or diseased cells inhibit PVRIG expression Anti-tumor response and anti-infiltrating immune response.
  • the method of the present disclosure is particularly suitable for the treatment of vascularized tumors.
  • a method for treating or preventing infection or sepsis in a subject comprising administering to the subject or subject the PVRIG binding protein, anti-PVRIG antibody (such as VHH), anti-PVRIG and TIGIT's bispecific antibody.
  • the infection is a pathogen infection, which is characterized by varying degrees of functional impairment of virus-specific T cell responses, such as HIV, HCV, and HBV.
  • the sepsis includes severe sepsis, septic shock, systemic inflammatory response syndrome (SIRS), bacteremia, sepsis, toxemia, and sepsis syndrome.
  • the above-mentioned PVRIG binding protein, PVRIG/TIGIT binding protein, anti-PVRIG antibody or antigen binding fragment, polynucleotide, or composition of the present disclosure is provided for use in treating or delaying diseases, preferably, The disease is a proliferative disease;
  • the proliferative disease is cancer
  • the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, stomach cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma , Kidney cancer, squamous cell carcinoma, blood system cancer, or any other disease or condition characterized by uncontrolled cell growth.
  • a method for diagnosing a disease including:
  • the tissue can be a blood sample, a solid tumor biopsy sample.
  • the PVRIG binding protein or anti-PVRIG antibody can be labeled, and further, the second labeled antibody bound to the PVRIG binding protein or anti-PVRIG antibody can be contacted with the aforementioned sample.
  • the PVRIG binding protein or anti-PVRIG antibody is labeled, including radioisotopes, dyes (such as with biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules, and Enhancers for magnetic resonance imaging (MRI) (for example, paramagnetic ions).
  • the disease or condition is cancer or tumor, infection or sepsis as described above.
  • the present disclosure provides the detection use of PVRIG binding protein.
  • the present disclosure provides a composition for detecting PVRIG, the composition comprising a PVRIG binding protein or an anti-PVRIG antibody.
  • the present disclosure also provides methods, systems or devices for detecting PVRIG in vivo or in vitro, which include the use of PVRIG binding proteins or anti-PVRIG antibodies.
  • the in vitro detection method, system or device may, for example, include (1) contacting the sample with PVRIG binding protein or anti-PVRIG antibody; (2) detecting the complex formed between the PVRIG binding protein or anti-PVRIG antibody and the sample ; And/or (3) contacting a reference sample (for example, a control sample) with the antibody; and (4) determining the degree of complex formation between the antibody and the sample by comparing with the reference sample.
  • a change in complex formation in the sample or subject indicates the presence of PVRIG in the sample.
  • the in vivo detection method, system or device may include: (1) administering a PVRIG binding protein or anti-PVRIG antibody to the subject; and (2) detecting the difference between the PVRIG binding protein or anti-PVRIG antibody and the subject The formation of inter-complex. Detection can include determining the location or time of formation of the complex.
  • Antibodies that bind PVRIG can be directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibodies. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances.
  • the formation of complexes between PVRIG binding protein or anti-PVRIG antibody and PVRIG can be detected by measuring or visualizing antibodies that bind or do not bind to PVRIG.
  • Conventional detection assays can be used, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry.
  • the sample is analyzed for the presence of PVRIG by a competitive immunoassay that uses a standard labeled with a detectable substance and an unlabeled PVRIG binding protein or anti-PVRIG antibody.
  • the living body sample to be detected or measured may be tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid or culture fluid.
  • the PVRIG binding protein or anti-PVRIG antibody of the present disclosure may be labeled with a fluorophore and a chromophore.
  • kits which includes a PVRIG binding protein or an anti-PVRIG antibody, and may also include diagnostic instructions.
  • the kit may also contain at least one additional reagent, such as a marker or additional diagnostic agent.
  • the antibody can be formulated as a pharmaceutical composition.
  • the PVRIG antibodies, anti-PVRIG and TIGIT double antibodies provided in the embodiments of the present disclosure have high specificity and high affinity for PVRIG and/or TIGIT; the immunogenicity of the humanized antibodies is greatly reduced, while completely retaining excellent in vivo and in vitro activities ; It has good metabolic kinetic characteristics of rats and human body; has long half-life, high bioavailability; has good long-term stability, no obvious abnormal chemical modification, no obvious aggregation under high concentration, high purity and heat Stability; has a good effect of enhancing the activity of T cells and NK cells, and inhibiting the occurrence and development of tumors.
  • Figure 1 shows the results of the PVRIG reporter cell activity test of the anti-PVRIG antibody.
  • Figure 2 shows the test results of anti-PVRIG antibody activating NK cells in the NK cell killing experiment.
  • Figure 3 shows the test results of the activity of the anti-PVRIG antibody to activate T cells in the MLR experiment.
  • Figures 4A-4B show the results of detection of humanized anti-PVRIG antibody activity in PVRIG reporter gene cells.
  • Figures 5A-5B are the detection results of the humanized anti-PVRIG antibody activating NK cells in the NK cell killing experiment.
  • Figures 6A-6E respectively show the binding activity of the humanized anti-PVRIG/TIGIT bispecific antibody to human PVRIG recombinant protein, human PVRIG overexpressing cells, cynomolgus monkey PVRIG recombinant protein, and cynomolgus monkey PVRIG overexpressing cells, and The detection result of the activity of blocking the combination of human PVRIG and human PVRL2.
  • Figures 7A-7E respectively show the binding activity of the humanized anti-PVRIG/TIGIT bispecific antibody to human TIGIT recombinant protein, human TIGIT overexpressing cells, cynomolgus TIGIT recombinant protein, and cynomolgus TIGIT overexpressing cells, and The detection result of the activity of blocking the combination of human TIGIT and human PVR.
  • Figure 8 shows the results of the activity detection of humanized anti-PVRIG/TIGIT bispecific antibodies in the MLR experiment to activate T cells.
  • Figures 9A-9B respectively show the effect of anti-PVRIG/TIGIT bispecific antibody on mouse body weight and tumor volume in a mouse subcutaneous xenograft model of human melanoma A375 mixed with human PBMC.
  • Figures 10A-10B respectively show the effect of anti-PVRIG/TIGIT bispecific antibody on the body weight and tumor volume of the mouse subcutaneous transplanted tumor model of human melanoma A375 mixed with human PBMC.
  • PVRIG or “PVRIG protein” or “PVRIG polypeptide” may optionally include any such protein or variants, conjugates or fragments thereof, including but not limited to known or wild-type PVRIG as described herein, And any naturally occurring splice variants, amino acid variants or isoforms, and especially the soluble extracellular domain (ECD) fragment of PVRIG.
  • ECD extracellular domain
  • PVRIG binding protein means any protein capable of specifically binding PVRIG or any molecule containing the protein.
  • the PVRIG binding protein may include an antibody against PVRIG as defined in the present disclosure, an antigen binding fragment thereof, or a conjugate thereof.
  • PVRIG binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR grafted molecules.
  • the "PVRIG binding protein” of the present disclosure may comprise at least one immunoglobulin single variable domain (such as VHH) that binds to PVRIG.
  • a "PVRIG binding protein” may comprise 2, 3, 4, or more PVRIG-binding immunoglobulin single variable domains (such as VHH).
  • the PVRIG binding protein of the present disclosure may also include linkers and/or parts with effector functions, such as half-life extension parts (such as immunoglobulin single variable domains that bind serum albumin). Domain), and/or fusion partner (such as serum albumin) and/or conjugated polymer (such as PEG) and/or Fc region.
  • the "PVRIG binding protein" of the present disclosure also encompasses bi/multispecific antibodies, which contain immunoglobulins that bind to different antigens (such as a first antibody that binds to a first antigen (such as PVRIG) and a second antibody that binds to a second antigen).
  • the second antibody to the antigen (such as TIGIT) optionally includes a third antibody that binds to a third antigen, and further optionally includes a fourth antibody that binds to the fourth antigen.
  • TIGIT or "TIGIT protein” or “TIGIT polypeptide” may optionally include any such protein or variants, conjugates or fragments 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.
  • Binds to PVRIG refers to the ability to interact with PVRIG or its epitope, and the PVRIG or its epitope may be of human origin.
  • Binds to TIGIT refers to the ability to interact with TIGIT or its epitope, and the TIGIT or its epitope may be of human origin.
  • Antigen-binding site refers to a discrete three-dimensional site on an antigen that is recognized by the antibody or antigen-binding fragment of the present disclosure.
  • Antibody or “immunoglobulin” broadly encompasses traditional antibodies (antibodies with a four-peptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds), as well as antigens Binding active Fab, Fv, sFv, F(ab')2, linear antibody, single chain antibody, scFv, sdAb, sdFv, nanobody, peptide antibody peptibody, domain antibody (heavy chain (VH) antibody, light chain ( VL) antibody) and multispecific antibodies (bispecific antibody, diabody, triabody and tetrabody, tandem two-scFv, tandem three-scFv), therefore, the term "antibody” used in the present disclosure includes full-length antibodies, single The chain and any part, domain or fragment with antigen binding activity and the multispecific antibody (including but not limited to antigen binding domain or Fragments, such as VHH domains or VH/VL domains, respectively).
  • immunoglobulins are usually a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and sequence of the constant region of the heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE.
  • the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain. , ⁇ chain, and ⁇ chain.
  • the same type of Ig can be divided into different subclasses according to the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into a kappa chain or a lambda chain by the difference of the constant region.
  • Each of the five types of Ig can have a ⁇ (kappa) chain or a ⁇ (lambda) chain.
  • the antibodies of the present disclosure specifically or substantially specifically bind to PVRIG and/or TIGIT.
  • the "antibody” of the present disclosure includes but is not limited to: (i) Fab fragments composed of VL, VH, CL and CH1 domains; (ii) Fd fragments composed of VH and CH1 domains; (iii) F(ab' ) 2 fragment, a bivalent fragment containing two linked Fab fragments; (vii) single-chain Fv molecule (scFv), in which the VH domain and the VL domain are connected by a peptide linker, which allows The two domains combine to form an antigen binding site; (Bird et al., 1988, "Science” 242: 423-426; Huston et al., 1988, "Proc. Natl. Acad.
  • the antibodies of the present disclosure generally use the Kabat numbering system.
  • the EU numbering in Kabat is also generally used for constant domains and/or Fc domains.
  • the antibodies of the present disclosure may be polyclonal, monoclonal, heterologous, allogeneic, syngeneic, or modified forms thereof, and monoclonal antibodies are particularly suitable for use in various embodiments.
  • the antibodies of the present disclosure are recombinant antibodies.
  • "recombinant” generally refers to products such as cells or nucleic acids, proteins, or vectors, meaning that the cells, nucleic acids, proteins, or vectors have been modified by introducing heterologous nucleic acids or proteins or altering natural nucleic acids or proteins, or The cells are derived from cells so modified.
  • recombinant cells express genes that are not present in the natural (non-recombinant) cell form or express natural genes that are originally abnormally expressed, underexpressed, or not expressed at all.
  • Monoclonal antibody and “monoclonal antibody composition” refer to a population of antibody molecules containing only one species of antigen binding site capable of immunoreacting with a specific epitope, while “polyclonal antibody” and “polyclonal antibody A “composition” refers to a population of antibody molecules containing multiple species of antigen binding sites capable of interacting with a specific antigen. Monoclonal antibody compositions typically exhibit a single binding affinity for the specific antigen with which they are immunoreactive.
  • Antigen refers to a molecule used to immunize immunocompetent vertebrates, to generate antibodies that recognize the antigen, or to screen expression libraries (e.g., phage, yeast, or ribosome display libraries, among others).
  • antigens are defined more broadly and are generally expected to include target molecules specifically recognized by antibodies, and therefore include a part or mimic of molecules used in the immunization process for antibody production or library screening for selection of antibodies Things.
  • Sequence (for example, in terms such as “immunoglobulin sequence”, “antibody sequence”, “single variable domain sequence”, “VHH sequence” or “protein sequence”) is generally understood to include both related amino acid sequences , Also includes the nucleic acid sequence or nucleotide sequence encoding the sequence, unless the disclosure requires further limited explanation.
  • Polynucleotide or “nucleic acid” refers to a chain of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after chain assembly.
  • Polynucleotides may also contain similar forms of ribose or deoxyribose sugars generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'- Azido-ribose, carbocyclic sugar analogs, ⁇ - or ⁇ -anomeric sugars, epimeric sugars (such as arabinose, xylose or lyxose, pyranose, furanose, sedum heptulose) ), acyclic analogs and abasic nucleoside analogs such as methyl riboside.
  • ribose or deoxyribose sugars generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'- Azido-ribose, carbocyclic sugar analogs, ⁇ - or ⁇ -anomeric sugars, epimeric sugars
  • “Homology” or “identity” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides.
  • the positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position .
  • 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 positions compared ⁇ 100%. For example, in the optimal sequence alignment, if there are 6 matches or homology in 10 positions in the two sequences, then the two sequences are 60% homologous. Generally speaking, the comparison is made when two sequences are aligned to obtain the greatest percentage of homology.
  • domain of a polypeptide or protein refers to a folded protein structure that can maintain its tertiary structure independently of the rest of the protein. Generally speaking, 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 losing the rest of the protein and/or the function of the domain.
  • Immunoglobulin domain refers to a spherical region of an antibody chain (for example, a chain of an antibody with a conventional tetrapeptide chain structure or a chain of a heavy chain antibody), or refers to a polypeptide consisting essentially of such a spherical region.
  • the immunoglobulin domain is characterized in that it maintains the immunoglobulin folding characteristics of the antibody molecule, and it consists of a 2-layer sandwich of about 7 anti-parallel ⁇ -sheet strands arranged in two ⁇ -sheets, optionally stabilized by conservative disulfide bonds. .
  • Immunoglobulin variable domains are basically referred to in the art and hereinafter as “framework region 1" or “FR1”, “framework region 2" or “FR2”, “framework region 3” or “FR3”, respectively.
  • an immunoglobulin domain consisting of four “framework regions” of "framework region 4" or “FR4", wherein the framework region is referred to as “complementarity determining region 1" or “CDR1” in the art and hereinafter, respectively.
  • the three “complementarity determining regions” or “CDRs” of “complementarity determining region 2" or “CDR2” and “complementarity determining region 3" or “CDR3” are spaced apart.
  • an immunoglobulin variable domain can be expressed as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the immunoglobulin variable domains have antigen-binding sites that give them specificity for antigens.
  • Antibody framework (FR) refers to a part of a variable domain that serves as a scaffold for the antigen binding loop (CDR) of the variable domain.
  • CDR the deterministic description of CDR and the identification of residues containing the binding site of the antibody can be completed by resolving the structure of the antibody and/or resolving the structure of the antibody-ligand complex. This can be achieved by any of various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analysis 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, conformational definition.
  • the Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, for example, 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 loop regions. (See, for example, Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83).
  • the AbM numbering system uses a computer program integration suite produced by Oxford Molecular Group for modeling antibody structures (see, for example, Martin et al., 1989, Proc Natl 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 databases and ab initio methods to model the tertiary structure of antibodies from basic sequences (see Samudrala et al., 1999, in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach).
  • the contact definition is based on the analysis of available complex crystal structures (see, for example, MacCallum et al., 1996, J. Mol. Biol., 5:732-45).
  • the position of the CDRs can be identified as residues that make enthalpy contributions to antigen binding (see, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166).
  • other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a part of Kabat CDR, although they can be shortened or lengthened according to specific residues or residue groups that do not significantly affect the prediction of antigen binding or experimental results. .
  • CDR may refer to a CDR defined by any method (including a combination of methods) known in the art.
  • the methods used herein can utilize CDRs defined according to any of these methods.
  • the CDR can be defined according to any of Kabat, Chothia, extended, AbM, IMGT, contact, and/or conformational definitions.
  • Immunoglobulin single variable domain is usually used to mean that it can be used without interacting with other variable domains (for example, when there is no requirement between the VH and VL domains of a conventional four-chain monoclonal antibody).
  • an immunoglobulin variable domain (which can be a heavy chain or a 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 VL domains or derived from VL domains (such as dAbs TM ).
  • Immunoglobulin single variable domains based on and/or derived from heavy chain variable domains are generally preferred.
  • a specific example of an immunoglobulin single variable domain is a "VHH domain” (or simply "VHH") as defined below.
  • VHH domain also known as heavy chain single domain antibody, VHH, VHH antibody fragment, VHH antibody, nanobody, is an antigen-binding immunoglobulin called "heavy chain antibody” (ie, "antibody lacking light chain”)
  • the variable domains of proteins 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)).
  • the term “VHH domain” is used to compare the variable domain with the heavy chain variable domain (which is referred to as "VH domain” in the present disclosure) and light chain variable domains present in conventional tetrapeptide chain structure antibodies.
  • variable domain (which is referred to as the "VL domain" in this disclosure) distinguishes it.
  • the VHH domain specifically binds to the epitope without the need for other antigen-binding domains (this is in contrast to the VH or VL domain in conventional tetrapeptide chain antibodies, in which case the epitope is recognized by the VL domain and the VH domain together) .
  • the VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain.
  • VHH domain includes, but is not limited to, natural antibodies produced by camelid animals, and can also be humanized antibodies produced by camelid animals, or obtained through phage display technology screening.
  • the total number of amino acid residues in each CDR may be different, and may not correspond to the total number of amino acid residues indicated by Kabat numbering (ie one according to Kabat numbering). Or multiple positions may be unoccupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by Kabat numbering). This means that in general, the 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 rules include Chothia, IMGT, and AbM.
  • 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 noted that smaller and longer sequences may also be suitable for the purposes described in this disclosure.
  • VHH domain (alone or as part of a larger polypeptide) offers many significant advantages over the use of conventional VH and VL domains, scFv or conventional antibody fragments (e.g. Fab- or F(ab')2-fragments):
  • VHH domain can be expressed by a single gene and does not require post-translational folding or modification
  • -VHH domain can be easily transformed into a multivalent and multispecific format
  • -VHH domain is highly soluble and has no tendency to aggregate
  • VHH domain is highly stable to heat, pH, proteases and other denaturants or conditions, and therefore can be prepared, stored or transported without using refrigeration equipment, thereby saving cost, time and environment;
  • -VHH domain is relatively small compared with conventional tetrapeptide chain structure antibody and its antigen-binding fragment (approximately 15kDa or 1/10 of the size of conventional IgG), so compared to conventional tetrapeptide chain structure antibody and its antigen-binding fragment , Shows higher tissue permeability and can be administered at higher doses;
  • -VHH domain can show so-called cavity binding properties (especially due to its extended CDR3 loop compared with conventional VH domain), so that it can reach targets and epitopes that are inaccessible to conventional tetrapeptide chain structure antibodies and antigen-binding fragments thereof.
  • Fc variant or “variant Fc” means a protein containing amino acid modifications in the Fc domain.
  • the Fc variants of the present disclosure are defined according to the amino acid modifications constituting them.
  • S228P or 228P is an Fc variant with a proline substitution at position 228 relative to the parent Fc polypeptide, where the numbering is based on the EU index.
  • the identity of the WT amino acid may not be specified, in this case the aforementioned variant is called 228P.
  • humanization examples include the VHH domain derived from Camelidae by replacing the amino acid sequence of the original VHH sequence with one or more amino acid residues present at the corresponding position in the VH domain of a human conventional tetrapeptide chain structure antibody “Humanization” (also referred to as “sequence optimization” in the present disclosure. In addition to humanization, “sequence optimization” can also cover one or more amino acid residues that provide improved properties of VHH Mutations make other modifications to the sequence, such as removing potential post-translational modification sites).
  • the humanized VHH domain may contain one or more fully human framework region sequences, and in some embodiments, may contain the human framework region sequence of IGHV3.
  • humanization includes transplanting mouse CDR sequences into human antibody variable region frameworks, that is, antibodies produced from different types of human germline antibody framework sequences. It can overcome the strong variable antibody response induced by the chimeric antibody due to the large amount of mouse protein components.
  • Humanization methods such as protein surface amino acid humanization (resurfacing) and antibody humanization universal framework grafting (CDR grafting to a universal framework), that is, CDR “grafting” to other "stents” (including but not limited to human stents or Non-immunoglobulin scaffold). Stents and techniques suitable for the CDR grafting are known in the art.
  • the germline DNA sequences of the human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/vbase), as well as in Kabat, EA, etc. People, 1991 Sequences of Proteins of Immunological Interest, found in the 5th edition.
  • the humanized antibodies of the present disclosure also include humanized antibodies that are further subjected to affinity maturation of CDRs by phage display.
  • the human antibody variable region framework sequence can be subjected to minimal reverse mutations or back mutations to maintain activity.
  • Affinity matured antibodies have one or more changes in one or more CDRs that result in an increase in affinity for the antigen compared to their respective parent antibodies.
  • Affinity matured antibodies can be prepared, for example, by methods known in the art as described below: Marks et al., 1992, Biotechnology 10: 779-783 or Barbas et al., 1994, Proc. Nat. Acad. Sci, USA 91: 3809-3813.; Shier et al., 1995, Gene 169: 147-155; Yelton et al., 1995, Immunol.155: 1994-2004; Jackson et al., 1995, J. Immunol.154(7): 3310-9; and Hawkins et al., 1992, J. MoI. Biol. 226(3): 889896; KS Johnson and RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.
  • the antibodies of the present disclosure will be such as to Biacore or KinExA assay measured Fortibio or preferably 10-7 to 10-10 moles / liter (M), more preferably from 10-8 to 10-10 mol / l, and even more preferably 10 -9 to 10 -10 or lower dissociation constant (KD), and/or at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, more preferably at least 10 -10 M
  • the association constant (KA) binds to the antigen to be bound (ie PVRIG). Any KD value greater than 10 -4 M is generally regarded as indicative of non-specific binding.
  • the specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, the surface plasmon resonance (SPR) assay, Scatchard assay, and/or competitive binding assay described in this disclosure (for example, radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competition assay.
  • SPR surface plasmon resonance
  • RIA radioimmunoassay
  • EIA enzyme immunoassay
  • sandwich competition assay sandwich competition assay.
  • Epitope or “antigenic determinant” used interchangeably refers to any epitope on the antigen to which the paratope of an antibody binds.
  • Antigenic determinants usually contain chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics.
  • an epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which can be "linear "Epitope” or "Conformational” epitope.
  • a linear epitope all interaction points between a protein and an interacting molecule (for example, an antibody) exist linearly along the protein's primary amino acid sequence.
  • Specific binding and “selective binding” refer to the binding of an antibody to an epitope on a predetermined antigen.
  • a predetermined antigen Generally, when recombinant human PVRIG, TIGIT or its epitope is used as an analyte and an antibody is used as a ligand, when measured by surface plasmon resonance (SPR) technology in the instrument, the antibody is approximately lower than 10 -7 M or even more.
  • the small equilibrium dissociation constant (K D ) binds to the predetermined antigen or its epitope, and its binding affinity to the predetermined antigen or its epitope is that of the predetermined antigen (or its epitope) or closely related antigens.
  • the binding affinity of foreign non-specific antigens such as BSA, etc.
  • Antibody that recognizes antigen can be used interchangeably with “antibody that specifically binds” herein.
  • Binding affinity is used herein as a measure of the strength of a non-covalent interaction between two molecules (eg, an antibody or part thereof and an antigen), and is used to describe a monovalent interaction (intrinsic activity).
  • the binding affinity between two molecules can be quantified by determining the dissociation constant (K D ).
  • K D can be determined by measuring the kinetics of complex formation and dissociation using, for example, a 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 a well-known method (see Caceci et al., 1984, Byte 9:340-362; Wong & Lohman, 1993, PNAS 90: 5428-5432).
  • Other standard assays for assessing the ability of antibodies to bind to target antigens include, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere herein.
  • the specificity of the interaction can be determined and compared by determining and comparing the K D value of the target interaction (for example, the specific interaction between the antibody and the antigen) and the non-target interaction (for example, a control antibody that is not known to bind PVRIG). K D value is evaluated.
  • the anti-PVRIG antibody of the present disclosure can bind to its target with at least 2-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold greater affinity than it binds to another non-PVRIG molecule. Times, 1,000 times, or 10,000 times, this is not a restrictive definition.
  • conservative modifications apply to amino acid and nucleotide sequences.
  • conservative modification refers to the mutual substitution of those nucleic acids that encode the same or substantially the same amino acid sequence, or in the case where the nucleotide does not encode the amino acid sequence, it refers to the substantially same nucleotide sequence .
  • conservative modification means that other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.) replace amino acids in the protein, so that frequent changes can be made without changing The biological activity of the protein.
  • amino acid mutations include amino acid substitutions, deletions, insertions, modifications and any combination thereof to realize the final construct so that the final construct possesses the desired characteristics, such as enhanced stability and increased activity.
  • Amino acid sequence deletions and insertions include amino and/or carboxy terminal deletions and amino acid insertions.
  • Preferred amino acid mutations are amino acid substitutions.
  • non-conservative amino acid substitutions can be made, that is, one amino acid can be replaced with another amino acid having a different structure and/or chemical properties.
  • Preferred amino acid substitutions include the substitution of hydrophilic amino acids for hydrophobic amino acids.
  • Amino acid substitutions include non-naturally-occurring amino acids or naturally-occurring amino acid derivatives of 20 standard amino acids (e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine Amino acid) substitution.
  • Amino acid mutations can be generated using genetic or chemical methods known in the art, including methods such as site-directed mutagenesis, PCR, gene synthesis, and chemical modification. Amino acid mutations can occur in the CDR, FR or Fc region of the antibody.
  • mutations can be introduced into the wild-type Fc sequence of the antibody of the present disclosure to change Fc-mediated related activities, and the mutations include but are not limited to: a). Changes in Fc-mediated CDC activity Mutations; b). Mutations that change Fc-mediated ADCC activity; or c). Mutations that change FcRn-mediated half-life in vivo (see Leonard G Presta, Current Opinion in Immunology 2008, 20: 460-470; Esohe E. Idusogie et al., J Immunol 2000, 164: 4178-4184; RAPHAEL A. CLYNES et al., Nature Medicine, 2000, Volume 6, Number 4: 443-446; Paul R.
  • Hinton et al., J Immunol, 2006, 176: 346-356 includes: modifying the hinge region of CH1 so that the number of cysteine residues in the hinge region is changed, for example, increased or decreased (see US 5,677,425, which is incorporated in its entirety here).
  • mutations that enhance binding to Fc ⁇ RIIIa to cause enhanced ADCC
  • mutations that weaken binding to Fc ⁇ RIIb such as 236A, 239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F , 236A/332E, 239D/332E/330Y, 239D, 332E/330L, 299T and 297N (see US11/124,620, US6,737,056, which are incorporated in full here).
  • Carry out Fc modification to increase its biological half-life for example, one or more of the following mutations can be introduced: T252L, T254S, T256F (see US 6,277,375); in order to increase the biological half-life, the antibody can be changed in the CH1 or CL region to contain The rescue receptor binding epitope obtained from the two loops of the CH2 domain of the Fc region of IgG (see US5,869,046, US6,121,022); additional mutations used to increase the serum half-life, including 428L, 434A, 434S and 428L/ 434S (see US 8,883,973, US 6,737,056, US 7,371,826, which are incorporated in their entirety here).
  • the Fc region is changed by replacing at least one amino acid residue, thereby changing the effector function of the antibody.
  • one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 can be changed.
  • the effector ligand with altered affinity may be, for example, the Fc receptor or the C1 component of complement (see US 5,624,821, US 5,648,260, which are incorporated in their entirety here).
  • the Fc region is modified by modifying one or more amino acids at the following positions to improve the antibody's ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or increase the antibody's affinity for Fc ⁇ receptors: 238, 239, 248, 249 , 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298 , 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378 , 382, 388, 389,
  • mutations such as M252Y/S254T/T256E or M428L/N434S improve the binding to FcRn and increase the circulating half-life of the antibody (see Chan CA and Carter PJ (2010) “Nature Rev Immunol” 10:301 -316).
  • Modifications of the antibodies of the present disclosure include pegylation (PEGylation) or the addition of other water-soluble moieties, for example in order to enhance the half-life.
  • PEGylation refers to the attachment of at least one PEG molecule to another molecule, such as a therapeutic protein.
  • PEG is a linear or branched polyether attached to a hydroxyl group at one end, and has the following general structure: HO-(CH 2 CH 2 O) n -CH 2 CH 2 -OH.
  • PEG can be activated by preparing some or two derivatives of PEG with functional groups at the ends.
  • the common approach for PEG conjugation of proteins is to activate PEG with a functional group suitable for the reaction with lysine and the N-terminal amino acid group.
  • the common reactive group involved in conjugation is the alpha or epsilon amino group of lysine.
  • the reaction of the PEGylated linker with the protein can lead to the attachment of the PEG moiety mainly at the following positions: the alpha amino group on the N-terminus of the protein, the epsilon amino group on the side chain of the lysine residue, or the histidine residue Imidazolyl on the side chain. Since most recombinant proteins have a single ⁇ and many ⁇ amino groups and imidazole groups, many positional isomers can be produced according to the chemical properties of the linking group.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods.
  • the cDNA sequences encoding the heavy and light chains can be cloned and recombined into an expression vector.
  • the recombinant immunoglobulin expression vector can be stably transfected into CHO cells.
  • Mammalian expression systems can 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 the serum-free medium of the bioreactor to produce antibodies.
  • the antibody-secreted culture medium can be purified and collected by conventional techniques.
  • the antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.
  • the resulting product needs to be frozen immediately, such as -70°C, or lyophilized
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact, such as treatment, pharmacokinetics, diagnosis, research and experimental methods.
  • the treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells.
  • administering”, “administration” and “treatment” also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo. When applied to human, veterinary or research subjects, it refers to treatment, preventive or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent to a subject, such as containing any one of the antibodies of the present disclosure or a pharmaceutical composition thereof as a therapeutic agent, the subject has, is suspected of having, or is prone to There are one or more proliferative diseases or their symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • the therapeutic agent is administered in the subject or population to be treated in an amount effective to alleviate one or more symptoms of the disease, whether by inducing the regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent.
  • the amount of the therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the subject’s disease state, age and weight, and the amount of the drug that produces the desired therapeutic effect in the subject. ability. Through any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms, it can be evaluated whether the symptoms of the disease have been alleviated.
  • the embodiments of the present disclosure may be ineffective in alleviating the symptoms of the target disease in a subject, according to any statistical test methods known in the art such as Student's t test, chi-square test, and basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of subjects.
  • any statistical test methods known in the art such as Student's t test, chi-square test, and basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of subjects.
  • an “effective amount” includes an amount sufficient to ameliorate or prevent the symptoms or conditions of the medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a subject can vary depending on factors such as the condition to be treated, the general health of the subject, the method of administration and dosage, and the severity of side effects.
  • the effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.
  • the subject of the present disclosure may be an animal or a human subject.
  • “Host cell” includes individual cells or cell cultures, which may be or have been recipients of vectors for incorporation of polynucleotide inserts.
  • a host cell includes the progeny of a single host cell, and due to natural, accidental or deliberate mutations, the progeny may not necessarily be exactly the same as the original parent cell (in morphology or genomic DNA complement).
  • Host cells include cells transfected and/or transformed in vivo with polynucleotides of the present disclosure.
  • Cell Cell line
  • “cell culture” are used interchangeably, and all such names include their progeny. It should also be understood that due to deliberate or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Including mutant progeny with the same function or biological activity as screened in the original transformed cell.
  • Vector means a construct capable of being delivered in a host cell and in some embodiments expressing one or more genes or sequences of interest.
  • examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors combined with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes , And certain eukaryotic cells such as producer cells.
  • “Pharmaceutical composition” means a mixture containing one or more antibodies or antigen-binding fragments or physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components of the antibodies or antigen-binding fragments described herein, as well as other components such as physiological/pharmacological Medicinal carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
  • “Pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, emulsions such as oil/water emulsions, and various types of wetting agents.
  • the diluent for aerosol or parenteral administration is phosphate buffered saline (PBS) or physiological (0.9%) saline.
  • PBS phosphate buffered saline
  • Compositions containing such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, editor, Mack Publishing Co., Easton, PA, 1990; and R Remington, The Science and Practice of Pharmacy 20th Edition Mack Publishing, 2000).
  • the "PVRIG binding protein” or “PVRIG antibody” of the present disclosure may include one or more effector molecules in a conjugation manner, for example.
  • the "effector molecule” includes, for example, anti-tumor 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, in particular radioiodides, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds detectable by NMR or ESR spectroscopy.
  • the effector molecule when it is a polymer, it can usually be a synthetic or naturally occurring polymer, such as an optionally substituted linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymer or branched Polysaccharides or unbranched polysaccharides, such as homo- or hetero-polysaccharides.
  • Specific optional substituents that may be present on the aforementioned synthetic polymers include one or more hydroxy, 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, particularly optionally substituted poly(ethylene two Alcohol) such as methoxy poly(ethylene glycol) or its derivatives.
  • 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 method of conjugating the polymer to the PVRIG binding protein or PVRIG antibody can be achieved by conventional methods.
  • His-tagged human PVRIG (h-PVRIG-his) recombinant protein, mouse IgG2a Fc-tagged human PVRIG (h-PVRIG-mIgG2a Fc) recombinant protein, human IgG1 Fc-tagged mouse PVRIG (m- PVRIG-hIgG1 Fc) is a purified commercial protein reagent purchased from Acrobiosystems, and its sequence is shown in Table 1.
  • cyno-PVRIG-his The sequence of the cyno-PVRIG (cyno-PVRIG-his) recombinant protein with his tag is as follows:
  • the recombinant protein was expressed by transient transfection in HEK293 cells by conventional methods, and the supernatant was collected and purified by Ni-NTA. After testing, cyno-PVRIG-his was obtained.
  • Anti-human PVRIG monoclonal single domain antibodies are produced by immunizing camels.
  • the immune antigen is human PVRIG recombinant protein (h-PVRIG-his) with his tag.
  • Emulsification with Freund's adjuvant Sigma, Lot No.: F5881/F5506): First use Freund's complete adjuvant (CFA) CFA, and other booster immunizations with Freund's incomplete adjuvant (IFA).
  • CFA Complete adjuvant
  • IFA immunofluorization time
  • the single-domain antibody variable region gene was amplified by PCR and cloned into a phage vector to build a phage library of anti-human PVRIG single-domain antibody.
  • the phage library was diluted and blocked with BSA, incubated with magnetic beads Dynabeads (M-280, invitrogen), and the phage after the negative sieve incubation was collected.
  • the Dynabeads were coated and blocked with biotin-labeled human PVRIG with his tag, the phage suspension collected after the negative sieve was incubated with the Dynabeads, and the phage was eluted with trypsin. After 3 rounds of screening, 400 clones from the 3rd round of screening were selected for sequencing.
  • the heavy chain sequences of 5 single domain antibodies are shown in Table 2, and the CDRs with different coding rules are shown in Table 3.
  • the heavy chain variable regions of the five antibodies in Example 2 were connected to the human IgG4 heavy chain Fc region to form a full-length anti-PVRIG antibody.
  • the Fc region of the heavy chain includes the hinge region with S228P, F234A, L235A, and K447A mutations (Eu nomenclature system).
  • 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 does not bind 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 above sequence was synthesized, digested with BamHI and XhoI, and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20) through the BamHI/XhoI restriction site.
  • the expression vector and the transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1:2, and incubated in a CO 2 incubator 4- 5 days. After the expressed antibody is recovered by centrifugation, the antibody is purified according to the conventional method, and the target antibody is obtained after detection.
  • ELISA experiments were used to detect the binding properties of anti-PVRIG antibodies.
  • the PVRIG recombinant protein with his tag is directly coated. After the antibody is added, the activity of the antibody binding to the antigen is detected by adding the secondary antibody (HRP-conjugated anti-Fc antibody) and the HRP substrate TMB.
  • Human, cynomolgus or mouse PVRIG protein was coated with 96-well microtiter plate, 100 ⁇ L per well at a concentration of 1 ⁇ g/mL, and incubated overnight at 4°C. Wash the lotion three times, 250 ⁇ L per well. Shake for 10 seconds each time to ensure adequate cleaning. Add 300 ⁇ L/well blocking solution (PBS+0.05% Tween20+1% BSA) and incubate at room temperature for 1 hour. Wash the lotion three times, 250 ⁇ L per well. Shake for 10 seconds each time to ensure adequate cleaning. Add 100 ⁇ L of anti-PVRIG antibody to be tested diluted with diluent to each well. Incubate at 37°C for 1 hour. Wash the lotion three times, 250 ⁇ L per well.
  • Flow cytometry was used to detect the binding properties of the anti-PVRIG antibody. Construct a cell line that overexpresses human or cynomolgus PVRIG. After the antibody is added, the activity of the antibody binding to the antigen is detected by adding a secondary antibody.
  • the expression plasmid with human or cynomolgus monkey PVRIG gene sequence was transfected into HEK293 cells, and overexpression and stable transfection monoclonal cell lines were obtained by antibiotic selection and infinite dilution method.
  • a 96-well plate was seeded with 2 ⁇ 10 5 overexpression cells per well. 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.
  • the anti-PVRIG antibody screened out was tested to block the binding ability of human PVRIG and its ligand human PVRL2.
  • the specific method is to coat the human PVRIG recombinant protein (h-PVRIG-mIgG2a Fc) with mouse IgG2a Fc tag on a 96-well microtiter plate, add anti-PVRIG antibody to fully bind and occupy the epitope, and then add the his tag PVRL2 (PV2-H52E2, AcroBiosystem), by detecting the his tag, calculates the binding amount of PVRIG and PVRL2, and calculates the IC 50 value of the anti-PVRIG antibody blocking the active site of PVRIG.
  • h-PVRIG-mIgG2a Fc protein was coated on a 96-well microtiter plate, 100 ⁇ L per well at a concentration of 1 ⁇ g/mL, and incubated overnight at 4°C. Wash the lotion three times, 250 ⁇ L per well. Shake for 10 seconds each time to ensure adequate cleaning. Add 300 ⁇ L/well blocking solution and incubate at room temperature for 1 hour. Wash the lotion three times, 250 ⁇ L per well. Shake for 10 seconds each time to ensure adequate cleaning. Add 50 ⁇ L of diluted anti-PVRIG antibody to be tested and 50 ⁇ L of his-labeled ligand PVRL2 to each well, and incubate at 37°C for 1 hour. Wash the lotion three times, 250 ⁇ L per well.
  • the Protein A biosensor (Fortebio, #18-5010) was immersed in 200 ⁇ L of KB buffer (PBS, pH 7.4, 0.02% tween-20, 0.1% BSA) for 60 seconds, and then wetted. Then, use KB buffer to dilute the anti-PVRIG antibody to 10 ⁇ g/mL, place the sensor in 200 ⁇ L of this solution, and stop when the reading reaches 1.2 nm. Soak the sensor in KB buffer for 100 seconds to elute excess antibody.
  • the human PVRIG with his tag was diluted 2-fold with KB buffer to between 64nM-4nM. Place the sensor in the solution and bind for 300 seconds. Dissociate the sensor in KB buffer for 600 seconds. Using dynamic 1:1 binding, the affinity of anti-PVRIG antibody and human PVRIG is shown in Table 8.
  • plvx-OS8 (G418 resistant) plasmid, transfect 293F cells, select G418, use flow cytometry to detect the expression of OS8 in cloned cells and also detect the activation of OS8 on Jurkat cells, select the clone with moderate activation degree to obtain 293F -OS8 cell line; construct plvx-PVRL2 plasmid, use it to infect 293F-OS8 cells, use flow cytometry to screen out the clone with the highest expression of PVRL2, thereby obtaining 293F-OS8-PVRL2 cell line.
  • plvx-NFAT-Luc Hygromycin resistant
  • plvx-NFAT-Luc Hygromycin resistant
  • infect Jurkat E6.1 cells add Hygromycin to screen out resistant clones
  • use OKT3 to stimulate clones
  • screen out clones with medium Luciferase signal Obtain the Jurkat-NFAT-Luc cell line
  • construct the plvx-PVRIG Puromycin resistance
  • package it into a lentivirus infect Jurkat-NFAT-Luc cells, screen out the clone with the highest expression of PVRIG by flow cytometry, and obtain Jurkat -NFAT-Luc-PVRIG cell line.
  • PVRIG is expressed on NK cells, while PVRL2 is expressed on many tumor cells (including K562 cells).
  • Anti-PVRIG antibodies can block the binding of PVRL2 and PVRIG to relieve the inhibitory effect of tumor cells on NK cell activity.
  • NK92 cell line NK cells of human malignant non-Hodgkin's lymphoma patients
  • lotion including RPMI 1640, 5% FBS, 10ng/mL IL-2
  • NK92 cells NK cells of human malignant non-Hodgkin's lymphoma patients
  • lotion including RPMI 1640, 5% FBS, 10ng/mL IL-2
  • IL-2 resuspend to 2 ⁇ 10 6
  • the density of pieces/mL Add 50 ⁇ L (1 ⁇ 10 5 in total) NK92 cells to each well in a 96-well plate.
  • 50 ⁇ L of 20nM or 100nM antibody to be tested, and incubate at 37°C for 30 minutes. Wash twice with lotion and resuspend to a density of 2 ⁇ 10 5 cells/mL.
  • control groups which are a sample containing only culture medium (control group one), a sample containing only NK92 cells (control group two), and 150 ⁇ L of a sample containing only K562 cells (control group three), and perform the same operation.
  • 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 the control group 1 when AAF-Glo is added
  • E is the fluorescence value of the control group 2 when AAF-Glo is added
  • T is the control group 3 when AAF-Glo is added.
  • the fluorescence value of Glo is the fluorescence value of the control group 3 after adding the lysis solution
  • BGL is the fluorescence value of the control group after adding the lysis solution repeatedly.
  • PVRIG is expressed on T cells, while PVRL2 is expressed on dendritic cells (DC cells).
  • Anti-PVRIG antibodies can block the binding of PVRL2 and PVRIG, relieve the inhibitory effect of dendritic cells on T cells, and activate T cells.
  • PBMCs were isolated from the peripheral blood of the first individual, and the cells were cultured in RPMI 1640 medium containing 10% FBS at 50ng/mL GM-CSF (Peprotech, 300-03-100UG) and 50ng/mL IL- 4 (Peprotech, 200-04-100UG) add cytokine to the final concentration, add fresh medium containing cytokine every 2-3 days; after 6 days of culture, add 1 ⁇ g/mL LPS (Sigma, L2880-25MG) and incubate for 24 hours , Collect the DC cells obtained by differentiation and maturation.
  • GM-CSF 50ng/mL GM-CSF
  • IL- 4 50ng/mL IL- 4
  • PBMC peripheral blood-derived second, using EasySep human CD3 + T cell isolation kit (Stemcell, 17952) CD3 + T cells isolated therefrom. 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 120h, take the supernatant, and detect the IFN ⁇ content in the supernatant with an ELISA kit (R&D, DY202).
  • Stem EasySep human CD3 + T cell isolation kit
  • the heavy chain variable region sequence was compared with the antibody GermLine database to obtain a race with high homology Department template.
  • Replace the framework region of the camel single domain antibody with the heavy chain framework region of the human germline template retain the CDR (according to the Kabat numbering system), and combine it with the Fc region of human IgG (IgG4Fc with S228P, F234A, L235A, and K447A mutations). ) Reorganization.
  • antibodies 20H1-20H5 include CDR1 as shown in TDCMG (SEQ ID NO: 7), CDR2 as shown in HIDSDGIPRYVDSVKG (SEQ ID NO: 8), and CDR2 as shown in GFKFDEDYCAPND (SEQ ID NO: 150) CDR3.
  • antibody 30H1-30H5 contains CDR1 as shown in GDCMG (SEQ ID NO: 10), CDR2 as shown in TIDNAGRIKYADSVKG (SEQ ID NO: 11), and CDR3 as shown in GWTFGGQCSPAD (SEQ ID NO: 151) .
  • the above-mentioned humanized antibody heavy chain variable region is connected to the human IgG4 heavy chain Fc region to form a full-length anti-PVRIG antibody.
  • the Fc region of the heavy chain includes the hinge region with S228P, F234A, L235A, and K447A mutations.
  • the antibody is expressed and purified according to conventional methods, and the target antibody is obtained after detection.
  • Example 8 the activity of the humanized anti-PVRIG antibody in the reporter gene cell was detected.
  • the experimental results are shown in Figure 4A-4B and Table 19.
  • the antibodies listed in the table all have the ability to activate Jurkat cells.
  • Example 15 Experiment on the killing ability of activated NK cells of humanized anti-PVRIG antibody
  • Example 9 the ability of humanized anti-PVRIG antibody to activate NK cells was tested.
  • the experimental results are shown in Figure 5A-5B and Table 20-21.
  • the results show that the humanized anti-PVRIG antibodies of the present disclosure have obvious ability to activate NK cells and promote the killing of NK cells on the target cell K562.
  • the anti-PVRIG single domain antibody 151 was connected to the N of the heavy or light chain of the anti-TIGIT antibody 1708 through the GGGGSGGGGS (SEQ ID NO: 152) linker. Terminal or C terminal connected.
  • Anti-TIGIT antibody 1708 adopts human IgG4 subtype and carries a mutation of S228P (Eu nomenclature system).
  • the sequence of the anti-TIGIT antibody 1708 and the bispecific antibody formed with 151 is shown in Table 22 below.
  • the sequence information of anti-TIGIT antibodies is shown in Table 23-24.
  • the full text of the TIGIT antibody in WO2019062832A is hereby introduced.
  • Antibody number Expression amount (mg/L) SEC purity (%) 1708-151-1 140 97.6 1708-151-2 108 95.7 1708-151-3 160 94.7 1708-151-4 158 96.2
  • Example 5 and Example 6 the experiment was carried out, and the results are shown in Table 26.
  • the results showed that the binding of bispecific antibodies with different configurations to human PVRIG recombinant protein and over-expressing human PVRIG cells, as well as the blocking of PVRL2 binding to PVRIG, are basically the same and no difference.
  • Example 5 and Example 6 the corresponding receptors and ligands were replaced by human TIGIT and human PVR
  • the experiment was performed, and the results are shown in Table 27.
  • the results showed that the binding of bispecific antibodies and anti-TIGIT antibodies with different configurations to human TIGIT recombinant protein and over-expressing human TIGIT cells, as well as the blocking of TIGIT binding to its ligand PVR, are basically the same and no difference.
  • the connection mode of the anti-PVRIG antibody 151 has little effect on the binding of the anti-TIGIT antibody to TIGIT.
  • the antibody was transiently transfected, expressed and purified according to conventional methods, and the target double antibody was obtained after identification.
  • the humanized anti-PVRIG/TIGIT bispecific antibody was tested for binding to human and cyno TIGIT, and blocking the binding of human TIGIT to ligand, wherein the PVRIG protein was replaced with TIGIT , And replace PVRL2 with PVR.
  • the results are shown in Table 30 and Figures 7A-7E. The results showed that each double antibody can bind to human and cynomolgus TIGIT and block TIGIT from binding to PVR.
  • Biacore was used to detect the affinities of humanized bispecific antibodies with human PVRIG, cynomolgus PVRIG, and human TIGIT.
  • the humanized bispecific antibody was captured on the Protein A biosensor chip (GE lifesciences, 29127557) of the Biacore instrument (Biacore X100, GE), and then flowed through a series of human PVRIG antigen (AcroBiosystem , PVG-H52H4), Cynomolgus PVRIG antigen (SEQ ID NO:1), or human TIGIT antigen (AcroBiosystem, TIT-H52H3).
  • Biacore instrument (Biacore X100, GE) was used to detect the reaction signal in real time to obtain the binding and dissociation curves.
  • the data obtained from the experiment was fitted with BiacoreX100 evaluation software2.0GE software with a (1:1) Binding model, and the affinity value was obtained, as shown in Table 31.
  • Example 20 Mixed lymphocyte reaction (MLR) experiment of humanized anti-PVRIG/TIGIT bispecific antibody
  • the ability of the humanized anti-PVRIG/TIGIT bispecific antibody to activate T cells was tested.
  • the experimental results are shown in Figure 8 and Table 32.
  • the results show that the humanized anti-PVRIG/TIGIT bispecific antibody 1708-151H8 has obvious ability to activate T cells and promote the secretion of IFN ⁇ by T cells.
  • the bispecific antibody is more active than the anti-PVRIG antibody 151H8 alone and the anti-TIGIT antibody 1708 alone.
  • Example 21 Evaluation of anti-tumor effect of anti-PVRIG/TIGIT bispecific antibody in a mouse subcutaneous xenograft model of human melanoma A375 mixed with human PBMC
  • NCG mice female, 4-8 weeks old, weighing about 18-22 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. All NCG mice were cultured under the conditions of SPF animal room IVC constant temperature and pressure system.
  • A375 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS). A375 cells in the exponential growth phase were collected, and HBSS was resuspended to a suitable concentration for subcutaneous tumor inoculation in NCG mice. The A375 cells used for co-cultivation need to be treated with Mitomycin C for 2 hours, and then washed three times with PBS. Take normal human peripheral blood, separate human PBMC by density gradient centrifugation, and count. Then, PBMC was resuspended to a concentration of 3 ⁇ 10 6 cells/mL with 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-cultivation, PBMCs were collected, and freshly digested A375 cells were collected at the same time. Each mouse was inoculated: 5 ⁇ 10 5 PBMCs, 4 ⁇ 10 6 A375 cells; inoculation volume: 0.2 mL/mouse (containing 50% Matrigel); inoculated subcutaneously on the right side of female NCG mice. The mice were administered in groups randomly according to their body weight. The detailed administration method, dosage and route of administration are shown in Table 34. The day of group administration is day 0. Since the molecular weights of the anti-PVRIG antibody and the anti-TIGIT antibody are different, this 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; Dosing volume: Adjust the dosing volume (0.1mL/10g) according to the weight of the tumor-bearing mice.)
  • mice weight and tumor volume were measured twice a week.
  • the experimental results are shown in Tables 35-36 and Figures 9A-9B, respectively.
  • mice were administered in groups randomly according to their body weight.
  • the detailed administration method, dosage and route of administration are shown in Table 37.
  • the group administration day is the 0th day.
  • N Number of animals used; i.p.: intraperitoneal injection; Q2D: once every two days; Dosing volume: Adjust the dosing volume (0.1mL/10g) according to the weight of the tumor-bearing mice.)
  • mice weight and tumor volume were measured twice a week.
  • the experimental results are shown in Tables 38-39 and Figures 10A-10B, respectively.
  • the 1708-30H2IgG4 and 1708-151H7IgG4 double antibody groups can effectively inhibit tumor growth at low doses, which is significantly different from the control group (see Figure 10A and Figure 10B).

Abstract

PVRIG结合蛋白及其医药用途。具体而言，抗PVRIG单域抗体及抗PVRIG和TIGIT的双特异性抗体，含有所述抗体的药物组合物及治疗癌症的方法和制药用途。

Description

PVRIG结合蛋白及其医药用途
本公开要求2020年03月13日提交的中国专利申请(申请号CN202010174835.4)的优先权。
技术领域
本公开涉及PVRIG结合蛋白，例如抗PVRIG抗体及其与抗TIGIT抗体形成的双特异性抗体，及其作为药物用于治疗癌症。
背景技术
癌症是当今人类社会长期面临的最大健康挑战。传统的疗法如手术、化疗和放疗在治疗播散的实体肿瘤中收效甚微。肿瘤免疫治疗是肿瘤治疗领域一个热点，其中T细胞的肿瘤免疫治疗又处于其核心位置。肿瘤免疫治疗是充分利用、调动肿瘤患者体内的杀伤性T细胞，对肿瘤进行杀伤作用，它可能是最有效的也是最安全的***的途径。肿瘤免疫疗法目前对治疗几种不同类型的癌症，包括播散的转移性肿瘤已具有很好的前景。
人体内T细胞的活化采取了两条信号通路的***，除了需要通过抗原呈递细胞(APC)递呈MHC-抗原肽给T细胞提供第一信号外，还需要一系列协同刺激分子提供第二信号，进而才能使T细胞产生正常的免疫应答。这个双信号通路***对体内免疫***的平衡起着至关重要的作用，它严格调控机体对自身和非自身抗原产生不同的免疫应答。如果缺少协同刺激分子提供的第二信号，将会导致T细胞的无应答或持续特异性免疫应答，从而产生耐受。因此，第二信号通路在机体免疫应答的整个过程中起着非常关键的调节作用。
PVRIG，又名CD112R，是细胞表面表达的蛋白，与TIGIT、CD96和CD226等同属于B7/CD28超家族，在免疫***中其重要作用。它包含一个胞外区，一个跨膜区，和一个胞内区。当其配体PVRL2(又叫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在很多实体瘤中都有高表达，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，可以有效抑制肿瘤的生长，并与PD-1和TIGIT抑制剂发生协调作用。
另一个所关注的靶标TIGIT在淋巴细胞上高度表达，包括浸润不同类型肿瘤的肿瘤浸润淋巴细胞(TIL)和Treg。已经证明TIGIT序号传导与其同源配体PVR(也称为CD155)的接合通过其细胞质ITIM结构域直接抑制NK细胞的细胞毒性。PVR也在肿瘤中广泛表达，表明TIGIT-PVR信号轴可能是癌症的主要免疫逃逸机制。
但是，目前尚未有PVRIG/TIGIT双特异性抗体药物进入临床。Compugen公司的COM701是全球首个获FDA批准进入临床的针对PVRIG的人源化杂交瘤抗体，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。
现有技术中仍然缺乏能够抑制体内癌症或肿瘤生长的高亲合力、高选择性、高生物活性的抗PVRIG抗体、抗PVRIG/TIGIT双特异性抗体，本公开旨在提供这样的抗体，通过阻断PVRIG和/或TIGIT的抑制通路，活化免疫，以治疗癌症。
发明内容
本公开提供PVRIG结合蛋白、抗PVRIG抗体(例如VHH)及其与抗TIGIT抗体的双特异性抗体，及其编码核酸、载体、宿主细胞、药物组合物、用于治疗癌症的方法和制药用途。
第一方面，本公开提供PVRIG结合蛋白或抗PVRIG抗体。
一些实施方案中，PVRIG结合蛋白包含至少一个免疫球蛋白单一可变结构域，所述至少一个免疫球蛋白单一可变结构域包含三个互补决定区CDR1、CDR2和CDR3，其中：
CDR1选自SEQ ID NO：7、10、13、16、19、22、25、28、31、34、37、40、43、46、49、52、55、58、61、64任一所示的氨基酸序列或与之具有3、2、1个或更多氨基酸差异的氨基酸序列，和/或
CDR2选自SEQ ID NO：8、11、14、17、20、23、26、29、32、35、38、41、44、47、50、53、56、59、62、65任一所示的氨基酸序列或与之具有3、2、1个或更多氨基酸差异的氨基酸序列，和/或
CDR3选自SEQ ID NO：9、12、15、18、21、24、27、30、33、36、39、42、45、48、51、54、57、60、63、66、150、151任一所示的氨基酸序列或与之具有3、2、1个或更多氨基酸差异的氨基酸序列。
其中，SEQ ID NO：7-21、150、151是依据Kabat编码规则，SEQ ID NO：22-36是依据Chothia编码规则，SEQ ID NO：37-51是依据IMGT编码规则，SEQ ID NO：52-66是依据AbM编码规则。
一些实施方案中，PVRIG结合蛋白包含至少一个免疫球蛋白单一可变结构域，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO：2、75-79任一所示序列中的CDR1、CDR2、CDR3；或包含SEQ ID NO：3、80-84任一所示序列中的CDR1、CDR2、CDR3；或包含SEQ ID NO：4、86-90任一所示序列中的CDR1、CDR2、CDR3；或包含SEQ ID NO：5、91-95任一所示序列中的CDR1、CDR2、CDR3；或包含SEQ ID NO：6、96-100任一所示序列中的CDR1、CDR2、CDR3，所述CDR1、CDR2、CDR3是根据Kabat、IMGT、Chothia、AbM或Contact编号***定义的，一些具体实施方案中，CDR是根据Kabat编号规则确定的。
一些实施方案中，依据Kabat编码规则，PVRIG结合蛋白的免疫球蛋白单一可 变结构域包含三个互补决定区CDR1、CDR2和CDR3，其中：
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：7、8、9所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：7、8、150所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：10、11、12所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：10、11、151所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：13、14、15所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：16、17、18所示；或
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：19、20、21所示。
一些实施方案中，依据Chothia编码规则，PVRIG结合蛋白的免疫球蛋白单一可变结构域包含三个互补决定区CDR1、CDR2和CDR3，其中：
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：22、23、24所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：25、26、27所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：28、29、30所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：31、32、33所示；或
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：34、35、36所示。
一些实施方案中，依据IMGT编码规则，PVRIG结合蛋白的免疫球蛋白单一可变结构域包含三个互补决定区CDR1、CDR2和CDR3，其中：
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：37、38、39所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：40、41、42所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：43、44、45所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：46、47、48所示；或
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：49、50、51所示。
一些实施方案中，依据AbM编码规则，PVRIG结合蛋白的免疫球蛋白单一可变结构域包含三个互补决定区CDR1、CDR2和CDR3，其中：
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：52、53、54所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：55、56、57所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：58、59、60所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：61、62、63所示；或
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：64、65、66所示。
在一些实施方案中，提供了PVRIG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CDR1、CDR2和CDR3，其中根据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、8和9所示；或
如SEQ ID NO：7、8和150所示；或
如SEQ ID NO：10、11和12所示；或
如SEQ ID NO：10、11和151所示；或
如SEQ ID NO：13、14和15所示；或
如SEQ ID NO：16、17和18所示；或
如SEQ ID NO：19、20和21所示。
在一些实施方案中，本公开的PVRIG结合蛋白是抗体或其抗原结合片段，优选为VHH抗体，更优选为人源化的和/或经亲合力成熟的VHH抗体。
在一些实施方案中，本公开的PVRIG结合蛋白的免疫球蛋白单一可变结构域的氨基酸序列如SEQ ID NO：2-6中任一所示，或与之具有至少80％、至少90％、至少95％、至少98％或至少99％的序列同一性。一些实施方案中，PVRIG结合蛋白为变体蛋白，其在上述PVRIG结合蛋白的CDR1具有3、2、1个或更多氨基酸差异，和/或在CDR2具有3、2、1个或更多氨基酸差异，和/或在CDR3具有3、2、1个或更多氨基酸差异。
一些实施方案中，提供抗PVRIG抗体，其包含如上PVRIG结合蛋白中的CDR1、CDR2、CDR3。其可以人源化的和/或经亲合力成熟的。一些具体实施方案中，抗PVRIG抗体的氨基酸序列如SEQ ID NO：2-6、75-84、86-100任一所示，或与之具有至少80％、至少90％、至少95％、至少98％、至少99％序列同一性。一些具体实施方案中，抗PVRIG单域抗体与人IgG1、IgG2、IgG3、IgG4的Fc区连接，例如，其与有S228P、F234A、L235A和/或K447A突变的IgG4的Fc区连接(例如SEQ ID NO:101或153所示)。
一些实施方案中，本公开PVRIG结合蛋白中的免疫球蛋白单一可变结构域是单域抗体(VHH)，一些具体实施方案中，所述VHH是人源化的和/或经亲合力成熟的VHH。
一些实施方案中，本公开的PVRIG结合蛋白包含抗体。
一些实施方案中，本公开的PVRIG结合蛋白是抗体(例如，VHH)。
一些实施方案中，本公开的PVRIG结合蛋白是骆驼抗体、人源化抗体、全人抗体。
一些实施方案中，本公开的PVRIG结合蛋白或其中的免疫球蛋白单一可变结构域为骆驼抗体，其中VHH的氨基酸序列如SEQ ID NO：2-6任一所示，或与之具有至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％序列同一性。
一些实施方案中，本公开的PVRIG结合蛋白或其中的免疫球蛋白单一可变结构域为人源化抗体，抗体的框架区为人种系模板的重链框架区，如IGHV3-7，具体如IGHV3-7*01、IGHV3-30*02。
一些具体实施方案中，本公开的抗PVRIG人源化抗体的氨基酸序列如SEQ ID NO：75-84、86-100任一所示，或与之具有至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％序列同一性。
在一些实施方案中，本公开的PVRIG结合蛋白包含或为人源化抗体，其包含人种系模板的重链框架区。
在一些实施方案中，所述人种系模板的重链框架区为IGHV3-7*01或IGHV3-30*02。
在一些实施方案中，所述人源化抗体的免疫球蛋白单一可变结构域的氨基酸序列如SEQ ID NO：75-84和86-100中任一所示，或与之具有至少90％、至少95％、至少98％或至少99％序列同一性。
一些实施方案中，本公开的PVRIG结合蛋白还包含人免疫球蛋白Fc区，例如人IgG1、IgG2、IgG3、IgG4的Fc区。一些具体实施方案中，所述人免疫球蛋白Fc区是人IgG4的Fc区。一些具体实施方案中，所述人免疫球蛋白Fc区是人IgG1的Fc区。Fc区可以有突变，例如S228P、F234A、L235A和/或K447A的氨基酸突变(例如SEQ ID NO：101或153所示)。
一些实施方案中，本公开的PVRIG结合蛋白中，能够特异性结合PVRIG的免疫球蛋白单一可变结构域直接或通过接头与免疫球蛋白Fc区连接。所述接头可以是长1-20、1-30、1-40、1-50个或更多个氨基酸、无二级以上结构的非功能性氨基酸序列。所述接头可以是柔性接头，例如GS、GAP、ASGS、G 4S、(G 4S) 2、(G 4S) 3、(G 4S) 4、(G 4S) 5、(G 4S) 6、YGNGT、(YGNGT) 2、(YGNGT) 3、(YGNGT) 4、(YGNGT )5、(YGNGT )6等。
一些具体实施方案中，本公开的PVRIG结合蛋白中Fc区允许所述PVRIG结合蛋白形成包含两个或四个PVRIG结合结构域的二聚体分子。这样的PVRIG结合蛋白也称为二价或四价PVRIG结合蛋白。所述二聚体例如是同二聚体。
本公开的PVRIG结合蛋白或抗PVRIG抗体具有下述特征中的至少一个：
(a)结合人PVRIG的K D值小于1×10 -7M；
(b)阻断PVRIG与其配体(例如，PVRL2)的相互作用；
(c)解除树突状细胞对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；
(e)抑制肿瘤生长。
本公开的PVRIG结合蛋白或抗PVRIG抗体结合PVRIG的K D值可以小于1×10 -7M，小于1×10 -8M，小于1×10 -9M，小于1×10 -10M。
本公开的PVRIG结合蛋白或抗PVRIG抗体能够抑制肿瘤生长至少约10％，至少约20％，至少约30％，至少约40％，至少约50％，至少约60％，至少约70％，至少约80％。
本公开的PVRIG结合蛋白或抗PVRIG抗体可以是单体，和/或PEG化的，和/或糖基化的，和/或白蛋白缀合或融合的，和/或Fc融合的，和/或羟乙基化的，和/或去除O-糖基化的。
第二方面，本公开提供抗PVRIG的双特异性抗体。
一些实施方案中，提供双特异性抗体，包含第一抗原结合结构域和第二抗原结合结构域，其中，第一抗原结合结构域特异性结合PVRIG。
一些实施方案中，本公开的双特异性抗体的第一抗原结合结构域特异性结合 PVRIG，所述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(如VHH)，所述至少一个免疫球蛋白单一可变结构域(如VHH)包含三个互补决定区CDR1、CDR2和CDR3，其中：
CDR1选自SEQ ID NO：7、10、13、16、19、22、25、28、31、34、37、40、43、46、49、52、55、58、61、64任一所示的氨基酸序列或与之具有3、2、1个或更多氨基酸差异的氨基酸序列，和/或
CDR2选自SEQ ID NO：8、11、14、17、20、23、26、29、32、35、38、41、44、47、50、53、56、59、62、65任一所示的氨基酸序列或与之具有3、2、1个或更多氨基酸差异的氨基酸序列，和/或
CDR3选自SEQ ID NO：9、12、15、18、21、24、27、30、33、36、39、42、45、48、51、54、57、60、63、66、150、151任一所示的氨基酸序列或与之具有3、2、1个或更多氨基酸差异的氨基酸序列。
其中，SEQ ID NO：7-21是依据Kabat编码规则，SEQ ID NO：22-36是依据Chothia编码规则，SEQ ID NO：37-51是依据IMGT编码规则，SEQ ID NO：52-66是依据AbM编码规则。
一些实施方案中，双特异性抗体中特异性结合PVRIG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，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO：2、75-79任一所示序列中的CDR1、CDR2、CDR3；或包含SEQ ID NO：3、80-84任一所示序列中的CDR1、CDR2、CDR3；或包含SEQ ID NO：4、86-90任一所示序列中的CDR1、CDR2、CDR3；或包含SEQ ID NO：5、91-95任一所示序列中的CDR1、CDR2、CDR3；或包含SEQ ID NO：6、96-100任一所示序列中的CDR1、CDR2、CDR3，所述CDR1、CDR2、CDR3是根据Kabat、IMGT、Chothia、AbM或Contact编号***定义的，一些具体实施方案中，CDR是根据Kabat编号规则确定的。
一些具体实施方案中，依据Kabat编码规则，特异性结合PVRIG的第一抗原结合结构域(如VHH)包含三个互补决定区CDR1、CDR2和CDR3，其中：
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：7、8、9所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：7、8、150所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：10、11、12所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：10、11、151所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：13、14、15所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：16、17、18所示；或
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：19、20、21所示。
一些具体实施方案中，依据Chothia编码规则，特异性结合PVRIG的第一抗原结合结构域(如VHH)包含三个互补决定区CDR1、CDR2和CDR3，其中：
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：22、23、24所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：25、26、27所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：28、29、30所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：31、32、33所示；或
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：34、35、36所示。
一些具体实施方案中，依据IMGT编码规则，特异性结合PVRIG的第一抗原结合结构域(如VHH)包含三个互补决定区CDR1、CDR2和CDR3，其中：
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：37、38、39所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：40、41、42所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：43、44、45所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：46、47、48所示；或
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：49、50、51所示。
一些具体实施方案中，依据AbM编码规则，特异性结合PVRIG的第一抗原结合结构域(如VHH)包含三个互补决定区CDR1、CDR2和CDR3，其中：
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：52、53、54所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：55、56、57所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：58、59、60所示；
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：61、62、63所示；或
CDR1、CDR2、CDR3的氨基酸序列分别如SEQ ID NO：64、65、66所示。
一些实施方案中，本公开的双特异性抗体的第一抗原结合结构域(如VHH)包含SEQ ID NO：2-6、75-84、86-100任一所示氨基酸序列，或与之具有至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％同一性的序列。
一些实施方案中，本公开的双特异性抗体中：
第一抗原结合结构域为第一抗体，其是VHH；
第二抗原结合结构域为第二抗体，其包括重链(HC)和轻链(LC)；
所述VHH作为第一抗***于第二抗体的重链或轻链的N端和/或C端。
一些具体实施方案中，本公开的双特异性抗体包含1个第二抗体和2个VHH的第一抗体；所述第二抗体包括两条HC和两条LC，第二抗体的一条HC的VH与一条LC的VL形成抗原结合部位，另一条HC的VH与另一条LC的VL形成抗原结合部位。
一些具体实施方案中，本公开的双特异性抗体中的一个VHH的第一抗***于第二抗体的重链或轻链的N端，另一个VHH的第一抗***于第二抗体的重链或轻链的C端。
一些具体实施方案中，本公开的双特异性抗体中每个VHH的第一抗体分别位于第二抗体的两条重链或两条轻链的N端；或者，每个VHH的第一抗体分别位于第二抗体的两条重链或两条轻链的C端。
一些具体实施方案中，本公开的双特异性抗体中的每个VHH的第一抗体分别 位于第一抗体的两条重链的N端；或者，每个VHH的第一抗体分别位于第一抗体的两条重链的C端；
一些具体实施方案中，本公开的第一抗体可以连接有1、2、3、4、5、6、7、8个VHH的第二抗体，所述VHH的第二抗体可以是相同的或不同的，可以均连接在第一抗体的重链N端，或均连接在第一抗体的重链C端，或均连接在第一抗体的轻链N端，或均连接在第一抗体的轻链C端，或重链N端、重链C端、轻链N端、轻链C端的任意组合。
一些具体实施方案中，本公开的双特异性抗体中的VHH的第一抗体直接或通过连接子与第二抗体的每条重链的N端或C端连接。所述连接子选自：如(G mS n) x或(GGNGT) x或(YGNGT) x所示的氨基酸序列，其中m、n各自独立地选自1-8的整数(例如，1、2、3、4、5、6、7或8)，x独立地选自1-20的整数(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20)。例如，连接子为G 4S、(G 4S) 2、(G 4S) 3、(G 4S) 4、(G 4S) 5、(G 4S) 6所示的氨基酸序列。
一些实施方案中，本公开的双特异性抗体的第二抗体的重链包含重链可变区(VH)和重链恒定区(CH)，轻链包含轻链可变区(VL)和轻链恒定区(CL)。第二抗体可以为全长抗体。
一些实施方案中，本公开的双特异性抗体的第二抗体的重链为IgG同种型，例如IgG1、IgG2、IgG3或IgG4，例如为IgG1同种型；和/或，所述第二抗体的轻链为Kappa同种型。
一些实施方案中，本公开的双特异性抗体的第二抗体的两条HC包含相同的CDR和/或两条LC包含相同的CDR。一些具体实施方案中，第二抗体的两条HC包含相同的VH和/或两条LC包含相同的VL。一些具体实施方案中，第二抗体的两条HC具有相同的氨基酸序列和/或两条LC具有相同的氨基酸序列。
一些实施方案中，本公开的双特异性抗体的两个VHH的第一抗体具有相同或不相同的氨基酸序列。例如，两个所述VHH的第一抗体具有相同的氨基酸序列。
一些实施方案中，本公开的双特异性抗体包含两条第一多肽链和两条第二多肽链，其中对于每条多肽链：a)第一多肽链各自独立地包含VHH的第一抗体和第二抗体的重链(HC)；和b)第二多肽链各自独立地包含第二抗体的轻链(LC)；其中，VHH通过接头与第一抗体的HC的N端和/或C端相连。
或者，i)第一多肽链各自独立地包含第二抗体的重链(HC)；和ii)第二多肽链各自独立地包含VHH的第一抗体和第二抗体的轻链(LC)；其中，VHH直接或通过接头与第二抗体的LC的N端和/或C端相连。
一些具体实施方案中，本公开双特异性抗体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，本公开的双特异性抗体的第二抗原结合结构域为任意的抗TIGIT抗体。此处全文引入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)。
一些实施方案中，本公开双特异性抗体的第二抗原结合结构域为第二抗体。此处全文引入WO2019062832中的抗TIGIT抗体作为第二抗体。所述第二抗体中：
重链可变区包含分别如SEQ ID NO：115、116和117所示的HCDR1、HCDR2和HCDR3，轻链可变区包含分别如SEQ ID NO：118、119和120所示的LCDR1、LCDR2和LCDR3；或
重链可变区包含分别如SEQ ID NO：121、122和123所示的HCDR1、HCDR2和HCDR3；轻链可变区包含分别如SEQ ID NO：124、125和126所示的LCDR1、LCDR2和LCDR3；或
重链可变区包含分别如SEQ ID NO：127、128和129所示的HCDR1、HCDR2和HCDR3；轻链可变区包含分别如SEQ ID NO：130、131和132所示的LCDR1、LCDR2和LCDR3；或
重链可变区包含分别如SEQ ID NO：133、134和135所示的HCDR1、HCDR2和HCDR3；轻链可变区包含分别如SEQ ID NO：136、137和138所示的LCDR1、LCDR2和LCDR3；或
重链可变区包含分别如SEQ ID NO：139、140和141所示的HCDR1、HCDR2和HCDR3；轻链可变区包含分别如SEQ ID NO：142、143和144所示的LCDR1、LCDR2和LCDR3。
一些具体实施方案中，本公开双特异性抗体中的第一抗原结合结构域或第一抗体(如VHH)包含如SEQ ID NO：7、8、9所示的CDR1、CDR2、CDR3，或包含如SEQ ID NO：7、8、150所示的CDR1、CDR2、CDR3；第二抗原结合结构域或第二抗体的重链可变区包含分别如SEQ ID NO：121、122、123所示的HCDR1、HCDR2、HCDR3，轻链可变区包含分别如SEQ ID NO：124、125、126所示的LCDR1、LCDR2、LCDR3。
一些具体实施方案中，本公开双特异性抗体中的第一抗原结合结构域或第一抗体(如VHH)包含如SEQ ID NO：10、11、12所示的CDR1、CDR2、CDR3，或包含如SEQ ID NO：10、11、151所示的CDR1、CDR2、CDR3；第二抗原结合结构域或第二抗体的重链可变区包含分别如SEQ ID NO：121、122、123所示的HCDR1、HCDR2、HCDR3，轻链可变区包含分别如SEQ ID NO：124、125、126所示的LCDR1、LCDR2、LCDR3。
一些具体实施方案中，本公开双特异性抗体中的VHH的第一抗体包含如SEQ ID NO：6、79、81、92、98、99之一所示的氨基酸序列或与之具有至少70％、至 少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性；第二抗体包含如SEQ ID NO：145-147任一项所示的VH、SEQ ID NO：148-149任一项所示的VL或如SEQ ID NO：102所示的HC和SEQ ID NO：103所示的LC或与所述VH、HC或VL、LC具有至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性。
一些具体实施方案中，本公开双特异性抗体包含：
如SEQ ID NO：104所示第一多肽链，如SEQ ID NO：103所示第二多肽链；
如SEQ ID NO：105所示第一多肽链，如SEQ ID NO：103所示第二多肽链；
如SEQ ID NO：102所示第一多肽链，如SEQ ID NO：106所示第二多肽链；
如SEQ ID NO：102所示第一多肽链，如SEQ ID NO：107所示第二多肽链；
如SEQ ID NO：108-112、114任一所示第一多肽链，如SEQ ID NO：103所示所示第二多肽链；或
与上述第一多肽链和/或第二多肽链具有至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性的变体。
一些实施方案中，本公开的PVRIG结合蛋白、抗PVRIG抗体及其与抗TIGIT的双特异性抗体的Fc区有突变，包括选自以下的一个或多个氨基酸突变：
i)改变CH1的铰链区中的半胱氨酸残基的数量，以便于轻链和重链的组装或提高或降低抗体的稳定性；
ii)使得与FcγRIIIa的结合增强的突变，以引起增强的ADCC，使得与FcγRIIb的结合减弱的突变，例如236A、239D、239E、332E、332D、239D/332E、267D、267E、328F、267E/328F、236A/332E、239D/332E/330Y、239D、332E/330L、299T、297N或其任意组合；
iii)延长生物半衰期的突变，例如T252L、T254S、T256F、428L、434A、434S428L/434S或其任意组合；
iv)234、235、236、237、297、318、320和322位的一个或多个氨基酸突变或其任意组合，以改变抗体对效应配体的亲合力，同时保留亲本抗体的抗原结合能力；
v)329、331和322位的一个或多个氨基酸突变或其任意组合，使得抗体具有改变了的C1q结合和/或降低或消除补体依赖性细胞毒性(CDC)；
vi)231-239内的一个或多个氨基酸突变或其任意组合，使得抗体固定补体的能力改变；
vii)238、239、248、249、252、254、255、256、258、265、267、268、269、270、272、276、278、280、283、285、286、289、290、292、293、294、295、296、298、301、303、305、307、309、312、315、320、322、324、326、327、329、330、331、333、334、335、337、338、340、360、373、376、378、382、388、389、398、414、416、419、430、434、435、437、438或439中的一个或多 个氨基酸突变或其任意组合，以提高ADCC的能力和/或提高抗体对Fcγ受体的亲合力；
viii)S228P、F234A、L235A和/或K447A的氨基酸突变；
ix)S354C，E356D，M358L和/或T366W的氨基酸突变。
一些实施方案中，提供与本公开的PVRIG结合蛋白、PVRIG\TIGIT结合蛋白、抗PVRIG单域抗体、抗PVRIG和TIGIT的双特异性抗体竞争性结合相同表位的抗体。
在一些实施方案中，本公开提供了PVRIG/TIGIT结合蛋白，其包含特异性结合PVRIG的第一抗原结合结构域和特异性结合TIGIT的第二抗原结合结构域，
所述特异性结合PVRIG的第一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：3、80-84任一所示序列中的CDR1、CDR2、CDR3；或
SEQ ID NO：2、75-79任一所示序列中的CDR1、CDR2、CDR3；或
SEQ ID NO：4、86-90任一所示序列中的CDR1、CDR2、CDR3；或
SEQ ID NO：5、91-95任一所示序列中的CDR1、CDR2、CDR3；或
SEQ ID NO：6、96-100任一所示序列中的CDR1、CDR2、CDR3；
所述CDR1、CDR2、CDR3是根据Kabat、IMGT、Chothia、AbM或Contact编号***定义的。
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、8和9所示；或
如SEQ ID NO：7、8和150所示；或
如SEQ ID NO：10、11和12所示；或
如SEQ ID NO：10、11和151所示；或
如SEQ ID NO：13、14和15所示；或
如SEQ ID NO：16、17和18所示；或
如SEQ ID NO：19、20和21所示。
在具体的实施方案，所述的PVRIG/TIGIT结合蛋白的第一抗原结合结构域包含SEQ ID NO：2-6、75-84和86-100中任一所示或与之具有至少90％、至少95％、至少98％、至少99％序列同一性的氨基酸序列。
在具体的实施方案，所述的PVRIG/TIGIT结合蛋白的第二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：115、116和117所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：118、119和120所示的LCDR1、LCDR2和LCDR3；
所述重链可变区包含分别如SEQ ID NO：121、122和123所示的HCDR1、 HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：124、125和126所示的LCDR1、LCDR2和LCDR3；
所述重链可变区包含分别如SEQ ID NO：127、128和129所示的HCDR1、HCDR2和HCDR3,所述轻链可变区包含分别如SEQ ID NO：130、131和132所示的LCDR1、LCDR2和LCDR3；
所述重链可变区包含分别如SEQ ID NO：133、134和135所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：136、137和138所示的LCDR1、LCDR2和LCDR3；或
所述重链可变区包含分别如SEQ ID NO：139、140和141所示的HCDR1、HCDR2和HCDR3,所述轻链可变区包含分别如SEQ ID NO：142、143和144所示的LCDR1、LCDR2和LCDR3。
在具体的实施方案中，所述的PVRIG/TIGIT结合蛋白的第二抗原结合结构域包含全长重链(HC)和全长轻链(LC)；
具体地，所述全长重链为IgG1或IgG4同种型，所述全长轻链为Kappa同种型；
更具体地，所述重链序列为SEQ ID NO：102所示或与之具有至少90％序列同一性，所述轻链序列为SEQ ID NO：103所示或与之具有至少90％序列同一性。
在本公开的PVRIG/TIGIT结合蛋白的具体实施方案中，所述特异性结合PVRIG的第一抗原结合结构域的VHH位于特异性结合TIGIT的第二抗原结合结构域的重链可变区或全长重链的N端；
所述特异性结合PVRIG的第一抗原结合结构域的VHH位于特异性结合TIGIT的第二抗原结合结构域的重链可变区或全长重链的C端；
所述特异性结合PVRIG的第一抗原结合结构域的VHH位于特异性结合TIGIT的第二抗原结合结构域的轻链可变区或全长轻链的N端；和/或
所述特异性结合PVRIG的第一抗原结合结构域的VHH位于特异性结合TIGIT的第二抗原结合结构域的轻链可变区或全长轻链的C端。
在本公开的PVRIG/TIGIT结合蛋白的具体实施方案中，所述特异性结合PVRIG的第一抗原结合结构域的VHH与特异性结合TIGIT的第二抗原结合结构域直接或通过连接子相连接；
优选地，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的整数；
更优选地，所述连接子为(G 4S) 2、(G 4S) 3所示的氨基酸序列。
在一些实施方案中，本公开提供的PVRIG/TIGIT结合蛋白包含第一多肽链和第二多肽链，其中：
所述第一多肽链包含如SEQ ID NO：108-112和114中任一所示的氨基酸序列，所述第二多肽链包含如SEQ ID NO：103所示的氨基酸序列；或者
所述第一多肽链包含如SEQ ID NO：104或105所示的氨基酸序列，所述第二多肽链包含如SEQ ID NO：103所示的氨基酸序列；或者
所述第一多肽链包含如SEQ ID NO：102所示的氨基酸序列，所述第二多肽链包含如SEQ ID NO：106或107所示的氨基酸序列。
第三方面，本公开提供多核苷酸，其编码前述PVRIG结合蛋白、PVRIG/TIGIT结合蛋白、抗PVRIG抗体(如VHH)或抗PVRIG和TIGIT的双特异性抗体。所述多核苷酸可以是DNA或RNA。
一些实施方案中，提供多核苷酸组合物，包含：
第一核酸，编码包含本公开抗PVRIG和TIGIT的双特异性抗体的VH或HC；和第二核酸，编码包含本公开抗PVRIG和TIGIT的双特异性抗体的VL或LC。
第四方面，本公开提供含有如上所述的多核苷酸或多核苷酸组合物的表达载体或表达载体组合物，表达载体可以是真核表达载体、原核表达载体、病毒载体。
一些实施方案中，提供表达载体组合物，包含：
第一表达载体，包含上述多核苷酸组合物中第一核酸；和
第二表达载体，包含上述多核苷酸组合物中第二核酸。
第五方面，本公开提供用如上所述的表达载体、表达载体组合物转化的或含有所述表达载体、表达载体组合物的宿主细胞，其可以是真核细胞、原核细胞。
一些实施方案中，所述宿主细胞为细菌、酵母菌、哺乳动物细胞。一些具体实施方案中，所述宿主细胞为大肠杆菌、毕赤酵母、中国仓鼠卵巢(CHO)细胞或人胚肾(HEK)293细胞。
第六方面，本公开提供一种制备方法，包括：在如前所述的宿主细胞中表达PVRIG结合蛋白、抗PVRIG抗体(如VHH)、抗PVRIG和TIGIT的双特异性抗体，并自该宿主细胞中分离、回收PVRIG结合蛋白、抗PVRIG抗体(如VHH)、抗PVRIG和TIGIT的双特异性抗体。
在具体的实施方案中，本公开提供了制备PVRIG结合蛋白、PVRIG/TIGIT结合蛋白、或抗PVRIG抗体或其抗原结合片段的方法，包括：
在本公开的宿主细胞中本公开的多核苷酸，以及
从所述宿主细胞中分离表达的PVRIG结合蛋白、PVRIG/TIGIT结合蛋白或抗PVRIG抗体或其抗原结合片段。
第七方面，本公开提供组合物(如药物组合物)，其含有治疗有效量的如上所述的PVRIG结合蛋白、抗PVRIG抗体(如VHH)、抗PVRIG和TIGIT的双特异性抗体、或PVRIG/TIGIT结合蛋白，以及可药用的赋形剂、稀释或载体。
一些实施方案中，组合物(如药物组合物)中含有本公开的PVRIG结合蛋白或抗PVRIG抗体(如VHH)，和抗TIGIT抗体。所述TIGIT抗体可以是如前所述的任意抗TIGIT抗体，例如本公开表23、表24中的抗TIGIT抗体。以及，组合物种可以含有可药用的赋形剂、稀释或载体。
一些具体实施方案中，所述抗TIGIT抗体含有如SEQ ID NO：121、122、123所示的HCDR1、HCDR2、HCDR3，和轻链可变区包含分别如SEQ ID NO：124、 125、126所示的LCDR1、LCDR2、LCDR3。
在一些具体实施方式中，所述药物组合物单位计量中可含有0.01至99重量％的PVRIG结合蛋白、抗PVRIG抗体(如VHH)、抗PVRIG和TIGIT的双特异性抗体，或药物组合物单位剂量中含PVRIG结合蛋白、抗PVRIG抗体(如VHH)、抗PVRIG和TIGIT的双特异性抗体的量为0.1-2000mg，或1-1000mg。
第八方面，提供本公开的PVRIG结合蛋白、抗PVRIG抗体(如VHH)、抗PVRIG和TIGIT的双特异性抗体、PVRIG/TIGIT结合蛋白、编码多核苷酸本公开的PVRIG结合蛋白、抗PVRIG抗体(如VHH)、抗PVRIG和TIGIT的双特异性抗体、编码多核苷酸中的任一项或其任意组合，用于诊断、治疗、预防疾病的方法和制备药物、药物组合物的用途(例如，用于治疗或预防增殖性病症(例如癌症或肿瘤)或延缓相关病症进展)。
一些实施方案中，提供治疗、缓解受试者病症的方法，包含向所述受试者施用本公开的PVRIG结合蛋白、抗PVRIG抗体(如VHH)、抗PVRIG和TIGIT的双特异性抗体，所述病症是癌症。
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，包含向所述受试者施用本公开的PVRIG结合蛋白、抗PVRIG抗体(如VHH)、抗PVRIG和TIGIT的双特异性抗体，其中所述受试者的所述CTL的亚组被活化。
一些实施方案中，提供活化受试者的NK细胞的方法，包含向所述受试者施用本公开的PVRIG结合蛋白、抗PVRIG抗体(如VHH)、抗PVRIG和TIGIT的双特异性抗体，其中所述受试者的所述NK细胞的亚组被活化。
一些实施方案中，提供活化受试者的γδT细胞的方法，包含向所述受试者施用本公开的PVRIG结合蛋白、抗PVRIG抗体(如VHH)、抗PVRIG和TIGIT的双特异性抗体，其中所述受试者的所述γδT细胞的亚组被活化。
一些实施方案中，提供活化受试者的Th1细胞的方法，包含向所述受试者施用本公开的PVRIG结合蛋白、抗PVRIG抗体(如VHH)、抗PVRIG和TIGIT的双特异性抗体，其中所述受试者的所述Th1细胞的亚组被活化。
一些实施方案中，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，包括向所述受试者施用本公开的PVRIG结合蛋白、抗PVRIG抗体(如VHH)、抗PVRIG和TIGIT的双特异性抗体。
一些实施方案中，提供增加受试者体内的干扰素-γ产生和/或促炎性细胞因子分泌的方法，包括向所述受试者施用本公开的PVRIG结合蛋白、抗PVRIG抗体(如VHH)、抗PVRIG和TIGIT的双特异性抗体。
一些实施方案中，提供抑制受试者体内的PVRIG和PVLR2的相互作用的方法，包括向所述受试者施用本公开的PVRIG结合蛋白、抗PVRIG抗体(如VHH)、抗PVRIG和TIGIT的双特异性抗体。
一些实施方案中，提供治疗受试者的方法，包括向所述受试者或受试者施用本公开的PVRIG结合蛋白、抗PVRIG抗体(如VHH)、抗PVRIG和TIGIT的双特异性抗体。
一些具体实施方案中，上述受试者的病症为增殖性病症(例如癌症或肿瘤)或患有增殖性病症(例如癌症或肿瘤)。所述癌症或肿瘤选自以下或其组合：***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)。上述病症可以是与PVRIG和\或TIGIT异常表达相关的。一些具体实施方案中，所述癌症或肿瘤选自由以下癌症或其组合：三阴性乳腺癌、胃癌、肺癌(小细胞肺癌、非小细胞肺癌)、默克细胞癌、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症(MDS)。一些具体实施方案中，所述癌症或肿瘤选自由以下癌症或其组合：三阴性乳腺癌、胃癌、肺癌(小细胞肺癌、非小细胞肺癌)、默克细胞癌和高MSI癌。
一些实施方案中，上述受试者具有与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。本公开的方法尤其适合于治疗血管化肿瘤。
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，包括向所述受试者或受试者施用本公开的PVRIG结合蛋白、抗PVRIG抗体(如VHH)、抗PVRIG和TIGIT的双特异性抗体。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。
在一些实施方案中，提供了本公开上述的PVRIG结合蛋白、PVRIG/TIGIT结合蛋白、抗PVRIG抗体或其抗原结合片段、多核苷酸、或组合物，其用于治疗或延缓疾病，优选地，所述疾病为增殖性疾病；
更有选地，所述增殖性疾病为癌症；
更优选地，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，提供诊断疾病的方法，包含：
a)使来自受试者的组织与本公开的PVRIG结合蛋白或抗PVRIG抗体接触；并且
b)判定所述组织中PVRIG的过表达的存在作为疾病或病症存在的指示。
所述组织可以是血液样品、实体肿瘤活检样品。所述PVRIG结合蛋白或抗PVRIG抗体可以经过标记，进一步地，可以使结合到所述PVRIG结合蛋白或抗PVRIG抗体的第二标记抗体与前述样品接触。一些具体实施方案中，所述PVRIG结合蛋白或抗PVRIG抗体是被标记的，包括放射性同位素、染料(如具有生物素-抗生蛋白链菌素复合体)、造影剂、荧光化合物或分子以及用于磁共振成像(MRI)的增强剂(例如，顺磁性离子)。一些具体实施方案中，所述疾病或病症是如上所述的癌症或肿瘤、感染或脓毒症。
第九方面，本公开提供PVRIG结合蛋白的检测用途。
本公开提供检测PVRIG的组合物，所述组合物包含PVRIG结合蛋白或抗PVRIG抗体。本公开还提供用于体内或体外检测PVRIG的方法、***或装置，其包括使用PVRIG结合蛋白或抗PVRIG抗体。
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PVRIG结合蛋白或抗PVRIG抗体接触；(2)检测在PVRIG结合蛋白或抗PVRIG抗体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)与抗体接触；和(4)通过与参比样品比较，确定抗体和样品之间复合物形成的程度。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PVRIG。
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PVRIG结合蛋白或抗PVRIG抗体；和(2)检测在PVRIG结合蛋白或抗PVRIG抗体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。结合PVRIG的抗体可以直接或间接地用可检测物质标记以促进所结合的或未结合的抗体的检测。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PVRIG结合或不结合的抗体或使其可视化，检测在PVRIG结合蛋白或抗PVRIG抗体和PVRIG之间的复合物形成。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。一些实施方案中，通过竞争免疫测定法分析样品中PVRIG的存在，所述竞争免疫测定法使用以可检测物质标记的标准物和未标记的PVRIG结合蛋白或抗PVRIG抗体。检测或测定的活体样品可以是组织细胞、血液、血浆、血清、胰液、尿液、粪便、组织液或培养液。
一些实施方案中，出于检测目的，本公开的PVRIG结合蛋白或抗PVRIG抗体可以用荧光团和发色团标记。
一些实施方案中，还提供试剂盒，所述试剂盒包含与PVRIG结合蛋白或抗PVRIG抗体，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，抗体可以配制为药物组合物。
本公开实施例提供的PVRIG抗体、抗PVRIG和TIGIT双抗对PVRIG和/或TIGIT具有高特异性、高亲合力；人源化抗体的免疫原性大大降低，同时完全保留了优异的体内外活性；具有良好的大鼠和人体的代谢动力学特性；具有长半衰期、高生物利用度；具有良好的长期稳定性，无明显异常化学修饰，高浓度下无明显聚集，有较高的纯度和热稳定性；具有良好的增强T细胞和NK细胞活性、抑制 肿瘤发生发展的效果。
附图说明
图1为抗PVRIG抗体的PVRIG报告基因细胞活性检查结果。
图2为抗PVRIG抗体在NK细胞杀伤实验中激活NK细胞的活性检测结果。
图3为抗PVRIG抗体在MLR实验中激活T细胞的活性检测结果。
图4A-图4B为人源化的抗PVRIG抗体在PVRIG报告基因细胞活性检测结果。
图5A-图5B为人源化的抗PVRIG抗体在NK细胞杀伤实验中激活NK细胞的活性检测结果。
图6A-图6E分别显示人源化的抗PVRIG/TIGIT双特异性抗体对人PVRIG重组蛋白、人PVRIG过表达细胞、食蟹猴PVRIG重组蛋白、食蟹猴PVRIG过表达细胞的结合活性、以及阻断人PVRIG与人PVRL2结合的活性检测结果。
图7A-图7E分别显示人源化的抗PVRIG/TIGIT双特异性抗体对人TIGIT重组蛋白、人TIGIT过表达细胞、食蟹猴TIGIT重组蛋白、食蟹猴TIGIT过表达细胞的结合活性、以及阻断人TIGIT与人PVR结合的活性检测结果。
图8为人源化的抗PVRIG/TIGIT双特异性抗体在MLR实验中激活T细胞的活性检测结果。
图9A-图9B分别显示抗PVRIG/TIGIT双特异性抗体在人黑色素瘤A375混合人PBMC的小鼠皮下移植瘤模型中对小鼠体重和肿瘤体积的影响。
图10A-图10B分别显示抗PVRIG/TIGIT双特异性抗体在人黑色素瘤A375混合人PBMC的小鼠皮下移植瘤模型中对小鼠体重和肿瘤体积的影响。
具体实施方式
一、术语
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。除显而易见在本文件中的它处另有明确定义，否则本文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。
“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中的。完整的人类PVRIG序列可以GenBank登录号AAH73861.1找到。
“PVRIG结合蛋白”意指任何能够特异性结合PVRIG的蛋白或包含所述蛋白的任何分子。PVRIG结合蛋白可以包括针对PVRIG的如本公开定义的抗体、其抗原结合片段或其缀合物。PVRIG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“PVRIG结合蛋白”可以包含至少一个结合PVRIG 的免疫球蛋白单一可变结构域(如VHH)。在一些实施方案中，“PVRIG结合蛋白”可以包含2、3、4或更多个结合PVRIG的免疫球蛋白单一可变结构域(如VHH)。本公开的PVRIG结合蛋白除包含PVRIG的免疫球蛋白单一可变结构域外，也可包含接头和/或具有效应器功能的部分，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“PVRIG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PVRIG)的第一抗体和结合第二抗原(如TIGIT)的第二抗体，可选的，包括结合第三抗原的第三抗体，进一步可选的，包括结合第四抗原的第四抗体。
“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。
“抗体”或“免疫球蛋白”广义上涵盖传统的抗体(由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构抗体)，以及具有抗原结合活性的Fab、Fv、sFv、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(重链(VH)抗体、轻链(VL)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)，因而，本公开中使用的术语“抗体”包括全长抗体、其单个的链及其任意具有抗原结合活性的部分、结构域或片段以及包含其单个的链及任意具有抗原结合活性的部分、结构域或片段的多特异性抗体(包括但不限于抗原结合结构域或片段，分别例如VHH结构域或VH/VL结构域)。传统的抗体或免疫球蛋白通常是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。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中每类Ig都可以有κ(kappa)链或λ(lambda)链。在一些实施例中，本公开的抗体特异性地或基本上特异性地结合到PVRIG和/或TIGIT。
本公开的“抗体”包括但不限于：(i)由VL、VH、CL和CH1结构域组成的Fab片段；(ii)由VH和CH1结构域组成的Fd片段；(iii)F(ab’)2片段，一种包含两个连接着的Fab片段的二价片段；(vii)单链Fv分子(scFv)，其中VH结构域和VL结构域通过肽连接子连接，所述肽连接子允许两个结构域结合形成抗原结合位点；(Bird等人，1988，《科学(Science)》242：423-426；Huston等人，1988，《美国国 家科学院院刊(Proc.Natl.Acad.Sci.U.S.A.)》85：5879-5883)242，通过引用完全并入本文中)；(iv)“双功能抗体”或“三功能抗体”，通过基因融合构造的多价或多特异性片段(Tomlinson等人，2000，《酶学方法(Methods Enzymol.)》326：461-479；WO94/13804；Holliger等人，1993，《美国美国国家科学院院刊》90：6444-6448，全部通过引用完全并入本文中)；(v)“结构域抗体”或“dAb”(有时称为“免疫球蛋白单一可变结构域”)，包括来自其它物种的免疫球蛋白单一可变结构域，如啮齿动物(例如，如WO00/29004中所公开)、护士鲨和骆驼科V-HH dAb；(vi)SMIP(小分子免疫药物)、骆驼抗体、纳米抗体以及IgNAR；(vii)上述(i)-(vi)的人源化抗体。
在未特殊指明的情况下，本公开的抗体通常使用Kabat编号***。Kabat中的EU编号一般也用于恒定结构域和/或Fc结构域。
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，或所述细胞来源于如此修饰的细胞。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。
“单克隆抗体”和“单克隆抗体组合物”是指仅含有一个物种的能够与特定抗原表位发生免疫反应的抗原结合位点的抗体分子群体，而“多克隆抗体”和“多克隆抗体组合物”是指含有多个物种的能够与特定抗原相互作用的抗原结合位点的抗体分子群体。单克隆抗体组合物典型地对与其发生免疫反应的特定抗原展现出单一的结合亲合力。
“抗原”指用于免疫接种免疫活性的脊椎动物的分子，以产生识别抗原的抗体，或筛选表达文库(例如尤其是噬菌体、酵母或核糖体展示文库)。在本文中，抗原被更广义地定义，并且一般预期包括由抗体特异性识别的靶分子，因此包括用于产生抗体的免疫接种过程或用于选择抗体的文库筛选中使用的分子的一部分或模拟物。
“序列”(例如在“免疫球蛋白序列”、“抗体序列”、“单一可变结构域序列”、“VHH序列”或“蛋白序列”等的术语中)一般应理解为既包括相关氨基酸序列，又包括编码所述序列的核酸序列或核苷酸序列，除非本公开需要进一步限定的解释。
“多核苷酸”或“核酸”指任何长度的核苷酸链，包括DNA和RNA。核苷酸可为脱氧核糖核苷酸、核糖核苷酸、经修饰的核苷酸或碱基和/或其类似物、或者可通过DNA或RNA聚合酶掺入链内的任何底物。多核苷酸可包含经修饰的核苷酸，例如甲基化的核苷酸及其类似物。如果存在的话，可在链组装之前或链组装之后赋予对核苷酸结构的修饰。多核苷酸还可含有本领域一般已知的核糖或脱氧核糖糖的类似形式，包括例如2′-O-甲基-、2′-O-烯丙基、2′-氟-或2′-叠氮基-核糖、碳环糖类似物、α-或β-异头糖、差向异构糖(如***糖、木糖或来苏糖、吡喃糖、呋喃糖、景天庚酮糖)、无环类似物和无碱基核苷类似物如甲基核糖苷。
“同源性”或“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碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，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。
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、移除或转移至其他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(例如常规四肽链结构抗体的链或重链抗体的链)的球形区域，或是指基本上由这类球形区域组成的多肽。免疫球蛋白结构域的特征在于其维持抗体分子的免疫球蛋白折叠特征，其由排列在两个β折叠中任选由保守二硫键稳定的约7个反平行β折叠股的2层夹层组成。
“免疫球蛋白可变结构域”是指基本上由本领域及下文中分别称为“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”组成的免疫球蛋白结构域，其中所述框架区由本领域及下文中分别称为“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”间隔开。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。
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CDR的确定性描绘和包含抗体的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，尽管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，它们可缩短或延长。如本文使用的，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。本文使用的方法可利用根据这些方法中的任一种定义的CDR。对于包含超过一个CDR的任何给定实施例，可根据Kabat、Chothia、延伸的、AbM、IMGT、接触和/或构象定义中的任一个来定义CDR。
“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、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”以将所述可变结构域与存在于常规四肽链结构抗体中的重链可变结构域(其在本公开中称为“VH结构域”)以及轻链可变结构域(其在本公开中称为“VL结构域”)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(此与常规四肽链结构抗体中的VH或VL结构域相反，在该情况下表位由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”、“VHH结构域”、“VHH抗体片段”、“VHH抗体”以及“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。
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。其它的编号*** 或编码规则包括Chothia、IMGT、AbM。
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。
VHH结构域(单独或作为较大多肽的一部分)提供许多优于使用常规VH及VL结构域、scFv或常规抗体片段(例如Fab-或F(ab’)2-片段)的显著优势：
-仅需要单一结构域以高亲合力及高选择性结合抗原，从而使得既不需要存在两个单独结构域，也不需要确保该两个结构域以适当空间构象及构型存在(例如scFv一般需要使用经特别设计的接头)；
-VHH结构域可由单一基因表达且不需要翻译后折叠或修饰；
-VHH结构域可容易地改造成多价及多特异性格式；
-VHH结构域高度可溶且无聚集趋势；
-VHH结构域对热、pH、蛋白酶及其他变性剂或条件高度稳定，且因此可在制备、储存或运输中不使用冷冻设备，从而达成节约成本、时间及环境；
-VHH结构域易于制备且相对廉价，甚至在生产所需的规模上亦如此；
-VHH结构域与常规四肽链结构抗体及其抗原结合片段相比相对较小(大约15kDa或大小为常规IgG的1/10)，因此相比于常规四肽链结构抗体及其抗原结合片段，显示较高的组织渗透性且可以较高剂量给药；
-VHH结构域可显示所谓腔结合性质(尤其由于与常规VH结构域相比其延长的CDR3环)，从而可到达常规四肽链结构抗体及其抗原结合片段不可到达的靶及表位。
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。
“Fc变异体”或“变异体Fc”意指在Fc结构域中包含氨基酸修饰的蛋白质。本公开的Fc变异体根据构成其的氨基酸修饰来定义。因此，举例来说，S228P或228P是相对于亲本Fc多肽在位置228处具有脯氨酸取代的Fc变异体，其中编号是根据EU索引。WT氨基酸的身份可以不指明，在此情况下前述变异体称为228P。
“人源化”的例子包括可将源自骆驼科的VHH结构域通过以人常规四肽链结构抗体VH结构域中相应位置处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他修饰，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，且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小鼠蛋白成分，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”于其他“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(在因特网www.mrccpe.com.ac.uk/vbase可获得)，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进行亲合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。
“亲合力成熟”的抗体，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合力相比于其各自的亲本抗体有所增加。亲合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。
通常，本公开的抗体将以如于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(KD)，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PVRIG)。任何大于10 -4M的KD值一般都视为指示非特异性结合。抗原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”或可互换使用的“抗原决定簇”指抗体的互补位所结合的抗原上的任何抗原决定簇。抗原决定簇通常包含分子的化学活性表面基团，例如氨基酸或糖侧链，并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸，其可以是“线性”表位或“构象”表位。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。可使用本领域中熟知的许多表位定位技术鉴别给定抗原的表位(例如Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)，US4708871)。可使用本领域技术人员已知的常规技术，就与相同表位的结合竞争性筛选抗体。例如，可进行竞争和交叉竞争研究，以获得彼此竞争或交叉竞争与抗原结合的抗体(高通量筛选方法如参见WO03/48731)。因此，可使用本领域技术人员已知的常规技术，获得与本公开的抗体分子竞争结合PVRIG上的相同表位的抗体及其抗原结合片段。
“特异性结合”、“选择性结合”是指抗体与预定的抗原上的表位结合。通常，当使用重组人PVRIG、TIGIT或其表位作为分析物并使用抗体作为配体，在 仪器中通过表面等离子体共振(SPR)技术测定时，抗体以大约低于10 -7M或甚至更小的平衡解离常数(K D)与预定的抗原或其表位结合，并且其与预定抗原或其表位结合的亲合力是其与预定抗原(或其表位)或紧密相关的抗原之外的非特异性抗原(如BSA等)结合的亲合力的至少两倍。“识别抗原的抗体”在本文中可以与“特异性结合的抗体”互换使用。
“结合亲合力”在本文中用作两个分子(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，用于描述单价相互作用(固有活性)。两个分子之间的结合亲合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(参见Caceci等人，1984，Byte 9：340-362；Wong&Lohman，1993，PNAS 90：5428-5432)。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、以及本文其它地方例举的其它测定。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PVRIG的对照抗体)的K D值进行评价。在一些实施方案中，本公开的抗PVRIG抗体能够与其靶结合的亲合力比它与另一种非PVRIG分子结合的亲合力大至少2倍、10倍、50倍、100倍、200倍、500倍、1,000倍或10,000倍，这里不属于限制性定义。
“保守修饰”适用于氨基酸和核苷酸序列。对于特定的核苷酸序列，保守修饰是指编码相同或基本相同的氨基酸序列的那些核酸的相互置换，或在核苷酸不编码氨基酸序列的情况下，是指基本上相同的核苷酸序列。对于氨基酸序列，保守修饰是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)MolecμLar Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。
“氨基酸突变”包括氨基酸取代、缺失、***、修饰及其任意组合，以实现最终构建体，使得最终构建体拥有期望的特性，例如增强的稳定性、提高的活性。氨基酸序列缺失和***包括氨基和/或羧基端缺失和氨基酸***。优选的氨基酸突变是氨基酸取代。为了改变例如抗PVRIG抗体的结合特性，可以将非保守性的氨基酸取代，即将一个氨基酸用具有不同结构和/或化学特性的另一种氨基酸替换。优选的氨基酸取代包括用亲水性氨基酸替换疏水性氨基酸。氨基酸取代包括由非天然存在的氨基酸或由20种标准氨基酸的天然存在的氨基酸衍生物(例如4-羟脯氨酸、3-甲基组氨酸、鸟氨酸、高丝氨酸、5-羟赖氨酸)替换。可以使用本领域中公知的遗传或化学方法生成氨基酸突变，包括定点诱变、PCR、基因合成、化学修饰等方法。氨基酸突变可以发生在抗体的CDR区、FR区或Fc区。
对于Fc区的氨基酸突变，可以在本公开的抗体野生型的Fc序列上引入突变用于改变Fc介导的相关活性，所述突变包括但不限于：a).改变Fc介导的CDC活性的突变；b).改变Fc介导的ADCC活性的突变；或c).改变FcRn介导的体内半衰期的突变(参见Leonard G Presta，Current Opinion in Immunology 2008，20：460-470；Esohe E.Idusogie et al.，J Immunol 2000，164：4178-4184；RAPHAEL A.CLYNES et al.，Nature Medicine，2000，Volume 6，Number 4：443-446；Paul R.Hinton et al.，J Immunol，2006，176：346-356)。具体地，包括：对CH1的铰链区进行修饰以使得铰链区中的半胱氨酸残基的数量发生改变，例如增加或减少(参见US5,677,425，此处全文引入)。引入使得与FcγRIIIa的结合增强的突变(以引起增强的ADCC)，使得与FcγRIIb的结合减弱的突变，例如236A、239D、239E、332E、332D、239D/332E、267D、267E、328F、267E/328F、236A/332E、239D/332E/330Y、239D、332E/330L、299T以及297N(参见US11/124,620、US6,737,056，此处全文引入)。进行Fc修饰以增加其生物半衰期，例如可以引入以下突变中的一种或多种：T252L、T254S、T256F(参见US6,277,375)；为了增加生物半衰期，可以在CH1或CL区内改变抗体以含有从IgG的Fc区的CH2结构域的两个环获得的挽救受体结合表位(参见US5,869,046、US6,121,022)；用于增加血清半衰期的额外突变，包括428L、434A、434S和428L/434S(参见US8,883,973号、US6,737,056、US7,371,826，此处全文引入)。通过将至少一个氨基酸残基替换来改变Fc区，从而改变抗体的效应子功能，例如可以将一个或多个选自氨基酸残基234、235、236、237、297、318、320和322的氨基酸替换，以使得抗体对效应配体的亲合力改变，但是保留亲本抗体的抗原结合能力。亲合力改变了的效应配体可以是例如补体的Fc受体或C1组分(参见US5,624,821、US5,648,260，此处全文引入)。改变氨基酸位置231和239内的一个或多个氨基酸残基，从而改变抗体固定补体的能力(参见WO 94/29351，此处全文引入)。通过修饰以下位置处的一个或多个氨基酸来修饰Fc区以提高抗体介导抗体依赖性细胞毒性(ADCC)的能力和/或提高抗体对Fcγ受体的亲合力：238、239、248、249、252、254、255、256、258、265、267、268、269、270、272、276、278、280、283、285、286、289、290、292、293、294、295、296、298、301、303、305、307、309、312、315、320、322、324、326、327、329、330、331、333、334、335、337、338、340、360、373、376、378、382、388、389、398、414、416、419、430、434、435、437、438或439(参见WO00/42072，全文引入)。此外，人类IgG1上关于FcγRI、FcγRII、FcγRIII和FcRn的结合位点已经映射并且具有改良结合的变异体已有描述(参见Shields，R.L.等人(2001)《生物化学杂志(J.Biol.Chem.)》276：6591-6604)。在位置256、290、298、333、334和339处的特异性突变显示可改良与FcyRIII的结合。另外，以下组合突变体显示可改良FcγRIII结合：T256A/S298A、S298A/E333A、S298A/K224A和S298A/E333A/K334A。 此外，如M252Y/S254T/T256E或M428L/N434S等突变改良了与FcRn的结合并且增加了抗体循环半衰期(参见Chan CA和Carter PJ(2010)《自然综述免疫学(Nature Rev Immunol)》10：301-316)。
本公开的抗体的修饰包括聚乙二醇化(PEG化)或添加其它水溶性部分，以例如为了增强半衰期。“PEG化”是指至少一个PEG分子与另一个分子(例如治疗性蛋白)连接。例如，PEG是在一端与羟基连接的直链或支链聚醚，并且具有下列常规结构：HO-(CH 2CH 2O) n-CH 2CH 2-OH。为了使PEG与分子(多肽、多糖、多核苷酸和小的有机分子)偶联，可以通过制备一些或两个末端具有官能团的PEG的衍生物来活化PEG。蛋白的PEG缀合的常见途径是用官能团活化PEG，该官能团适合与赖氨酸和N-末端氨基酸基团的反应。尤其是，参与缀合的常见反应基团是赖氨酸的α或ε氨基。聚乙二醇化连接基与蛋白的反应可导致PEG部分主要在下列位点处的连接：蛋白的N-末端的α氨基、赖氨酸残基侧链上的ε氨基、或组氨酸残基侧链上的咪唑基。由于大部分重组蛋白质具有单个α和许多ε氨基和咪唑基，可以根据连接基团的化学性质，产生许多位置异构体。
本公开工程化的抗体或抗原结合片段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。重组的免疫球蛋白表达载体可以稳定地转染CHO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。可溶的混合物和多聚体，也可以用常规方法去除，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。
“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，诸如包含本公开的任一种抗体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 治疗方法或制品)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。
“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***片段的载体的受体。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，子代可不一定与原始亲本细胞完全相同(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，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。
“载体”意指能够在宿主细胞中递送并且在一些实施例中表达一种或多种目的基因或序列的构建体。载体的例子包括但不限于病毒载体、裸露DNA或RNA表达载体、质粒、粘粒或噬菌体载体、与阳离子缩合剂结合的DNA或RNA表达载体、包封在脂质体中的DNA或RNA表达载体、以及某些真核细胞如生产细胞。
“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地场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本文所述抗体或抗原结合片段或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
“药学可接受的载体”或“药学可接受的赋形剂”包括当与活性成分组合时，允许该成分保留生物学活性并且不与受试者的免疫***反应的任何材料。例子包括但不限于任何标准药物载体，例如磷酸盐缓冲盐水溶液、水、乳剂如油/水乳剂、和各种类型的润湿剂。在一些实施例中，用于气雾剂或肠胃外施用的稀释剂是磷酸盐缓冲盐水(PBS)或生理(0.9％)盐水。包含此类载体的组合物通过众所周知的常规方法配制(参见例如Remington′s Pharmaceutical Sciences，第18版，A.Gennaro，编辑，Mack PublishingCo.，Easton，PA，1990；以及R Remington，The Science and Practice of Pharmacy第20版Mack Publishing，2000)。
本公开的“PVRIG结合蛋白”或“PVRIG抗体”可以例如缀和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PVRIG结合蛋白或PVRIG抗体的缀和方式可以通过常规方法实现。
以下结合实施例用于进一步描述，但这些实施例并非限制的范围。
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.PVRIG蛋白质序列及制备
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)为购买自Acrobiosystems公司的纯化商业蛋白试剂，其序列见表1。
表1.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 PCTCN2021080470-appb-000001
在HEK293细胞中通过常规方法进行瞬时转染表达重组蛋白，收集上清并通过Ni-NTA进行纯化。经检测，获得cyno-PVRIG-his。
实施例2.抗人PVRIG单域抗体的产生
抗人PVRIG单克隆单域抗体通过免疫骆驼产生。免疫抗原为带his标签的人PVRIG重组蛋白(h-PVRIG-his)。用弗氏佐剂(Sigma，Lot No.：F5881/F5506) 乳化：首次用弗氏完全佐剂(CFA)CFA，其余加强免疫用弗氏不完全佐剂(IFA)。免疫注射时间为第0，14，28，42天。于第56天采血进行血检，用ELISA方法检测骆驼血清，确定骆驼血清中的抗体滴度。
取200mL骆驼外周血，分离其中的PBMC，用Trizol提取细胞中的RNA，并反转录成cDNA。用PCR的方法扩增单域抗体可变区的基因，克隆到噬菌体载体中，从而建立抗人PVRIG单域抗体的噬菌体文库。
将噬菌体文库用BSA稀释封闭，与磁珠Dynabeads(M-280,invitrogen)共孵育，收集负筛孵育后的噬菌体。用生物素标记的带有his标签的人PVRIG包被封闭Dynabeads，将负筛后收集的噬菌体悬液与所述Dynabeads孵育，用胰酶洗脱噬菌体。经过3轮筛选，挑选第3轮筛选获得的400个克隆进行测序，其中5株单域抗体的重链序列如表2所示，不同编码规则的CDR如表3所示。
表2.抗PVRIG抗体重链可变区(HCVR)序列
Figure PCTCN2021080470-appb-000002
表3.抗PVRIG抗体的重链互补决定区(CDR)序列
Figure PCTCN2021080470-appb-000003
Figure PCTCN2021080470-appb-000004
实施例3.全长抗PVRIG抗体的制备
将实施例2中5个抗体的重链可变区与人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突变(Eu命名***)。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。
5个抗体和阳性抗体的全长序列如表4所示。
表4.全人源抗PVRIG抗体的重轻链全长序列
Figure PCTCN2021080470-appb-000005
Figure PCTCN2021080470-appb-000006
(注：下划线为重链的Fc结构域)
合成上述序列，用BamHI和XhoI消化后，通过BamHI/XhoI酶切位点***到pcDNA3.1表达载体(Life Technologies Cat.No.V790-20)中。将表达载体和转 染试剂PEI(Polysciences，Inc.Cat.No.23966)以1:2的比例转染HEK293细胞(Life Technologies Cat.No.11625019)，并置于CO 2孵育箱中孵育4-5天。表达的抗体通过离心回收后，按常规方法进行抗体纯化，经检测，获得目的抗体。
实施例4.抗PVRIG抗体与PVRIG重组蛋白的结合实验
ELISA实验被用来检测抗PVRIG抗体的结合特性。用直接包被带his标签的PVRIG重组蛋白，抗体加入后，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孔酶标板，按1μg/mL浓度每孔100μL，4℃孵育过夜。洗液洗三遍，每孔250μL。每次洗涤震荡10秒以保证充分清洗。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250μL。每次洗涤震荡10秒以保证充分清洗。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250μL。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250μL。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的结合EC 50值。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5.抗PVRIG抗体对不同种系PVRIG重组蛋白的结合实验结果
Figure PCTCN2021080470-appb-000007
实施例5.抗PVRIG抗体与表达PVRIG的细胞结合实验
用流式细胞仪(FACS)来检测抗PVRIG抗体的结合特性。构建过表达人或食蟹猴PVRIG的细胞株，抗体加入后，通过加入二抗检测抗体与抗原结合的活性。
将带有人或食蟹猴PVRIG基因序列的表达质粒在HEK293细胞中进行转染，并用抗生素筛选和无限稀释法获得过表达稳定转染单克隆细胞株。在96孔板中每孔接种2x10 5个过表达细胞。300g离心5分钟，去除上清，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6.抗PVRIG抗体对不同种系PVRIG的细胞结合实验结果
Figure PCTCN2021080470-appb-000008
(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。)
实施例6.抗PVRIG抗体阻断PVRIG和PVRL2结合实验
本实验中，通过体外阻断实验，检测所筛选出来的抗PVRIG抗体阻断人PVRIG和其配体人PVRL2的结合能力。具体方法是将带小鼠IgG2a Fc标签的人PVRIG重组蛋白(h-PVRIG-mIgG2a Fc)包被到96孔酶标板上，加入抗PVRIG抗体充分结合占据表位后，再加入带his标签的PVRL2(PV2-H52E2，AcroBiosystem)，通过检测his标签，来计算PVRIG与PVRL2的结合量，计算抗PVRIG抗体对PVRIG活性位点阻断的IC 50值。
h-PVRIG-mIgG2a Fc蛋白包被96孔酶标板，按1μg/mL浓度每孔100μL，4℃孵育过夜。洗液洗三遍，每孔250μL。每次洗涤震荡10秒以保证充分清洗。加入300μL/孔封闭液室温孵育1小时。洗液洗三遍，每孔250μL。每次洗涤震荡10秒以保证充分清洗。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250μL。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250μL。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。
结果显示，所有检测抗体均可以强烈抑制人PVRIG与人PVRL2的结合。
表7.抗体对人PVRIG/PVRL2结合的阻断实验
抗体编号 ELISA IC 50(nM)
20 1.18
30 1.11
38 0.93
39 0.76
151 0.37
Tab5 1.16
IgG4 无阻断
实施例7.抗PVRIG抗体与PVRIG的亲合力测定
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。将传感器置于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的亲合力如表8所示。
结果显示，所有检测抗体均具有与人PVRIG的高亲合力。
表8.抗PVRIG抗体与人PVRIG的亲合力
抗体编号 Kon(1/Ms) Koff(1/s) KD(M)
20 3.43E+05 8.07E-05 2.36E-10
30 2.84E+05 2.05E-04 7.23E-10
38 1.32E+05 2.87E-04 2.17E-09
39 2.42E+05 1.69E-04 6.96E-10
151 2.61E+05 5.22E-05 2.00E-10
Tab5 7.37E+05 1.61E-05 2.19E-10
实施例8.抗PVRIG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。实验结果如图1和表9所示。
结果显示，所有检测抗体均有较强的激活Jurkat细胞中Luciferase的能力，活性是阳性抗体的3.7-18.5倍，证明这些抗体可以结合PVRIG并阻断PVRL2与PVRIG的结合。
表9.抗PVRIG抗体报告基因细胞活性实验结果
抗体株编号 PVRIG报告基因细胞活性实验EC 50(nM)
20 0.04
30 0.06
38 0.20
39 0.06
151 0.04
Tab5 0.74
IgG4 无结合
实施例9.抗PVRIG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。
将培养的NK92细胞系(人恶性非霍奇金淋巴瘤患者的NK细胞)用洗液(包括RPMI 1640，5％FBS，10ng/mL IL-2)洗涤两次，并重悬至2×10 6个/mL的密度。在96孔板中每孔加入50μL(总计1×10 5个)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，裂解孔中所有细胞，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)，150μL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。
实验结果如图2和表10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10.抗PVRIG抗体的NK细胞杀伤实验
Figure PCTCN2021080470-appb-000009
实施例10.抗PVRIG抗体的混合淋巴细胞反应(MLR)实验
PVRIG在T细胞上表达，而PVRL2在树突状细胞(DC细胞)中表达。抗PVRIG抗体可以通过阻断PVRL2与PVRIG的结合，解除树突状细胞对T细胞的抑制作用，活化T细胞。
混合淋巴细胞反应，是指两个无关个体、功能正常的淋巴细胞在体外混合培养时，由于主要组织相容性抗原不同，可相互刺激对方的T细胞发生增殖。从第一个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)的终浓度添加细胞因子，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120h，取上清，用ELISA试剂盒(R&D，DY202)检测上清中的IFNγ含量。
实验结果如图3和表11所示，表明，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，本公开的抗体较之阳性对照Tab5的效果更优。
表11.抗PVRIG抗体混合淋巴细胞反应IFNγ分泌量
Figure PCTCN2021080470-appb-000010
实施例11.抗PVRIG抗体的人源化改造
在所获得的骆驼单域抗体20、抗体30、抗体38、抗体39和抗体151的VH典型结构的基础上，将重链可变区序列与抗体GermLine数据库比较，获得同源性高的人种系模板。将骆驼单域抗体的框架区替换为人种系模板的重链框架区，CDR(根据Kabat编号***)保留，再与人源IgG的Fc区域(带有S228P，F234A，L235A，K447A突变的IgG4Fc)重组。以骆驼单域抗体的三维结构为基础，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，产生一系列人源化单域抗体。各个单域抗体的人种系模板以及人源化抗体重链可变区序列如表12-16所示。
表12.抗体20的人种系模板和人源化抗体序列
Figure PCTCN2021080470-appb-000011
Figure PCTCN2021080470-appb-000012
根据表12，抗体20H1-20H5包含如TDCMG(SEQ ID NO：7)所示的CDR1，如HIDSDGIPRYVDSVKG(SEQ ID NO：8)所示的CDR2，和如GFKFDEDYCAPND(SEQ ID NO：150)所示的CDR3。
表13.抗体30的人种系模板和人源化抗体序列
Figure PCTCN2021080470-appb-000013
根据表13，抗体30H1-30H5包含如GDCMG(SEQ ID NO：10)所示的CDR1，如TIDNAGRIKYADSVKG(SEQ ID NO：11)所示的CDR2，如GWTFGGQCSPAD(SEQ ID NO：151)所示的CDR3。
表14.抗体38的人种系模板和人源化抗体序列
Figure PCTCN2021080470-appb-000014
Figure PCTCN2021080470-appb-000015
表15.抗体39的人种系模板和人源化抗体序列
Figure PCTCN2021080470-appb-000016
表16.抗体151的人种系模板和人源化抗体序列
Figure PCTCN2021080470-appb-000017
Figure PCTCN2021080470-appb-000018
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突变。
>人IgG4重链Fc区域(S228P/F234A/L235A/K447A)
Figure PCTCN2021080470-appb-000019
>人IgG4重链Fc区域(S228P/K447A)
Figure PCTCN2021080470-appb-000020
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12.人源化抗PVRIG抗体与表达PVRIG的细胞结合实验
依照实施例5的方法，用流式细胞仪检测人源化抗PVRIG抗体与人或食蟹猴PVRIG的结合。实验结果如表17所示。
表17.人源化抗PVRIG单域抗体对不同种系PVRIG的FACS结合实验结果
Figure PCTCN2021080470-appb-000021
Figure PCTCN2021080470-appb-000022
(注：N.T.，not tested，未测试。)
实施例13.人源化抗PVRIG抗体与PVRIG的亲合力测定
依照实施例7的方法，检测人源化抗PVRIG抗体与人PVRIG的亲合力。结果如表18所示。表中列出的所有抗体均具有与人PVRIG的高亲合力。
表18.人源化抗PVRIG抗体与人PVRIG的亲合力
抗体编号 Kon(1/Ms) Koff(1/s) KD(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
39H1 2.64E+05 8.65E-04 3.28E-09
39H2 1.80E+05 1.24E-04 6.92E-10
39H4 1.89E+05 9.11E-05 4.82E-10
151H7 1.57E+05 1.88E-04 1.20E-09
实施例14.人源化抗PVRIG抗体报告基因细胞活性实验
依照实施例8的方法，检测人源化抗PVRIG抗体在报告基因细胞中的活性。实验结果如图4A-图4B和表19所示。表中列出的抗体均具有激活Jurkat细胞的能力。
表19.人源化抗PVRIG抗体报告基因细胞活性实验
Figure PCTCN2021080470-appb-000023
实施例15.人源化抗PVRIG抗体的活化NK细胞杀伤能力实验
依照实施例9的方法，检测人源化抗PVRIG抗体对NK细胞的活化能力。实验结果如图5A-图5B和表20-表21所示。结果显示，本公开人源化抗PVRIG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20.人源化抗PVRIG抗体的NK细胞杀伤实验
Figure PCTCN2021080470-appb-000024
表21.人源化抗PVRIG抗体的NK细胞杀伤实验
Figure PCTCN2021080470-appb-000025
Figure PCTCN2021080470-appb-000026
实施例16.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单域抗体151通过GGGGSGGGGS(SEQ ID NO：152)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22所示。抗TIGIT抗体序列信息如表23-24所示。此处全文引入WO2019062832A中的TIGIT抗体。
表22.抗PVRIG/TIGIT双特异性抗体的第一、第二多肽链序列
Figure PCTCN2021080470-appb-000027
Figure PCTCN2021080470-appb-000028
Figure PCTCN2021080470-appb-000029
表23.抗TIGIT抗体重链及轻链CDR区序列(Kabat编号规则)
Figure PCTCN2021080470-appb-000030
Figure PCTCN2021080470-appb-000031
表24.抗TIGIT抗体重链VH及轻链VL序列
Figure PCTCN2021080470-appb-000032
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。双特异性抗体的表达量和纯度如表25所示。将纳米抗体偶联到普通单克隆抗体上，无论是重、轻链的N端还是C端，均显示了良好的表达量和纯度。
表25.抗PVRIG/TIGIT双特异性抗体的表达量和纯度
抗体编号 表达量(mg/L) SEC纯度(％)
1708-151-1 140 97.6
1708-151-2 108 95.7
1708-151-3 160 94.7
1708-151-4 158 96.2
实施例17.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实验
A)不同构型的双特异性抗体与人PVRIG的结合及对配体PVRL2的阻断
依照实施例4、实施例5和实施例6的方法，进行实验，结果如表26所示。结果表明，不同构型的双特异性抗体，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，基本一致、无差异。
表26.双特异性抗体对PVRIG的结合和配体阻断
Figure PCTCN2021080470-appb-000033
B)不同构型的双特异性抗体与人TIGIT的结合及对配体PVR的阻断
依照实施例4、实施例5和实施例6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27所示。结果表明，不同构型的双特异性抗体和抗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，基本一致、无差异。抗PVRIG抗体151的连接方式对于抗TIGIT抗体与TIGIT的结合基本无影响。
表27.双特异性抗体对TIGIT的结合和配体阻断
Figure PCTCN2021080470-appb-000034
综合表24-25的数据，我们发现抗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。
实施例18.人源化抗PVRIG/TIGIT双特异性抗体的制备
将不同的人源化抗PVRIG抗体(20H5，30H2，39H2，151H7，151H8) 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，构建双抗，序列如表28所示。
表28.人源化抗PVRIG/TIGIT双特异性抗体的第一和第二多肽链全长序列
Figure PCTCN2021080470-appb-000035
Figure PCTCN2021080470-appb-000036
Figure PCTCN2021080470-appb-000037
按常规方法进行抗体的瞬时转染、表达和纯化，经鉴定，得到目的双抗。
实施例19.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4、5、6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和图6A-6E所示。结果表明，各个人源化双特异性抗体，均可以结合人PVRIG，阻断PVRIG结合PVRL2。1708-151H8对食蟹猴PVRIG结合较弱。
表29.人源化双特异性抗体对PVRIG的结合和配体阻断
Figure PCTCN2021080470-appb-000038
与实施例4、5、6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和图7A-图7E所示。结果表明，各双抗均可以结合人和食蟹猴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 PCTCN2021080470-appb-000039
Figure PCTCN2021080470-appb-000040
利用Biacore检测人源化双特异性抗体与人PVRIG，食蟹猴PVRIG，和人TIGIT的亲合力。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)，食蟹猴PVRIG抗原(SEQ ID NO:1)，或人TIGIT抗原(AcroBiosystem，TIT-H52H3)。利用Biacore仪器(Biacore X100，GE)实时检测反应信号从而获得结合和解离曲线。实验得到的数据用BiacoreX100 evaluation software2.0GE软件以(1:1)Binding模型进行拟合，得出亲合力数值，见表31。
表31.人源化双特异性抗体与人PVRIG，食蟹猴PVRIG，和人TIGIT的亲合力
Figure PCTCN2021080470-appb-000041
实施例20.人源化抗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10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8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 PCTCN2021080470-appb-000042
实施例21.抗PVRIG/TIGIT双特异性抗体在人黑色素瘤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，除了上述IgG4亚型的双特异性抗体，我们也合成了相应的IgG1亚型的抗体，用于动物药效试验。在该实验中用到的其他之前未描述的抗体序列如表33。
表33.IgG1型人源化抗PVRIG/TIGIT双特异性抗体的第一和第二多肽链全长序列
Figure PCTCN2021080470-appb-000043
Figure PCTCN2021080470-appb-000044
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34，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34.给药方案
Figure PCTCN2021080470-appb-000045
Figure PCTCN2021080470-appb-000046
(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35-36和图9A-图9B。
表35.抗PVRIG/TIGIT双特异性抗体对小鼠体重的影响
Figure PCTCN2021080470-appb-000047
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 PCTCN2021080470-appb-000048
(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单药组没有明显差异。抗TIGIT抗体1708-IgG1单药组，抗PVRIG抗体151与抗TIGIT抗体1708-IgG1联用组，1708-151-IgG1双抗组，肿瘤体积下降。而1708-151-IgG4双抗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9B)。
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37，分组给药当天为第0天。
表37.给药方案
Figure PCTCN2021080470-appb-000049
Figure PCTCN2021080470-appb-000050
(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38-39和图10A-图10B。
表38.抗P RIG/TIGIT双特异性抗体对小鼠体重的影响
Figure PCTCN2021080470-appb-000051
表39.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 PCTCN2021080470-appb-000052
(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，1708-30H2IgG4与1708-151H7IgG4双抗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10A和图10B)。
虽然以上描述了本公开的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本公开的保护范围由所附权利要求书限定。

Claims (21)

  1. PVRIG结合蛋白，其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、80-84任一所示序列中的CDR1、CDR2和CDR3；或
    SEQ ID NO：2、75-79任一所示序列中的CDR1、CDR2和CDR3；或
    SEQ ID NO：4、86-90任一所示序列中的CDR1、CDR2和CDR3；或
    SEQ ID NO：5、91-95任一所示序列中的CDR1、CDR2和CDR3；或
    SEQ ID NO：6、96-100任一所示序列中的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，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：10、11和12或151所示；或
    如SEQ ID NO：7、8和9或150所示；或
    如SEQ ID NO：13、14和15所示；或
    如SEQ ID NO：16、17和18所示；或
    如SEQ ID NO：19、20和21所示。
  2. 如权利要求1所述的PVRIG结合蛋白，其中所述PVRIG结合蛋白的免疫球蛋白单一可变结构域为VHH，
    优选为人源化的和/或经亲合力成熟的VHH；
    更优选地，所述人源化的和/或经亲合力成熟的VHH包含人种系模板的重链框架区IGHV3-7*01或IGHV3-30*02。
  3. 如权利要求1或2所述的PVRIG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分别如
    SEQ ID NO：3、80-84中任一所示；或
    SEQ ID NO：2、75-79中任一所示；或
    SEQ ID NO：4、86-90中任一所示；或
    SEQ ID NO：5、91-95中任一所示；或
    SEQ ID NO：6、96-100中任一所示；或
    与前述序列任一具有至少80％、至少90％、至少95％、至少98％或至少99％的序列同一性。
  4. 如权利要求1-3中任一项所述的PVRIG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或IgG4的Fc区；
    更优选地，所述人IgG4的Fc区具有S228P、F234A、L235A和/或K447A突变。
  5. PVRIG/TIGIT结合蛋白，其包含特异性结合PVRIG的第一抗原结合结构域和特异性结合TIGIT的第二抗原结合结构域，所述特异性结合PVRIG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、80-84任一所示序列中的CDR1、CDR2和CDR3；或
    SEQ ID NO：2、75-79任一所示序列中的CDR1、CDR2和CDR3；或
    SEQ ID NO：4、86-90任一所示序列中的CDR1、CDR2和CDR3；或
    SEQ ID NO：5、91-95任一所示序列中的CDR1、CDR2和CDR3；或
    SEQ ID NO：6、96-100任一所示序列中的CDR1、CDR2和CDR3；
    所述CDR1、CDR2、CDR3是根据Kabat、IMGT、Chothia、AbM或Contact编号***定义的；
    优选地，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：10、11和12或151所示；或
    如SEQ ID NO：7、8和9或150所示；或
    如SEQ ID NO：13、14和15所示；或
    如SEQ ID NO：16、17和18所示；或
    如SEQ ID NO：19、20和21所示。
  6. 如权利要求5所述的PVRIG/TIGIT结合蛋白，其中所述第一抗原结合结构域中的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、80-84中任一所示；或
    SEQ ID NO：2、75-79中任一所示；或
    SEQ ID NO：4、86-90中任一所示；或
    SEQ ID NO：5、91-95中任一所示；或
    SEQ ID NO：6、96-100中任一所示；或
    与前述序列任一具有至少80％、至少90％、至少95％、至少98％或至少99％的序列同一性。
  7. 如权利要求5或6中任一项所述的PVRIG/TIGIT结合蛋白，其中所述特异性结合TIGIT的第二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：121、122和123所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：124、125和126所示的LCDR1、LCDR2和LCDR3；或
    所述重链可变区包含分别如SEQ ID NO：115、116和117所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：118、119和120所示的LCDR1、LCDR2和LCDR3；或
    所述重链可变区包含分别如SEQ ID NO：127、128和129所示的HCDR1、HCDR2和HCDR3,所述轻链可变区包含分别如SEQ ID NO：130、131和132所示的LCDR1、LCDR2和LCDR3；或
    所述重链可变区包含分别如SEQ ID NO：133、134和135所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：136、137和138所示的LCDR1、LCDR2和LCDR3；或
    所述重链可变区包含分别如SEQ ID NO：139、140和141所示的HCDR1、HCDR2和HCDR3,所述轻链可变区包含分别如SEQ ID NO：142、143和144所示的LCDR1、LCDR2和LCDR3。
  8. 如权利要求7所述的PVRIG/TIGIT结合蛋白，其中，所述特异性结合TIGIT的第二抗原结合结构域的重链可变区包含如SEQ ID NO：145-147中任一所示或与之具有至少90％、至少95％、至少98％、至少99％序列同一性的氨基酸序列，轻链可变区包含如SEQ ID NO：148-149中任一所示或与之具有至少90％、至少95％、至少98％、至少99％序列同一性的氨基酸序列。
  9. 如权利要求8所述的PVRIG/TIGIT结合蛋白，其中，所述特异性结合TIGIT的第二抗原结合结构域包含全长重链(HC)和全长轻链(LC)；
    优选地，所述全长重链为IgG1或IgG4同种型，所述全长轻链为Kappa同种型；
    更优选地，所述重链序列为SEQ ID NO：102所示或与之具有至少90％序列同一性，所述轻链序列为SEQ ID NO：103所示或与之具有至少90％序列同一性。
  10. 如权利要求5-9中任一项所述的PVRIG/TIGIT结合蛋白，所述特异性结合TIGIT的第二抗原结合结构域包含重链可变区(VH)和轻链可变区(VL)，其中：
    所述特异性结合PVRIG的第一抗原结合结构域的免疫球蛋白单一可变结构域位于特异性结合TIGIT的第二抗原结合结构域的重链可变区的N端；
    所述特异性结合PVRIG的第一抗原结合结构域的免疫球蛋白单一可变结构域位于特异性结合TIGIT的第二抗原结合结构域的重链可变区的C端；
    所述特异性结合PVRIG的第一抗原结合结构域的免疫球蛋白单一可变结构域位于特异性结合TIGIT的第二抗原结合结构域的轻链可变区的N端；和/或
    所述特异性结合PVRIG的第一抗原结合结构域的免疫球蛋白单一可变结构域位于特异性结合TIGIT的第二抗原结合结构域的轻链可变区的C端。
  11. 如权利要求10所述的PVRIG/TIGIT结合蛋白，其中，特异性结合PVRIG的第一抗原结合结构域的免疫球蛋白单一可变结构域与特异性结合TIGIT的第二抗原结合结构域直接或通过连接子相连接；
    优选地，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的整数；
    更优选地，所述连接子为(G 4S) 2、(G 4S) 3所示的氨基酸序列。
  12. 如权利要求5-11中任一项所述的PVRIG/TIGIT结合蛋白，其包含第一多肽链和第二多肽链，其中：
    所述第一多肽链包含如SEQ ID NO：108-112和114中任一所示的氨基酸序列，所述第二多肽链包含如SEQ ID NO：103所示的氨基酸序列；或者
    所述第一多肽链包含如SEQ ID NO：104或105所示的氨基酸序列，所述第二多肽链包含如SEQ ID NO：103所示的氨基酸序列；或者
    所述第一多肽链包含如SEQ ID NO：102所示的氨基酸序列，所述第二多肽链包含如SEQ ID NO：106或107所示的氨基酸序列。
  13. 抗PVRIG抗体或其抗原结合片段，其含有权利要求1-3或5-6中任一项所述的免疫球蛋白单一可变结构域；
    优选地，其还包含权利要求7中所述的特异性结合TIGIT的第二抗原结合结构域。
  14. 多核苷酸，其编码权利要求1-4任一项所述的PVRIG结合蛋白、权利要求5-12任一项所述的PVRIG/TIGIT结合蛋白或权利要求13所述的抗PVRIG抗体或其抗原结合片段。
  15. 宿主细胞，其包含权利要求14所述的多核苷酸。
  16. 制备PVRIG结合蛋白、PVRIG/TIGIT结合蛋白、或抗PVRIG抗体或其抗原结合片段的方法，包括：
    在权利要求15所述的宿主细胞中表达权利要求14所述的多核苷酸，以及
    从所述宿主细胞中分离表达的PVRIG结合蛋白、PVRIG/TIGIT结合蛋白或抗PVRIG抗体或其抗原结合片段。
  17. 药物组合物，其含有权利要求1-4任一项所述的PVRIG结合蛋白、权利要求5-12任一项所述的PVRIG/TIGIT结合蛋白、或权利要求13所述的抗PVRIG抗体或其抗原结合片段，以及，可药用的赋形剂、稀释剂或载体。
  18. 治疗疾病或延缓疾病进展的方法，包括：
    向受试者施用治疗或延缓疾病有效量的权利要求1-4任一项所述的PVRIG结合蛋白、权利要求5-12任一项所述的PVRIG/TIGIT结合蛋白、或权利要求13所述的抗PVRIG抗体或其抗原结合片段、权利要求14所述的多核苷酸、或权利要求17所述的药物组合物，或其任意组合，
    优选地，所述疾病为增殖性疾病；
    更有选地，所述增殖性疾病为癌症；
    更优选地，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***癌症或者任何特征在于不受控细胞生长的其它疾病或病症。
  19. 活化NK细胞、γδT细胞和/或Th1细胞的方法，包含向有需要的受试者施用有效量的权利要求1-4任一项所述的PVRIG结合蛋白、权利要求5-12任一项所述的PVRIG/TIGIT结合蛋白、或权利要求13所述的抗PVRIG抗体或其抗原结合片段、权利要求14所述的多核苷酸、或权利要求17所述的药物组合物或其任意组合。
  20. 增加受试者体内的干扰素-γ产生和/或促炎性细胞因子分泌的方法，包含向有需要的受试者施用有效量的权利要求1-4任一项所述的PVRIG结合蛋白、权利要求5-12任一项所述的PVRIG/TIGIT结合蛋白、或权利要求13所述的抗PVRIG抗体或其抗原结合片段、权利要求14所述的多核苷酸、或权利要求17所述的药物组合物或其任意组合。
  21. 根据权利要求1-4任一项所述的PVRIG结合蛋白、权利要求5-12任一项所述的PVRIG/TIGIT结合蛋白、或权利要求13所述的抗PVRIG抗体或其抗原结合片段、权利要求14所述的多核苷酸、或权利要求17所述的药物组合物，用于治疗或延缓疾病，优选地，所述疾病为增殖性疾病；
    更有选地，所述增殖性疾病为癌症；
    更优选地，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***癌症或者任何特征在于不受控细胞生长的其它疾病或病症。
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